PENICILLINS

β-lactam antibiotics- Penicillins, cephalosporins, carbapenemes, Monobactums

HISTORY

 1928: Alexander Fleming observed that Penicillium mould contaminating

staphylococci colonies were surrounded by a clear zone of free growth. He suspected
some substance released by mould inhibited the growth which he named as
‘penicillin’.

 1933: ‘Florey and Chain’ worked on the physical, chemical and pharmacological
properties of penicillin.

 1942: Large quantity of penicillin was produced and was used clinically.

SOURCE
 Penicillium notatum, P. chrysogenum
 Mutant strains obtained by x-ray exposure of organism.
 Natural Penicillin, Penicillin-G (Benzyl Penicillin)
 Penicillin O and Penicillin V obtained by adding precursors to the fermentation broth.
 Natural penicillins can be produced depending on the chemical composition of the
fermentation medium used to culture Penicilliums. Natural Penicillins are produced
by mould cultures, then extracted and purified.
 6-aminopenicillanic acid obtained from cultures of Penicillium chrysogenum depleted
of side chain precursors could be developed into semi synthetic penicillins.
 Side chains can be added that after the susceptibility of the resultant compounds to
inactivating enzymes (β-lactamases) and that change the antibacterial activity and the
pharmacological properties of the drug.

CHEMISTRY

 Amorphous white crystalline powder.

 Benzyl penicillin which is Na or K salt of the acid.
 Soluble in water insoluble in oil. Solutions stable only for 7 days at 4°C. Citrate
buffering at pH 6 – 6.5 increases stability.

 Basic structure of Penicillin has:

(i) Thiazolidine ring [A] (five membered) - stable
(ii) β- lactam ring [B] (four membered) - unstable

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 (iii) Side-chain attached to β- lactam ring

 Penicillin nucleus is essential for the antimicrobial activity. Metabolic transformation

or chemical alteration of the molecule causes loss of all significant antibacterial
activity.
 β- lactam ring can be easily broken and difficult to synthesize.
 Gastric HCl hydrolyses most penicillins, rest hydrolysed by alkalinity in the jejunum.
 Stable at pH 6 to 7, phosphates and citrates buffer used for stability.
 Solubility depends on structure of the side chain and on the salt used.
 Interacts with heavy metal such as Hg, Cu, Zn; EDTA used as preservative.
 Also inactivated by oxidising and reducing agents, alcohols and certain thiol
compounds.

PENICILLIN UNITS
1 Unit = 0.6 μg of penicillin G, Na.
1667 Units = 1 mg of penicillin G, Na.
1595 Units = 1 mg of penicillin G, K.
Semi-synthetic penicillins are measured in gms or mgs.

ANTIBACTERIAL SPECTRUM
 Penicillins are effective against:-
Streptococcus, Staphylococcus, Corynebacterium, Bacillus anthracis,
Clostridium, Listeria, Erysipelothrix.
 Semi-synthetic penicillins are effective also against Gram -ve like Salmonella,
Proteus, E. coli, Hemophilus, Pseudomonas, Enterobacter.
 Gm-ve organism possesses an extra layer (lipopolysachharide) which renders the cells
less easily penetrated.
 In Gram +ve bacteria, the cell wall is 50-100 molecules thick, but is only 1-2
molecules (peptidoglycan) thick in Gram –ve.

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  Gram –ve bacteria have thick outer membrane and have pores more difficult to
penetrate.
 Drugs effective against Gram –ve bacteria are also those that can rapidly penetrate the
outer membrane of the cell wall.
 In Gram –ve bacteria, PBPs are situated within the outer membrane.

MECHANISM OF ACTION
Cell wall of bacteria consists of peptidoglycan which are linear strands of two
alternate sugars N- acetylglucosamine and N-acetylmuramic acid (pyranoside residues of
amino sugars) cross linked by peptide chains. N-acetylmuramic acid residues are substituted
with a short chain of 4 amino acids and the glycan chains joined together by bridges formed
between these peptide chains.

