Polypeptide Antibiotics

3.8 Polypeptide Antibiotics

Interestingly, a plethora of polypeptides of bacterial origin that are found to comprise of D- and L-amino acids, do exert a marked and pronounced antibiotic activity. It is, however, pertinent to mention here that these specific antibiotics have two inherent major anomalies, namely: first, very poor absorption from the intestinal tract; and secondly, possess high degree of nephrotoxicity* when used systemically. Generally, the polypeptide antibiotics exert a predominantly Gram-positive spectrum; however, there are a few-exceptions that are solely active against Gram-negative organisms, such as: the strongly basic polymyxins.

It has been observed that these polypeptide antibiotics have a tendency to occur as mixtures of very close structurally related compounds. Nevertheless, the exact composition of commercial mixtures depend to a great extent upon the skilful usage of selected strains of producing organisms.

Besides, a precise and reliable strength of therapeutic response against certain susceptible organisms is exclusively based on the quantitative microbial assay.

The various important members of ‘polypeptide antibiotics’ are, namely: cycloserine; polymyxin-B; colistin (polymixin-E), bacitrasin; vanomycin; and teichoplanin, which shall now be treated separately as under:

3.8.1 Cycloserine

Synonyms Closina; Farmiserina; Micoserina; Orientomycin; Oxamycin; Seromycin; PA-94.

Biological Sources It is a polypeptide antibiotic substance produced by Streptomyces garyphalus sive orchidaceus.**

Preparation It may also be synthesized by the method of Stammer et al.***

Chemical Structure

D-4-Amino-3-isoxazolidinone; C₃H₆N₂O₂.

Characteristic Features

1. It is obtained as crystals that decompose at 155-156°C.

2. Its specific optical rotations are: [α]_D²³ + 116° (C = 1.17); [α]²⁵₅₄₆ + 137° (C = 5 in 2N NaOH).

3. It has uv_max : 226 nm (E^1%_1cm 402).

4. Its aqueous solutions have a pH 6.

5. It is fairly soluble in water; and slightly soluble in methanol and propylene glycol.

6. It is found to form salts readily with acids and bases.

7. Its aqueous solutions buffered to pH 10 with Na₂CO₃ may be stored without any loss of activity upto a duration of one week between 0-10°C (i.e., at refrigerated temperatures).

Uses

1. It exhibits a fairly broad spectrum of activity; however, its therapeutic efficacy is exclusively associated with its inherent inhibitory effect on Mycobacterium tuberculosis.

2. It precisely inhibits alanine racemase, which action precludes the incorporation of D-alanine strategically into the pentapeptide side-chain of the specific murein* component of bacterial cell walls. Perhaps this unique features is solely responsible for its antibiotic activity.

3. It is invariably regarded as an ‘antibiotic of second choice’; and is frequently used in conjunction with isoniazid in the control, management and treatment of tuberculosis who usually fail to respond to the first-line agents.**

4. It is readily absorbed orally and is subsequently exerted quickly through the kidneys (i.e., newly 50% without any metabolic alteration whatsoever).

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* A toxic substance that causes damage to kidney tissues.

** Kuehl, Jr. et al. J. Am. Chem. Soc., 77, 2344, (1955).

*** Stammer et al. J. Am. Chem. Soc., 77, 2346, (1955).

3.8.2 Polymixin B

Polymyxins represent a group of cycle polypeptide antibiotics produced by various species of Bacillus. However, polymyxins A to E were primarily isolated from Bacillus polymyxa. Subsequently, it was shown that both polymyxin B and polymycin E (or colistin) were mixtures of two components each. The structures of polymyxin B₁ and polymyxin B₂; and polymyxin E₁ (colistin A) and polymyxin E₂ (colistin B) are as given below:

Actually, these molecules essentially contain ten amino acids, of which six happen to be L-α-, γ-diaminobutyric acid (L-Dab), having a fatty acid*** strategically bonded to the N-terminus; besides, a cyclic peptide portion meticulously designed via an amide bond located in between the γ-amino of one of the Dab-residues and the carboxyl terminus. Interestingly, the γ-amino functions

of the remaining Dab residues distinctly attribute a rather strong basic property to the various

antibiotics. This particular characteristic feature confers detergent-like properties and perhaps permits

them to either get bound or cause damage to bacterial membranes.

Characteristic Features

Polymyxin Hydrochloride

1. It is obtained as nearly colourless powder that gets decomposed at 228-230°.

2. It has specific optical rotation [α]_D²³-40° (C = 1.05).

3. It is very soluble (> 40%) in water and methanol; the solubility decreases considerably in higher alcohols; and almost insoluble in ethers, esters, ketones, hydrocarbons and the chlorinated solvents.

