DESC:RELATED PATENT APPLICATIONS

This application claims priority to Indian Patent Application No. 2426/MUM/2014 filed on Jul 26, 2014, the disclosures of which are incorporated herein by reference in its entirety as if fully rewritten herein.

FIELD OF THE INVENTION

The invention relates to a method of treating bacterial infections in a diabetic subject.

BACKGROUND OF INVENTION

Diabetes mellitus is a metabolic disorder of multiple aetiology, characterized by chronic hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism, resulting from defects in insulin secretion, insulin action, or both. The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of different organs, especially the eyes, kidneys, nerves, heart, and blood vessels. Symptoms of marked hyperglycemia include polyuria, polydipsia, weight loss, sometimes with polyphagia, and blurred vision. Acute, life-threatening consequences of uncontrolled diabetes are hyperglycemia with ketoacidosis or the nonketotic hyperosmolar syndrome. Long-term complications of diabetes include retinopathy with potential loss of vision; nephropathy leading to renal failure; peripheral neuropathy with risk of foot ulcers, amputations, and Charcot joints; and autonomic neuropathy causing gastrointestinal, genitourinary, and cardiovascular symptoms and sexual dysfunction.

Individuals with diabetes mellitus exhibit a greater frequency and severity of fungal, viral or bacterial infections. The reasons for this include incompletely defined abnormalities in cell-mediated immunity and phagocyte function associated with hyperglycemia as well as diminished vascularization secondary to long-standing diabetes. Many common infections are more frequent and severe in the diabetic population, whereas several rare infections are seen almost exclusively in the diabetic population (e.g. rhinocerebral mucormycosis and malignant otitis externa, which is usually secondary to P. aeruginosa infection in the soft tissue surrounding the external auditory canal). The most common infections are respiratory tract infections, urinanry tract infections, acute bacterial cystitis, acute pyelonephritis, bacteriuria, soft tissue infections, foot ulcers, Streptococcal infections, etc.

Disease states with special considerations for antimicrobial use are the situations in which pathophysiological changes may alter the pharmacokinetic behavior of antimicrobial agents. Thus the pharmacological properties such as absorption, protein binding and metabolism and elimination affecting the pharmacokinetic profile of any drug must be evaluated. Diabetes induced physiological and metabolic changes alter the pharmacokinetics of the various drugs like: Amoxicillin (Kurji et al. International Research Journal of Pharmacy, 2011, 2(12), 261-263), Halofantrine (Daniyan et al. Afr. Journal of Biotechnology 2008, 7(9), 1226-1234), Rifampin, pyrazinamide and Ethambutol (Ruslami et al. Antimicrobial Agents and Chemotherapy 2010, 54(3), 1068-1074), Telithromycin (Lee et al. Pharmaceutical Research 2008, 25(8), 1915-1924).

The inventors have now surprisingly observed the clinical implications of the pharmacokinetic alterations associated with oral and intravenous administration of Cephalosporin antibacterial agent in a diabetic subject.

SUMMARY OF THE INVENTION

Accordingly, there is provided a method of treating bacterial infections in a diabetic subject, said method comprising administering Cephalosporins or pharmaceutically acceptable derivatives thereof.

In one general aspect, there is provided a method of treating bacterial infections in a diabetic subject, said method comprising administrating high dose of Cephalosporin or pharmaceutical acceptable derivative thereof, wherein said Cephalosporin exhibits altered pharmacokinetic parameters in a diabetic subject.

In another general aspect, there is provided a method of treating bacterial infections in a diabetic subject, said method comprising administrating high dose of Cefpodoxime or pharmaceutical acceptable derivative thereof, wherein said Cefpodoxime exhibits reduction in dose proportional pharmacokinetic parameters in a diabetic subject.

In another general aspect, there is provided a method of treating bacterial infections in a diabetic subject, said method comprising high dose of Cefpodoxime or pharmaceutical acceptable derivative thereof, wherein said dose is in an amount 20-50% greater than that administered in non-diabetic subject.