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Biosynthesis of peptidoglycan involves 3 stages and 30 enzymes.
 First stage is precursor formation (uridine nucleotide) which takes place in the
cytoplasm (UDP brings together NAM and NAG).
Uridine nucleotides : UDP- acetylmuramyl pentapeptide,
UDP- acetylglucosamine.
 The uncross linked uridine nucleotide intermediate of peptidoglycan synthesis is
called “Park nucleotide”.
 Park nucleotide gets accumulated if the subsequent synthesis stages are inhibited
(Park nucleotide – name given after the discoverer J.J. Park).
 Accumulation of Park nucleotide was major step is elucidating the mechanism of
action of penicillin.
 The last reaction in the first stage is addition of dipeptide D-alanine-D-alanine to
UDP N-muramyl tripeptide.
 Synthesis of the dipeptide involves prior racemization of L-alanine and
condensation catalyzed by D-alanyl-D-alanine synthetase. D-Cycloserine (an
antitubercular agent) is a structural analog of D-alanine and acts as a competitive
inhibitor of both the racemase and the synthetase.
 In the Second stage UDP- acetylmuramyl pentapeptide and UDP- acetylglucosamine
are linked to from a long polymer. Linking takes place on the cell membrane bound
phospholipids.
 The completed strand of peptidoglycan is cleaved from phospholipids, a reaction
inhibited by Vancomycin and Bacitracin.
 Third and final stage comprises of cross linking of short peptides (attached to N-
acetylmuramic acid). This occurs by a bridge provided by pentaglycine peptide unit.
This transpeptidation reaction involves linking of terminal glycine residue of
pentaglycine bridge with the 4th residues (D-alanine) of pentapeptide side chain
releasing the 5th amino acid (also D-alanine) free. This reaction is catalysed by
transpeptidase enzyme.
 The cross linking gives the completed peptidoglycan its tensile strength and gives
support to cell membrane.
 Penicillins and cephalosporins inhibit transpeptidase enzyme.
 Conformation of penicillin is similar to that of D-alanyl-D-alanine. Penicillin
provides similar –CO–N– bond in its β-lactam ring as in the D-alanyl-D-alanine
residues of pentapeptide chain.
 Transpeptidase is composed of penicillin binding protein (PBP). Penicillin inhibits
(binds) these PBPs.
 Due to this, final cross linking remains uncompleted and the final rigidity needed
by bacterial cell is not provided. When the pressure increases in the cells, these
burst and lyse.
 High osmotic pressure development in bacterial cell. The ingress of water with
consequent lysis is prevented by the cell wall. Penicillin interferes with the
formation of cell wall of the developing microorganism and thus exposes it to the

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 lytic action of any solution whose osmotic pressure is less than that of the cells
own content.

Penicillin binding proteins (PBPs)

PBP - 1, 2, 3, 4 are affected by β-lactams. Variation in spectrum of action and
bactericidal action of β-lactam antibiotics can be related to their affinity for different PBPs.
e.g. Inhibition of PBP-1a and 1b causes lysis, PBP-2 results in rounded cells called
spheroblasts and PBP-3 produces long filamentous forms. S. aureus has 4 PBPs.
Carbapenems (Imipenem & meropenem) causes rapid lysis and are most bactericidal and
have highest affinity for PBP-1.
Carbapenemes are effective against gram negative as they affect PBP 1& 2 and
produce Post antibiotic effect not seen by other β-lactams. Rapid bactericidal activity is less
likely to induce release of endotoxin from gram negative sepsis.
β-lactam antibiotics are time dependent in their activity as they are slowly bactericidal
and the time of antibiotic concentration above MIC (T>MIC).