4. It usually gives rise to water insoluble salts with the help of a host of precipitants, such as: helianthic acid (C₇H₉O₄) picric acid; and Reinacke salt.

Polymyxin B: It is a mixture of Polymyxins B₁ and B₂. The mixture also contains minimal amounts of the more toxic polymyxins A, C and D. Both polymyxins B₁ and B₂ essentially possess a cyclopeptidic structure and comprise of six residues of α, γ-diaminodutyric acid (DABs). However, the latter characteristic feature affords an exceptionally strong basic property to the polymixin antibiotics.

It has specific optical rotation [α]₅₄₆₁-106.3° (1N. HCl).

Uses

1. It is used topically in ointments (usually 5000 or 10,000 Units/g) and ophthalmic solutions (10,000 Units/ml).

2. It was employed formerly for control, management and treatment of infections of the intestinal tract caused by Shigella, Pseudomonas aeruginosa, and E. coli.

Polymyxin B Sulphate [Synonyms Aerosporin; Mastimyxin;]: It is the sulphate salt of a substance produced by the growth of Bacillus polymyxa (Prazmowski) Mignla belonging to the natural order Bacillaceae. It has a potency of not less than 600 Units of polymyxin B. mg^–1, calculated on the anhydrous basis.

Preparation The filtered broth obtained from the fermentation process (section 3.7) is eventually treated with a ‘certified dye’, and the resulting polymyxin B-dye salt complex thus precipitated is collected by means of filtration, washed with water and finally treated with an alcoholic solution of a lower aliphatic amine sulphate. The polymyxin B sulphate thus produced is filtered off and subsequently purified and lypholized. Polymyxin B is a mixture of polymyxin B₁ (C₅₆H₉₈N₁₆O₁₃), and polymyxin B₂ (C₅₅H₉₆N₁₆O₁₃) the only vital point of difference is nothing but the composition of the N-acyl moiety (see Section 3.8.2).

Characteristic Features

1. It is a white to buff-coloured powder; either odourless or having a very faint odour.

2. It has dissociation constant pKa 8 to 9.

3. Its solutions are either slightly acidic or are neutral to litmus (pH 5 to 7.5).

4. It is found to be freely soluble in water; and slightly soluble in alcohol.

Uses

1. The antimicrobial spectrum of activity of polymyxin B sulphate for its in vitro and in vivo profile is solely restricted to Gram-negative organisms, namely: Aerobacter, Escherichia, Haemophilus, Klebsiella, Pasteurella, Pseudomonas, Salmonella, Shigella, most Vibrio and Yesinia; all strains of Pr. providencia and most of Serratio marceseens are found to be unaffected by this antibiotic.

2. It is used topically either for the treatment or the prevention and treatment of external ocular infections caused by susceptible microorganisms, especially Ps aeruginosa.

3. In topical therapy, it is invariably combined with neomycin, gramicidin and bacitracin.

4. It also forms an integral component in glucocorticoid ophthalmological topical preparations.

Note: Substances like soap, which is a triglyceride of fatty, acids, and hence specifically antagonize cationic surface-active agents, is found to impair the activity of the antibiotic.

Polymyxin B Sulphate mixture with Trimethoprim [Synonyms Polytrim]: The combination of polymyxin B sulphate with trimethoprim enhances the overall antibacterial profile rather than each one used alone.

Polymycin B₁ [C₅₆H₉₈N₁₆O₁₃]

Polymyxin B₁ Pentahydrochloride [C₅₆H₉₈N₁₆O₁₃.5HCl]: It is obtained as a white powder. It has specific optical rotation [α]_D²⁵-85.11° (C = 2.33 in 75% ethanol).

Polymycin B₂ [C₅₅H₉₆N₁₆O₁₃]: It has specific optical rotation [a]₂₂⁵⁴⁶¹-112.4° (2% acetic acid).

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* Murien: Chloride (Cl–).

** Rifampin and Rifabutin.

*** 6-Methyloctanoic acid; or 6-Methylheptanoic acid.

3.8.3 Colistin

Synonyms Polymyxin E; Colimycin; Coly-Mycin; Colisticina; Totazina.

Biological Source It is a cyclopolypeptide antibiotic produced by Bacillus colistinus (Aerobacillus colistinus) first isolated from Japansese soil). It is comprised of colistins A, B and C.*

DAB = α,β-diamobutyric acid

Polymyxin E₁ (Colistin A)       :  R = (+)-6-Methyloctanoyl

Polymyxin E2                                              :   R = 6-Methylheptanoyl

Uses This antibiotic has more or less the same spectrum and therapeutic application as that of polymyxin B.