The details of one or more embodiments of the invention are set forth in the description below. Other features, objects and advantages of the invention will be apparent from the following description including claims.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made to the exemplary embodiments, and specific language will be used herein to describe the same. It should nevertheless be understood that no limitation of the scope of the invention is thereby intended. Alterations and further modifications of the inventive features illustrated herein, which would occur to one skilled in the relevant art and having possession of this disclosure, are to be considered within the scope of the invention. It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. All references including patents, patent applications, and literature cited in the specification are expressly incorporated herein by reference in their entirety.

The inventors have surprisingly discovered a method of treating bacterial infections in a diabetic subject, said method comprising administrating high dose of Cephalosporin wherein said Cephalosporin exhibits altered pharmacokinetic parameters in a diabetic subject.

The term “Cmax” as used herein refers to the maximum concentration reached by a given dose of drug in a biological sample.

The term “AUC” as used herein refers to the mean area under the plasma concentration-time curve value after administration given dose of drug in a biological sample.
The term “t1/2” or “half life” as used herein refers to the time required to reduce the plasma concentration to one half its initial value.

The term “Cl” or “Clearance” as used herein refers to the volume of plasma in the vascular compartment cleared of drug per unit time by the processes of metabolism and excretion.

The term “pharmaceutically acceptable derivative” as used herein refers to and includes any pharmaceutically acceptable salt, pro-drugs, metabolites, esters, ethers, hydrates, polymorphs, solvates, complexes, enantiomers or adducts of a compound described herein which, upon administration to a subject, is capable of providing (directly or indirectly) the parent compound. For example, the term “antibacterial or a pharmaceutically acceptable derivative thereof” includes all derivatives of the antibacterial agent (such as salt, prodrugs, metabolites, esters, ethers, hydrates, polymorphs, solvates, complexes, enantiomers or adducts) which, upon administration to a subject, is capable of providing (directly or indirectly) the antibacterial compound.

The term “infection” or “bacterial infection” as used herein includes presence of bacteria, in or on a subject, which, if its growth were inhibited, would result in a benefit to the subject. As such, the term “infection” in addition to referring to the presence of bacteria also refers to normal floras, which are not desirable. The term “infection” includes infection caused by bacteria.
The term “treat”, “treating” or “treatment” as used herein refers to administering a medicament, including a pharmaceutical composition, or one or more pharmaceutically active ingredients, for prophylactic and/or therapeutic purposes. The term “prophylactic treatment” refers to treating a subject who is not yet infected, but who is susceptible to, or otherwise at a risk of infection (preventing the bacterial infection). The term “therapeutic treatment” refers to administering treatment to a subject already suffering from infection. The terms “treat”, “treating” or “treatment” as used herein also refer to administering compositions or one or more of pharmaceutically active ingredients discussed herein, with or without additional pharmaceutically active or inert ingredients, in order to: (i) reduce or eliminate either a bacterial infection or one or more symptoms of the bacterial infection, or (ii) retard the progression of a bacterial infection one or more symptoms of the bacterial infection, or (iii) reduce the severity of a bacterial infection or of one or more symptoms of the bacterial infection, or (iv) suppress the clinical manifestation of a bacterial infection, or (v) suppress the manifestation of adverse symptoms of the bacterial infection.

The term “pharmaceutically effective amount” or “therapeutically effective amount” or “effective amount” as used herein refers to an amount, which has a therapeutic effect or is the amount required to produce a therapeutic effect in a subject. For example, a therapeutically or pharmaceutically effective amount of an antibacterial agent or a pharmaceutical composition is the amount of the antibacterial agent or the pharmaceutical composition required to produce a desired therapeutic effect as may be judged by clinical trial results, model animal infection studies, and/or in vitro studies (e.g. in agar or broth media). The pharmaceutically effective amount depends on several factors, including but not limited to, the microorganism (e.g. bacteria) involved, characteristics of the subject (for example height, weight, sex, age and medical history), severity of infection and the particular type of the antibacterial agent used. For prophylactic treatments, a therapeutically or prophylactically effective amount is that amount which would be effective in preventing a microbial (e.g. bacterial) infection.