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RESISTANCE
Microorganisms may be intrinsically resistant because of the structural differences in
PBPs. Sensitive strain acquires resistance by development of high molecular weight PBPs
that have decreased affinity for antibiotics.
Methicillin resistant Staphylococcus aureus (MRSA) colonized in hospitals. Not in
veterinary science. Now seen.
Mutation in the enzyme, produced by resistance gene (Mec A) produces PBP-2a. PBP
that resists binding of the β-lactam drugs and renders bacteria with this gene resistant. The
PBP-2a target is the basis for resistance to methicillin and oxacillin, known as MRSA.
Overall, resistance of microorganisms can be due to following mechanisms: –
1) Production of β-lactamase – Major mechanism
2) Decreased penetration through the outer membrane to access cell wall enzymes.
3) Resistance of target (PBP) to binding by β-lactam antibiotics.

PREPARATIONS OF PENICILLINS
 For achieving rapid blood level – aqueous solution.
 For sustained response – repository form.
Aqueous solution
 Most common salts are K and Na. Should be given by IV but can be given by SC or
IM also.
 Buffered with 4 – 5% sodium citrate to keep pH to 5.0 to 7.5. Stable at refrigeration
temperature for 5-7 days.
 Dry powders stable at room temperature upto 3yrs.
 Blood levels last for 4 – 5 h.
Repository form
 Insoluble salts.
 Designed for deep IM injection so that drug is absorbed over a period of 12 h or
longer.
 IV, SC or IP routes are contraindicated.
(a) Procaine Penicillin G suspension in water
 Soluble in water (only 4 mg/ ml or 0.4%).
 Contains 1008 unit/mg.
 Due to procaine moiety, there is no pain.
 Procaine + Penicillin G
(1 molecule) + (1 molecule)
(41.5%) + (940 units/ mg)
 Other Preparations: These preparations are given as aqueous suspensions with
buffering and suspending agents, absorption is slow.
- Procaine Penicillin G in oil (vegetable/ mineral): One injection – 24 to 48 h.
- Procaine Penicillin G with aluminium monostearate (Insoluble penicillins are
suspended in oil): One injection – 3 to 4 days.
(b) Benzathine Penicillin G
 Very low solubility (0.2 mg/ml or 0.02%).
 Chemically salt is dibenzyl ethylene diamine.

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  Given by only IM route.
 Blood level maintained for 7-10 days.
[Note – Procaine penicillin and benzathine penicillin are not semi-synthetic penicillins.]

PHARMACOKINETICS

Route of administration
(i) Parenteral (IV, IM, SC and constant IV)
 For most Gram +ve organisms, intermittent IV regimen gives better
therapeutic results than constant IV regimen.
 Oily preparations should be given IM.
 SC route is better than IM because of lesser residual problem with comparable
blood levels.
 Repository preparations (Procaine Penicillin G, Benzathine Penicillin) - Only
IM, never IV or SC.
(ii) Oral
 Penicillin suppresses the growth of microflora in the digestive tract of
herbivores.
 Oral route to be used in young herbivores and for acid resistant penicillins like
penicillin V, phenethicillin and ampicillin.
(iii) Topical
 Penicillin may be used by this route as an ointment, solution and powder.
 This route may cause allergic reaction and induction of resistance.
(iv) Ophthalmic
 Only penicillin G is non irritant, ointment to treat superficial eye infections.
(v) Intramammary
 In mastitis, both intramammary and systemic route is used.
 Milk should be discarded for 96 hrs following treatment.

