FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule13)
1. TITLE OF THE INVENTION:
NOVEL ANTIBACTERIAL COMBINATION FOR ENHANCED EFFICACY
2. APPLICANT (S)
(a) NAME: WOCKHARDT LTD.
(b) NATIONALITY: INDIAN
(c) ADDRESS: Wockhardt Towers, Bandra-Kurla Complex, Bandra
(East), Mumbai - 400 051.
3. PREAMBLE TO THE DESCRIPTION
The present invention provides a novel antibacterial combination for enhanced efficacy and method for inhibiting bacterial resistance in Extended Spectrum Beta-Lactamase (ESBL) producing strains, so as to have better control over the therapy and thus achieve reduced hospital stay.
The following specification particularly describes the invention and the manner in which it is to be performed.
4. DESCRIPTION
The present invention provides a novel antibacterial combination for enhanced efficacy and method for inhibiting bacterial resistance in Extended Spectrum Beta-Lactamase (hereinafter referred as ESBL) producing strains, so as to have better control over the therapy and thus achieve reduced hospital stay.

Cefepime is a semi-synthetic, broad spectrum, fourth generation cephalosporin antibiotic. Cefepime is commercially available as hydrochloride salt (Formula I) under the trade name of Maxipime®. It is chemically 1-[[(6R, 7R)-7-[2-(2-amino-4-thiazolyl) -glyoxylamido] -2-carboxy-8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-en-3-yl] methyl]-1-methylpyrrolidinium chloride, 72 - (Z)-(O-methyloxime), monohydrochloride monohydrate.
## STR(

)##
Cefepime exerts antibacterial functions on G+ve bacteria, such as Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus pneumoniae and other hemolytic Streptococci. It also has good antibacterial functions on G -ve bacteria, such as Pseudomonas aeruginosa, Escherichia coli, Klebsiella, Proteus vulgaris, Hemophilus, Neisseria, Salmonella, Serratia, Shigaella, and Yersinia. In addition, it has good activity on anaerobic bacteria, such as Bacteroid and Clostridium perfringens.
Cefepime follows linear pharmacokinetics over the range of 250 mg-2g (IV) and 500 mg- 2g (IM). Its average steady state Vd is 18.0 (± 2.0) L and serum protein binding is approximately 20%. It is principally eliminated via renal excretion, average (±SD) half-life of cefepime is 2.0 (± 0.3) hours and total body clearance is 120.0 (± 8.0) mL/min. It is metabolized to N-methylpyrrolidine (NMP), which is rapidly converted to the N-oxide (NMP-N-oxide). Cefepime hydrochloride is indicated in the treatment of infections like Pneumonia
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(moderate to severe), Uncomplicated and Complicated Urinary Tract Infections (including pyelonephritis), Uncomplicated Skin and Skin Structure and Complicated Intra-abdominal Infections (used in combination with metronidazole).
Sulbactam, a derivative of the basic penicillin nucleus, is an irreversible beta-lactamase inhibitor. Sulbactam is commercially available as sodium salt (Formula II) in combination with ampicillin (3 lactam antibiotic) under the trade name of Unasyn. It is chemically (2S, 5R)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylate 4,4-dioxide. The mean serum half-life of sulbactam is approximately 1 hour and approximately 75 to 85% of sulbactam is excreted unchanged in the urine. It is given in combination with beta-lactam antibiotics to overcome beta-lactamase enzyme that destroys the antibiotics. ## STR (
)##

CN Publication No 1565456A discloses an antibacterial combination composed of cefepime and beta-lactamase depressant in the weight ratio from 1:2 ~ 1:0.1. The combination of beta-lactamase depressant and cefepime described in this invention has obvious combined antibacterial functions. The various dose combinations of cefepime with sulbactam that have been exemplified in this publication are given in Table 1.
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•
Table 1: The various dose combinations of cefepime with sulbactam.