The term “administration” or “administering” includes delivery of a composition or one or more pharmaceutically active ingredients to a subject, including for example, by any appropriate methods, which serves to deliver the composition or its active ingredients or other pharmaceutically active ingredients to the site of the infection. The method of administration may vary depending on various factors, such as for example, the components of the pharmaceutical composition or the type/nature of the pharmaceutically active or inert ingredients, the site of the potential or actual infection, the microorganism involved, severity of the infection, age and physical condition of the subject and a like. Some non-limiting examples of ways to administer a composition or a pharmaceutically active ingredient to a subject according to this invention includes oral, intravenous, topical, intrarespiratory, intraperitoneal, intramuscular, parenteral, sublingual, transdermal, intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal, gene gun, dermal patch, eye drop or mouthwash. In case of a pharmaceutical composition comprising more than one ingredient (active or inert), one of the way of administering such composition is by admixing the ingredients (e.g. in the form of a suitable unit dosage form such as tablet, capsule, solution, powder or like) and then administering the dosage form. Alternatively, the ingredients may also be administered separately (simultaneously or one after the other) as long as these ingredients reach beneficial therapeutic levels such that the composition as a whole provides a synergistic and/or desired effect.

The term “pharmaceutically inert ingredient” or “carrier” or “excipient” refers to a compound or material used to facilitate administration of a compound, for example, to increase the solubility of the compound. Solid carriers include, e.g., starch, lactose, dicalcium phosphate, sucrose, and kaolin. Liquid carriers include, e.g., sterile water, saline, buffers, non-ionic surfactants, and edible oils such as oil peanut and sesame oils. In addition, various adjuvants commonly used in the art may be included. These and other such compounds are described in the literature, e.g., in the Merck Index, Merck & Company, Rahway, N.J. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Gilman et al. (Eds.) (1990); Goodman and Gilman’s: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press., which is incorporated herein by reference in its entirety.

The term “subject” as used herein refers to vertebrate or invertebrate, including a mammal. The term “subject” includes human, animal, a bird, a fish, or an amphibian. Typical, non-limiting examples of a “subject” includes humans, cats, dogs, horses, sheep, bovine cows, pigs, lambs, rats, mice and guinea pigs.

In one general aspect, there is provided a method of treating bacterial infections in a diabetic subject, said method comprising administrating high dose of Cephalosporin antibacterial agent or pharmaceutical acceptable derivative thereof, wherein said Cephalosporin exhibits altered pharmacokinetic parameters in a diabetic subject.

Surprisingly, methods according to the invention are also effective in preventing or treating bacterial infections that are caused by bacteria producing one or more beta-lactamase enzymes. In some embodiments, there is provided a method of treating bacterial infections in a diabetic subject, said method comprising administrating high dose of Cephalosporin antibacterial agent or pharmaceutical acceptable derivative thereof, wherein said Cephalosporin exhibits altered pharmacokinetic parameters in a diabetic subject, and said bacterial infections are caused by bacteria producing one or more beta-lactamase enzymes.

Typical, non-limiting examples of Cephalosporin antibacterial agents include Cefazolin, Cefacetrile, Cefadroxil, Cefalexin, Cefaloglycin, Cefalonium, Cefaloridine, Cefalotin, Cefapirin, Cefatrizine, Cefazedone, Cefazaflur, Cefradine, Cefroxadine, Ceftezole, Cefaclor, Cefamandole, Cefminox, Cefonicid, Ceforanide, Cefotiam, Cefprozil, Cefbuperazone, Cefuroxime, Cefuzonam, Cephamycin, Cefdaloxine, Cefoxitin, Cefotetan, Cefmetazole, Carbacephem, Cefixime, Ceftazidime, Ceftriaxone, Cefcapene, Cefdaloxime, Cefdinir, Cefditoren, Cefetamet, Cefmenoxime, Cefodizime, Cefoperazone, Cefotaxime, Cefpimizole, Cefpiramide, Cefpodoxime, Cefsulodin, Cefteram, Ceftibuten, Cefiolene, Ceftizoxime, Oxacephem, Cefepime, Cefozopran, Cefpirome, Cefquinome, Ceftobiprole, Cetiofur, Cefquinome, Cefovecin, CXA-101, Ceftaroline, Ceftobiprole, Cefoselis, Cefluprenam, Cefclidin, Loracarbacef, Ceftolozane, Flomoxef, Latamoxef and the like.