Absorption
(i) Oral route
 Not absorbed from stomach or jejunum (destroyed at pH 2.0 or higher than
8.0).
 1/4th of the oral dose is absorbed from GI tract of monogastric animals.
 Peak blood level is achieved within 30-60 min.
 Food in GI tract retards absorption.
 Oral dose should be 5 times the IM route.
(ii) IM & SC route
 Absorption influenced by dose, vehicle, solubility and concentration of the
drug used.
 Peak blood concentration is reached within 20-30 min. Soluble salts give
higher and rapid blood levels than aqueous suspension.
 Salts with high molecular wt. slow absorption rate.
 Absorption is more prolonged by using vegetable oil as vehicle or benzathine
penicillin (remains in blood for 26 days). Given deep IM – slowly release
penicillin forming depot.
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 (iii) Intramammary route
 Through blood, penicillins can pass from one quarter of the under to others.
 After systemic administration penicillin is excreted in milk.
 Drug is excreted slowly from infected quarters than healthy quarters.
 Milk out times varies with preparation: 24 h - aqueous vehicle, 48 h - oil water
emulsion, 72 h - vegetable or mineral oil.
Distribution
 After IM or SC route significant level is reached in blood, liver, bile, lungs etc.
 At equilibrium highest concentration present in kidney.
 Distribution depends on the difference between blood and tissue concentration.
 Very low concentration is attained in joints, ocular, pericardial, pleural and
peritoneal fluid.
 Diffuses in milk (Ratio in milk: plasma varies from 0.2 to 1.7).
 Very small amount crosses blood brain barrier, however concentration is more
(5%) during inflammation.
 Only fraction passes placenta. Therapeutic concentration is not reached in foetus
with usual therapeutic dose.
 Minimum blood concentration is 0.02-0.03 μg/ml.
 Volume of distribution (Vd) is 50% of total body water and Plasma Protein
Binding (PPB) is 65%.
Metabolism
 Not easily destroyed in the body.
 In blood, 90% is in plasma and 10% in erythrocytes.
 90% excreted unchanged, 10% metabolised by unknown mechanisms.

Excretion
 60-90% of Penicillin G is recovered in urine after injection of aqueous
preparation.
 Renal excretion comprises of 80% tubular secretion and 20% by glomerular
filtration.
 Some penicillins like ampicillin excreted in bile.
 Excretion of penicillin can be delayed by:-
i) Depression of excretion- Probenecid (competes for excretory system), para-
aminohippuric acid (PAH)
ii) Slowing absorption of Penicillin- Benzathine
Clinical applications of Penicillin G Na
Choice of initial treatment in horses and cattle, little in small animals because of high
incidence of resistance.
 Bovine mastitis – 300,000 units/ quarter q24h x 3.
 Anthrax – Cattle, sheep, goat, pigs and dogs – 100,000 units/ kg q12h x 3.
 Erysipelothrix infection in pigs & sheep – 4000 units/ kg q24h.
 Strangles – 100,000 units/ kg for 5days.

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  Clostridial infections.

TOXICITY OF PENICILLINS
 Minimal direct toxicity in animals.
 LD50 of pure penicillin G is 500,000 units/kg in cats; 3,500,000 units/kg in mice.
 Acute toxicity of parenterally given pure salt is mainly due to procaine moiety.
 Epileptic seizures occur when the concentration reaches 300 unit/ml in C.S.F.
 Anaphylaxis: The sensitizing substance is protein conjugates of penicillin. [Also by
prior exposure to penicillin from environment (foods of animal origin or from fungus
producing penicillin)]
 Cattle are more commonly affected than dogs & cats if penicillin preparations
have carboxy methyl cellulose as adjuvant.
 Antigenic intermediate is penicilloyl moiety (formed by opening of the β-
lactam ring).
 In cattle produces laboured breathing, salivation and oedema of head.
 Eye ointment containing penicillin can cause swollen conjunctiva and oedema.

CLASSIFICATION OF PENICILLINS
Natural Amino Penicillinase resistant Penicillins Broad Spectrum
Penicillins Penicillins (Antistaphylococcal Penicillins) Penicillins
Penicillin G Amoxicillin* Cloxacillin* Azlocillin
Penicillin V Ampicillin* Dicloxacillin Carbenicillin
Hetacillin Methicillin* Mezlocillin
Metampicillin Nafcillin* Piperacillin
Pirampicillin Oxacillin* Ticarcillin*
Epicillin Carindacillin
Bacampicillin Sulfacillin
Suncillin
Ureidopenicillin
* Veterinary use