Practical Examplenumbers as given inpublication No1565456A Content Per Vial
Cefepime/ Cefepimehydrochloride / Cefepimehydrochloride hydrate(gm) Sulbactam Sodium(gm)
5 1 0.1
6 1 1
7 0.5 1
8 1 0.75
15 1 0.5
16 1 2
26 1 2
27 1 2
28 1 0.2
29 1.11 0.33
CN Publication No 1565455A discloses a kind of antibacterial combination for curing the infection caused due to the bacteria responsible for extended spectrum beta-lactamase. It is composed of cefepime and tazobactam with their weight ratio from 20:1~1:2.
Antibacterials are the agents used to inhibit or kill the pathogenic bacteria and many a time these pathogenic bacteria's develop resistance against antibacterials by one or more mechanisms. As a result efficacy of antibacterials is severely compromised and therapy becomes ineffective. To overcome this problem, the antibacterial levels needs to be kept as high as possible and the resistance level should be reduced by some means so that resistant organisms become sensitive towards these antibacterials.
The combination of cefepime with sulbactam has solved the increasing problem of clinical pathogenic bacteria having drug resistance to cefepime. As both the drugs are excreted through urine in unchanged form, higher levels of both are available in kidney and bladder tissues, which can also be useful for treating severe resistant urinary tract infections.
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The present inventors while working on pharmaceutical antibacterial combination of cefepime with sulbactam have surprisingly found that when 2 g of cefepime i.e. maximum permissible dose is combined with 0.1- 4 g of sulbactam shows enhanced efficacy over 1 g cefepime and 0.1 - 2 g sulbactam combination. Also, it was surprisingly found that when 2 g of cefepime is combined with 0.1- 4 g sulbactam, it is effective in inhibiting the resistance in ESBL producing strains.
In one of the aspects of the present invention there is provided a pharmaceutical dosage form comprising 2 gm of cefepime in combination with 0.1- 4 g of sulbactam and pharmaceutical^ acceptable excipients.
The two components of dosage form; viz., cefepime and sulbactam can be lyophilized separately or simply powder filled in one vial or individually can be powder filled and kept in separate vials. Before administration to the patient in need thereof, the contents of the individual vial are reconstituted using suitable pharmaceutical^ acceptable diluent and administered. Cefepime can be present in the form of cefepime internal salt, cefepime hydrochloride or cefepime hydrochloride hydrate, or the addition agents like L-arginine and sulbactam is present as sodium or potassium salt. The in vitro studies show that the dosage form containing 2 gm of cefepime (maximum permissible dose) and 0.1- 4 g of sulbactam show enhanced bactericidal activity over 1 g cefepime with 0.1- 2g sulbactam combination. The bactericidal activity is found to increase proportionally with proportional increase in cefepime concentration. The pharmaceutical antibacterial dosage form also contains pharmaceutically acceptable excipients wherein pharmaceutically acceptable excipient is selected from a group comprising of one or more of antioxidants, buffers, preservatives, tonicity agents, chelating agents and the like. It is also noted that L-arginine when added to this composition increases the solubility of cefepime and therefore is recommended as excipient.
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Suitable antioxidants include one or more of butylated hydroxytoluene (BHT), Ascorbic Acid, Sodium bisulphitei, Sodium metabisulphite and the like.
Suitable buffers include one or more of citrates, acetates, borax, phosphate and the like.
Suitable preservatives include one or more of benzyl alcohol, methyl paraben, propyli paraben, benzyl paraben and the like.
Suitable tonicity agents include one or more of dextrose, sodium chloride, mannitol and the like.
Suitable chelating agents include one or more of sodium EDTA, citric acid and the like.
In yet another aspect of the present invention there is provided a method of inhibiting bacterial infections caused by resistant ESBL producing strains wherein the said method comprises of administering to a patient in need thereof a combination of 2 gm of cefepime and 0.1- 4 gm of sulbactam.
For establishing the efficacy of the combination of present invention several in-vitro microbiological studies have been preformed.
In one such study (refer Example 1) the inventors of the present invention have found that higher concentration of cefepime in the blood, which is achievable only by 2 gm dose for maximum of 6 hours in combination with fixed dose of sulbactam, is effectively capable of inhibiting resistant bacterial zone 25% more than 500 mg dose and 15% more than 1 gm dose in combination with fixed dose of sulbactam.
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In yet another study (refer Example 2) the present inventors have found that higher concentration of cefepime in the blood, which is achievable only by 2 gm dose for maximum of 6 hours in combination with fixed dose of sulbactam, inhibits the growth almost 500 times more efficiently than compared with 500 mg of cefepime and 92 times more effective over 1 gm of cefepime in combination with fixed dose of sulbactam.
While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention
EXAMPLE 1
Table 2: Effect of increasing concentrations of cefepime with constant sulbactam concentration on enhancement of antibacterial action against cefepime resistant E.coli 71 MP.