Colistin Sodium Methanesulphonate [C₅₈H₁₀₅N₁₆Na₅O₂₈S₅] [Synonyms Colistimethate sodium; Alficetin; Methacolimycin]: It is the injectable form of colistin. It is soluble in water and fairly stable in the dry form. It is inactive in itself but releases active polymyxin in the body.

Colistin Sulphate [Synonyms Malimyxin; Multimycine]: It is mostly used either orally or topically.

Colistin Formaldehyde-Sodium Bisulphite*: It is obtained as crystals that decompose between 290-295°. It is found to be soluble in water; and slightly soluble in methanol, ethanol, acetone and ether.

Polymyxin E₁ [C₅₃H₁₀₀N₁₆O₁₃] [Synonym Colistin A]: It has specific optical rotation [α]₂₂⁵⁴⁶¹-93.3° (2% acetic acid).

Polymyxin E₂ [C₅₂H₉₈N₁₆O₁₃]: It has specific optical rotation is [α]₂₂⁵⁴⁶¹-94.5° (2% acetic acid).

Note: The use of methacolimycin nowadays is rarely justified on account of the availability of less toxic alternative antibiotics.

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* Suzuki et al., J. Biodiem. (Tokyo), 54, 25 (1963).

3.8.4 Bacitracin

Synonyms Altracin; Ayfivin; Fortracin; Penitracin; Topitracin; Zutracin.

Biological Source It is a polypeptide antibiotic complex produced by Bacillus subtilis and licheniformis (family: Bacillaceae).** The commercial bacitracin is found to be a mixture of at least nine bacitracins. The purification of bacitracin may be affected by ‘carrier displacement method’.

Chemical Structure The major component of the mixture is ‘Bacitracin A’, which is essentially a dodecylpeptide having five of its amino-acid-residues arranged strategically in a cyclic structure as shown below:

Characteristic Features

1. It is obtained as a Grayish-white powder having a very bitter taste, odourless and hygroscopic in nature.

2. It is found to be soluble in water and ethanol; and almost insoluble in ether, chloroform, and acetone.

3. It is fairly stable in acid solution and unstable in alkaline solutions.

4. It affords a loss in potency most probably on account of the transformation of bacitracin A to bacitracin F, and the latter does not have any antimicrobial activity.

5. Its solutions undergo rapid deterioration at room temperature, and ultimately affords precipitation.

6. Its activity is significantly negated by salts of many of the heavy metals.

7. Its aqueous solutions invariably retain their potency for several weeks when stored in a refrigerator.

Uses

1. It is found to be effective exclusively against Gram-negative organisms.

2. Its applications are more or less limited to such infections only which may be treated either by topical application or by local infiltration.

3. It is significantly effective topically in the control, management and treatment of the following cutaneous bacterial infections where the pathogenic organism is specifically bacitracin-sensitive, such as: impetigo-contagiosa; falliculitis; pyoderma; ecthyma; furunculosis; decubitus, ulcer; infectious eczematoid dermatitis; scabies and dermatophytosis.

4. Bacitracin also finds its applications in the treatment of various ophthalmological conditions.

5. Its zinc salt invariably is preferred for topical therapy; and is the form most often incorporated into combinations.

6. It is mostly combined with neomycin and polymyxin B sulphate.

Note: 1. Due to the relatively high incidence of nephrotoxicity (albuminuria, cylindruria, azotemia, accumulation of drug) which essentially follows its parenteral administration precludes systemic usage except in life-endangering staphylococcal infections, such as: pneumonia, empyema, particularly in infants wherein other antibiotics have proved to be either ineffective or in the treatment of antibioticassociated (pseudomembranous)-enterocolitis caused by Cl difficile.

2. Development of bacterial resistance is much less frequent and slower for bacitracin as compared to penicillin, and for most organisms it is found to be almost nil.

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* Koyama et al. Japan. pat. 57, 4898 (1957).

** Anker et al. J. Bacteriol. 55, 249 (1948).

3.8.5 Vancomycin

Synonyms Vancocin; Vncoled; Lyphocin (Lyphomed).

Biological Source It is an amphoteric glycopeptide antibiotic substance produced by Streptomyces orientalis (Family: Streptomycetaceae) from Indonesian and Indian soil that essentially inhibits bacterial nucleopeptide biosynthesis by formation of complexes.

Chemical Structure The structure of the primary component of the mixture has been established beyond any reasonable doubt to be a complex tricyclic aglycone linked glycosidically to glucose and vincosamine functions. The vancomycin molecule essentially contains one free carboxylic acid moiety, two chloro substituted aromatic residues, and seven amide bonds, one of which is a prominent primary-amide.

The apparently novel feature of vancomycin is the tricyclic structure exclusively generated by three phenolic oxidative coupling reactions.

Preparation It is produced by the submerged fermentation process as described earlier under penicillins.