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Natural Penicillins
1. Phenoxy methyl penicillin (Penicillin V) or Phenethicillin
 Congener of benzyl penicillin. Variation in the culture medium and precursors
incorporated.
 Acid stable (because of phenoxymethyl group) – suitable for oral use.
 Inactivated by penicillinase, intermediate spectrum.
 Used in streptococcal meningitis in pigs.
 Dose – Dogs - 20,000 – 30,000 units/kg q6h.
 Other preparations: Penicillin V benzathine, Penicillin V hydrabamine.
2. Phenoxy ethyl penicillin
 Similar to penicillin V.
 Acid stable and inactivated by penicillinase.
 Preparation – Phenethicillin K.
Disadvantages of Penicillin G Na (Benzyl Penicillin)
(i) Low activity against Gram –ve bacteria.
(ii) Destroyed by acid.
(iii) Destroyed by β- lactamases.
(iv) Rapid renal excretion.
(v) Poor diffusion into CSF.
(vi) Hypersensitivity.

Semisynthetic Penicillins
Produced by chemically combining special side chains or by incorporating special precursors
in the mould cultures.
o Narrow spectrum similar to Penicillin G.
o Penicillinase stable Penicillins
o Broad spectrum – Gram +ve & Gram –ve, Pseudomonas.

1. Methicillin (Staphcillin)
 Penicillinase resistant penicillin.
 Used to treat Penicillin G resistant Staphylococcus aureus infection.
 Inactivated by gastric acid – so not given orally. IM, IV route is used.
 Water soluble, distributed in CSF, bile and excreted mainly in urine.
 Anti-staphylococcal penicillin.
 If Staphylococcus shows phenotypic resistance to methicillin, it is a marker for
resistance mediated by Mec A gene which codes for resistant PBP protein.
Oxacillin is used to test this, though the resistant strains are still referred as
‘methicillin resistant’.

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2. Isoxazole compounds (Oxacillin, Cloxacillin, Dicloxacillin)
 All are acid stable, so orally used.
 Penicillinase resistant.
 Duration of blood level longer.
 Narrow spectrum, short half life.
 Dose: SA- 10 to 20 mg/ kg, 2 - 3 times a day.
 Benzathine cloxacillin is used for mastitis treatment during dry period.
 Na cloxacillin in oil – 200 mg/ quarter, mastitis during lactation;
Benzathine cloxacillin in oil – 500 mg/ quarter, mastitis during dry period.
3. Nafcillin Na (Flucloxacillin)
 Limited spectrum.
 Resistance to penicillinase and acid hydrolysis.
 Mainly used to treat respiratory, soft tissue infection and osteomyelitis.
 Excreted through bile and has enterohepatic circulation.
 Oral, IV and IM route of administration. IM route irritating.

4. Aminopenicillins (Ampicillin, Amoxicillin)

(i) Ampicillin
 Ampicillin prodrugs – Pirampicillin, Talampicillin, Bacampicillin, Hetacillin.
 Broad spectrum penicillin. Effective against Gram +ve as well as Gram –ve
organism (E. coli, Salmonella, Proteus and Klebsiella).
 Not resistant to penicillinase.
 Acid stable – Can be given orally.
 Na salt aqueous soluble but unstable after dissolution in water so solution to
be used within 3 – 4 h. Suspension of ampicillin in oily base is used.
 Well absorbed orally, peak concentration – 2 h.
 Evenly distributed throughout the body, less protein bound.
 Excreted in bile, enterohepatic circulation – used in treatment of enteric
bacterial infections. Concentration 40 times higher in bile than plasma.
 Preferred some times over other drugs like chloramphenicol, tetracyclines etc.
 Clinical uses : Cattle – Calf scours, enteritis, pneumonia, foot rot, mastitis,
metritis, pyelonephritis.
Pigs – Enteritis, pneumonia, erysipelas, metritis.
Sheep – Contagious foot rot, mastitis, metritis, pneumonia.
Horse – Enteritis, septicaemia, metritis, respiratory infection.
Poultry – Enteritis.
Dogs – Enteritis, leptospirosis, respiratory infections,
urogenital infections.
 Dosage : Cattle – 5 mg/kg q 24 h IM.
Dogs & cats – 2.5 - 5 mg/kg twice daily IM or orally.