Cefepime meg/disc Zone ofinhibition(mm) Cefepime + sulbactam (meg/disc) Zone ofinhibition(mm)
0.5 Nil 0.5+5 10
1 Nil 1+5 11
2 Nil 2+5 12
4 Nil 4+5 13
8 Nil 8+5 14
16 Nil 16+5 15
Procedure: The experiment was performed by quantitative agar drug diffusion assay. 50 ml of fresh sterile molten Mueller Hinton Agar (MHA, Difco) was poured in sterile petri plate positioned on a leveled surface. Plates containing media were allowed to cool at 4°C. E.coli 71 MP inoculum was spread using sterile cotton swab, which was pre-dipped in a CFU (Colony forming units) adjusted bacterial suspension. The discs containing cefepime alone and cefepime with sulbactam were firmly placed on to culture seeded agar surface
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under sterile conditions. The plates were incubated at 37 C for 24 h. The diameter of zones of inhibition was measured and recorded.
EXAMPLE 2
Table 3: Mutant Prevention Concentration (MPC) of cefepime with and without sulbactam using cefepime resistant E.coli 71 MP

CefepimeCone, (mcg/ml) CefepimealoneType of growth No of colonies per agarplateCefepime + 4 mcg/ml ofsulbactam No of colonies per agarplateCefepime + 8 mcg/ml ofsulbactam
4 Mat growth* 564 92
8 Mat growth* 130 Nil
16 Mat growth* Nil Nil
* Mat growth refers to the profusely growing bacterial culture forming mat indicating lack of effectiveness of the drug.
Procedure: Overnight grown cultures of cefepime resistant Gram-negative bacteria were brought to a log phase and concentrated in normal saline to a cell density of 5X109 CFU/ml by centrifugation. 150 ml of this suspension was spread in triplicate on to a large petri plates containing Mueller Hinton agar (MHA, Difco, USA). Plates were prepared with 4, 8, and 16 mcg/ml cefepime alone and each of these concentrations in combination with 4 and 8 mcg/ml of sulbactam. Plates were incubated at 37°C for 48 hours and resistant colonies were enumerated.
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EXAMPLE 3-11

ExampI e No. Cefepime/ CefepimeHydrochloride/Cefepime HydrochlorideHydrate(Calculated on the basis offreeCefepime base in gm) Sulbactam sodium(gm) L-arginine(gm) Sterile water for injection/normalsaline/ 5%dextrose(mL)
3 20 2 14.5 q.s
4 20 5 14.5 q.s
5 20 10 14.5 q.s
6 20 15 14.5 q.s
7 20 20 14.5 qs
8 20 25 14.5 q.s
9 20 30 14.5 q.s
10 20 35 14.5 q.s
11 20 40 14.5 q.s
Procedure: As mentioned in the examples (i.e. Example Nos. 3-11), suitable quantity of Cefepime/ Cefepime Hydrochloride/ Cefepime Hydrochloride Hydrate, sulbactam sodium and L-arginine were mixed under aseptic and clean conditions and proceeded according to the preparation process sequence of dry powder injection preparation to prepare 10 units of powder injection to be reconstituted with suitable solvents like sterile water for injection, normal saline, 5% dextrose before administration.
EXAMPLE 12
4 units of cefepime hydrochloride for injection (dosage: 0.5g/bottle) and sulbactam sodium for injection (dosage: 2.0g/bottle) were dissolved in the same 250 ml glucose transfusion under aseptic and clean conditions for the intravenous administration of patients infected with sensitive bacteria.
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WE CLAIM:
1. A pharmaceutical dosage form comprising 2 g of cefepime in combination with 0.1- 4 g of sulbactam along with optional pharmaceutically acceptable excipient.
2. A pharmaceutical dosage form as per claim 1 wherein cefepime is present in the form of cefepime internal salt, cefepime hydrochloride or cefepime hydrochloride hydrate, or the addition agents like L-arginine.
3. A pharmaceutical dosage form as per claim 1 wherein sulbactam is present in the form of sulbactam sodium or potassium salt.
4. A pharmaceutical dosage form as per claim 1 for parenteral administration.
5. A pharmaceutical dosage form as per claim 4 wherein the pharmaceutically acceptable excipient is selected from a group comprising one or more of antioxidant, buffer, preservative, tonicity adjusting agent, chelating agent and the like.
6. A method of inhibiting bacterial infections caused by resistant ESBL producing strains wherein the said method comprises of administering to a patient in need thereof a combination of 2 gm of cefepime and 0.1-4 gm of sulbactam.
Dated this 28th day of April, 2006
For Wockhardt Limited

(Kodgule, Mandar Madhukar) Authorized Signatory
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