Characteristic Features

Vancomycin Monohydrochloride [C₆₆H₇₅Cl₂N₉O₂₄.HCl] [Synonyms Lyphocin; Vancor;]:

1. It is obtained as white solid, free-flowing powder, odourless and having a bitter-taste.

2. It has uvmax (H₂O): 282 nm (E^1%_1cm 40).

3. Its solubility in water is more than 100 mg. ml^–1.

4. It is found to be moderately soluble in dilute methanol; and insoluble in the higher alcohols, acetone and ether.

5. Its solubility in neutral aqueous solutions is enhanced by low concentrations of urea.

6. The acidic solutions precipitate out the antibiotic on addition of either NaCl or (NH4)2 SO4.

Uses

1. It has a Gram-positive antibacterial spectrum.

2. It specifically acts on bacterial cell walls by inhibiting murein biosynthesis by virtue of its complexation with the D-alanyl-D-alanine precursor and hence is bactericidal, which eventually renders it particularly useful in serious infections besides in the immunocompromised patients.

3. It also exerts to a certain extent the ‘secondary modes of action’ i.e., enhancing cytoplasmic membrane permeability and impairing RNA synthesis.

4. Vancomycin hydrochloride is widely recommended for the control, management and treatment of serious infections, such as: septicemia, endocarditis, wound infections caused by Gram-positive bacteria, specifically in those patients who are allergic to β-lactam antibiotics.

5. Vancomycin HCl is also found to be effective in Enterococcus faecalis strains that are inadequately controlled and managed by β-lactam antibiotics.

6. Vancomycin is not absorbed orally; however, oral administration is usually recommended for the treatment of staphylococcal-enterocolitis and antibiotic-associated pseudomembranous colitis produced by Clostridium difficile.

7. IM administration is rather painful and very often associated with local necrosis; therefore, systemic therapy with vancomycin makes use of IV-infusion extended over a span of 20 to 30 minutes.

Note:

 (i) It is irritating to tissue and hence may cause thrombophlebitis, or pain at the site of injection and neurosis takes place if extravasted; also produces chills, fever, occasional urticaria and maculopapular rashes with hypotension (Red Man’s Syndrome), nephrotoxicity and ototoxicity and, rarely, thrombocytopenia and neuropathy.

(ii) Recently, the plasmid-mediated resistant strains of enterococcus have virtually clamped restriction for the use of vancomycin in hospitals with a view to control the spread of resistance.

3.8.6 Teicoplanin

Synonyms Tiecoplanin A2; Teichomycin A2; Targocid; Targosid; MDL-507.

Biological Source It is a glycopeptide antibiotic complex produced by Actinoplanes teichomyceticus nov. sp.; structurally related to vancomycin and comprised of a mixture of five teicoplanins, which essentially differ only in the nature and length of the fatty acid-chain attached to the sugar residue.

Characteristic Features

1. It is obtained as an amorphous powder having mp 260°C (decomposes).

2. It has uvmax in 0.1 N HCl 278 (E^1%_1cm 53); and in 0.1 N NaOH: 297 (E^1%_1cm 74).

3. It is found to be soluble in aqueous solution at pH 7.0; partially soluble in methanol, ethanol; and insoluble in dilute mineral acids, and also in non-polar organic solvents.

The various physical parameters of the five major components of teicoplanin are as follows:

(i) Teicoplanin A2-1: (C₈₈H₉₅Cl₂N₉O₃₃): It is a white amorphous powder which darkens at 220°C and decomposes at 255°C.

(ii) Teicoplanin A2-2: (C₈₈H₉₇Cl₂N₉O₃₃): It is a white amorphous powder, darkens at 210°C and gets decomposed at 250°C.

(iii) Teicoplanin A2-3: (C₈₈H₉₇Cl₂N₉O₃₃): It is a white amorphous powder, darkens at 210°C and decomposed at 250°C.

(iv) Teicoplanin A2-4: (C₈₈H₉₉Cl₂N₉O₃₃): It is a white amorphous powder, darkens at 210°C and gets decomposed at 250°C.

(v) Teicoplanin A2-5: (C₈₉H₉₉Cl₂N₉O₃₃): It is a white amorphous powder, darkens at 210°C and gets decomposed at 250°C.

Uses

1. Teicoplanin has almost similar antibacterial profile to vancomycin (Section 9.3.8.5), but possesses a longer duration of action, and may be administered by IM as well as IV injection.

2. It is also employed against Gram-positive pathogens that are resistant to established antibiotics.

3. The ‘Red-Neck Syndrome’ as observed upon rapid administration of vancomycin is rarely seen, besides the incidence of autoxicity also seems to be reduced considerably.

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Source:Pharmacognosy And Pharmacobiotechnology By Ashutosh Kar