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 (ii) Amoxicillin

 Broad spectrum like ampicillin.

 Better penetration into the bacterial cell wall than ampicillin.
 More acid stable than ampicillin – better absorption orally.
 Half life longer than ampicillin.
 Used to treat urinary, respiratory, intestinal, cerebral, skin and other soft tissue
infections.
 Dose : 10 mg/kg oral, 7 mg/ kg parenteral.

5. Anti-pseudomonas penicillins

(i) Carbenicillin

 Not stable in acid, so not given orally. Not resistant to penicillinase.

 Broad spectrum, effective against Pseudomonas aeruginosa, E. coli, Proteus.
 Synergistic with gentamicin for treating Pesudomonas infection, however,
they are chemically antagonistic.
 Carbenicillin indamyl – Acid stable, can be given orally. Used in patients
sensitized to penicillin.
 Clinically used in pneumonia, metritis, nephritis, otitis, cyctitis, mastitis ans
septicaemia.
 Dose : 10 - 20 mg/kg IM.
 Toxicity : Nephrotoxicity, fever, skin rashes, agranulocytosis, haemolytic
anaemia, anaphylaxis.

(ii) Acylureido Penicillins (Mezlocillin, Azlocillin, Apalcillin, Piperacillin)

 Effective against Pseudomonas aeruginosa.

β-LACTAMASE INHIBITORS

1. Clavulanic acid

o Obtained from Streptomyces clavuligerus.

o Compound with potent inhibiting against β-lactamases.
o Structurally similar to penicillin.
o Irreversibly inhibits β –lactamases during which clavulanic acid is destroyed but
the enzyme is permanently inactivated (Suicide inhibition).

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 o Combined together mainly with amoxicillin (similarity of pharmacokinetic
properties of two).
o Combination used for treatment of skin and enteric infections.
o Dose : 2.5 mg/kg in combination with amoxicillin (10 mg/kg), twice daily.

2. Salbactum: Less potent inhibitor.

3. Tazobactum: Mainly combined with Piperacillin.

*****

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 Chemical stru
ucture and major properties of peniccillins
Spectrum Side chain Non- Absorption Resistance Useful
of activity (R) proprietary after oral to antimicrobial
name administratio penicillinase spectrum
n
Narrow Penicillin G Variable No Streptococcus
spectrum CH2 (Benzyl (Poor) sp., Neisseria
penicillin) sp., many
anaerobes,
spirochaetes,
others
Penicillin V Good No
OCH2 (PPhenoxymet
hyyl derivatice)
Phenethecillin Good No
Anti- OCH3 methicillin Poor Yes
staphyloc (not given
occal OCH3 orally)
Isoxazolyl penicillins
Oxacillin
(R1=R2=H)
Cloxacillin Good yes
(R1=Cl;
R2=H)
Dicloxacillin Staphylococcus
(R1=R2=Cl) aureus
Floxacillin
R1=Cl;
R1=Cl;R2=F
Nafcillin variable yes
y

Broad Ampicillin good yes

y Haemophilus
spectrum R CH (R=H) influenze,
A
Amoxicillin excellent Proteus,
NH3
(R=OH) mirabilis,
Escherichia coli,
Hetacillin good no
n
Neiserria species
C
Carbenicillin Poor no
n
(R-H) (not given
orally)
C
Carbenicillin good Above plus
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 CH pseudomonas
Indanyl sp., Enterobacter
COOR
(R= 5 sp., and
indanol) proteus(indole
positive)
ticarcillin poor No
azlocillin poor No

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