In some embodiments, there is provided a method of treating bacterial infections in a diabetic subject, said method comprising administrating high dose of Cefpodoxime or pharmaceutical acceptable derivative thereof, wherein said Cefpodoxime exhibits altered pharmacokinetic parameters in a diabetic subject. In some embodiments, the Cephalosporin antibacterial agent is Cefpodoxime Proxetil.

In another general aspect, there is provided a method of treating bacterial infections in a diabetic subject, said method comprising administrating high dose of Cefpodoxime or pharmaceutical acceptable derivative thereof, wherein said Cefpodoxime exhibits altered pharmacokinetic parameters, in a diabetic subject. In some embodiments, there is provided a method of treating bacterial infections in a diabetic subject, said method comprising administrating high dose of Cefpodoxime or pharmaceutical acceptable derivative thereof, wherein said Cefpodoxime exhibits dose proportional reduction of pharmacokinetic parameters, in a diabetic subject. In some embodiments, the pharmacokinetic parameters which exhibit dose proportional reduction in a diabetic subject are selected from the group consisting of Cmax and AUC. In some embodiments, there is provided a method of treating bacterial infections in a diabetic subject, said method comprising administrating high dose of Cefpodoxime or pharmaceutical acceptable derivative thereof, wherein said Cefpodoxime exhibits enhanced clearance, in a diabetic subject.

In some embodiments, Cefpodoxime exhibits dose proportional pharmacokinetic parameters, in a diabetic subject. In some embodiments, Cefpodoxime exhibits Cmax of 8-10 µg/ml and AUC of 17-20 µg.h/ml after administration of 200 mg of Cefpodoxime in a diabetic subject.

In another general aspect, there is provided a method of treating bacterial infections in a diabetic subject, said method comprising high dose of Cefpodoxime or pharmaceutical acceptable derivative thereof, wherein said dose is in an amount 20-50% greater than that administered in non-diabetic subject.

In some embodiments, there is provided a method of treating bacterial infections in a diabetic subject, said method comprising the dose of Cefpodoxime in range of 240 mg/day to 1200 mg/day.

In some embodiments, there is provided a method of treating bacterial infections in a diabetic subject, said method comprising the dose of Cefpodoxime in range of 240 mg/day to 300 mg/day, instead of dose of 200 mg/day to be administered in a non-diabetic subject.

In some embodiments, there is provided a method of treating bacterial infections in a diabetic subject, said method comprising the dose of Cefpodoxime in range of 480 mg/day to 600 mg/day, instead of dose of 400 mg/day to be administered in a non-diabetic subject.

In some embodiments, there is provided a method of treating bacterial infections in a diabetic subject, said method comprising the dose of Cefpodoxime in range of 960 mg/day to 1200 mg/day, instead of dose of 800 mg/day to be administered in a non-diabetic subject.

Cephalosporin antibacterial agents may be present in the composition in their free forms or in the form of their pharmaceutically acceptable salts, prodrugs, metabolites, esters, ethers, hydrates, polymorphs, solvates, complexes, enantiomers, adducts or in the form of their any other pharmaceutically acceptable derivative. The dose administered for Cephalosporin antibacterial agents is calculated on the basis of their free forms. For example, a dose of 200 mg Cefepodoxime Proxetil is equivalent to 200 mg of Cefpodoxime.

In some embodiments, there is provided a pharmaceutical composition comprising high dose of Cephalosporin antibacterial agent or pharmaceutically acceptable derivative thereof. In some embodiments, there is provided a pharmaceutical composition comprising high dose of Cefpodoxime or pharmaceutically acceptable derivative thereof. In some embodiments, there is provided a pharmaceutical composition comprising high dose of Cefpodoxime or pharmaceutically acceptable derivative thereof, for the treatment of bacterial infections in a diabetic subject.

The pharmaceutical compositions according to the invention may include one or more pharmaceutically acceptable carriers or excipients or the like. Typical, non-limiting examples of such carriers or excipients include mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, wetting agents, emulsifying agents, solubilizing agents, pH buffering agents, lubricants, preservatives, stabilizing agents, binding agents etc.

The pharmaceutical composition or the active ingredients according to the present invention may be formulated into a variety of dosage forms. Typical, non-limiting examples of dosage forms include solid, semi-solid, liquid and aerosol dosage forms; such as tablets, capsules, powders, solutions, suspensions, suppositories, aerosols, granules, emulsions, syrups, elixirs and a like.

The pharmaceutical compositions according to this invention can exist in various forms. In some embodiments, the pharmaceutical composition is in the form of a powder or a solution. In some other embodiments, the pharmaceutical compositions according to the invention are in the form of a powder that can be reconstituted by addition of a compatible reconstitution diluent prior to parenteral administration. Non-limiting example of such a compatible reconstitution diluent includes water.

In some other embodiments, the pharmaceutical compositions according to the invention are in the form of a frozen composition that can be diluted with a compatible diluent prior to parenteral administration.

In some other embodiments, the pharmaceutical compositions according to the invention are in the form ready to use for parenteral administration.
In the methods according to the invention, the pharmaceutical composition and/or other pharmaceutically active ingredients disclosed herein may be administered by any appropriate method, which serves to deliver the composition or its constituents or the active ingredients to the desired site. The method of administration can vary depending on various factors, such as for example, the components of the pharmaceutical composition and the nature of the active ingredients, the site of the potential or actual infection, the microorganism (e.g. bacteria) involved, severity of infection, age and physical condition of the subject. Some non-limiting examples of administering the composition to a subject according to this invention include oral, intravenous, topical, intrarespiratory, intraperitoneal, intramuscular, parenteral, sublingual, transdermal, intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal, gene gun, dermal patch, eye drop, ear drop or mouthwash.

In general, the pharmaceutical compositions and method disclosed herein are useful in preventing or treating bacterial infections. Advantageously, the compositions and methods disclosed herein are also effective in preventing or treating infections caused by bacteria that are considered to be less or not susceptible to one or more of known antibacterial agents or their known compositions. Some non-limiting examples of such bacteria known to have developed resistance to various antibacterial agents include Acinetobacter, E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterobacter, Klebsiella, Citrobacter and a like.

A wide variety of microbial infections can be treated using the compositions and methods according to this invention. Other non-limiting examples of infections that may be prevented or treated using the compositions and/or methods of the invention include: skin and soft tissue infections, septicemic infections, febrile neutropenia, urinary tract infection, intraabdominal infections, respiratory tract infections, pneumonia (nosocomial), bacteremia meningitis, surgical infections etc.

A wide variety of bacterial infections can be treated using the compositions and methods according to this invention Examples of bacterial infections which can be treated or prevented using methods and/or pharmaceutical compositions according to this invention include, without limitation, E. coli infections (e.g. urinary tract), Yersinia pestis (pneumonic plague), Staphylococcal infection, Mycobacteria infection, bacterial pneumonia, Snigella dysentery, Serrate infection, Candida infection, Cryptococcal infection, methicillin resistant Staphyloccus aurues, anthrax, tuberculosis or those caused by Pseudomonas aeruginosa etc.

It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. For example, those skilled in the art will recognize that the invention may be practiced using a variety of different compounds within the described generic descriptions.

EXAMPLES

The following examples illustrate the embodiments of the invention that are presently best known. However, it is to be understood that the following are only exemplary or illustrative of the application of the principles of the present invention. Numerous modifications and alternative compositions, methods and systems may be devised by those skilled in the art without departing from the spirit and scope of the present invention. The appended claims are intended to cover such modifications and arrangements. Thus, while the present invention has been described above with particularity, the following examples provide further detail in connection with what are presently deemed to be the most practical and preferred embodiments of the invention.

Induction of Diabetes in rats by Streptozotocin injection: Diabetes was induced experimentally in overnight fasted rats by a single intraperitonial injection of freshly prepared Streptozotocin (dissolved in freshly prepared citrate buffer pH-4.5) at standardized dose of 45 mg/kg. The rats exceeding blood glucose levels of 200 mg/dl were considered as diabetic and were included in the study. Induction of Diabetes mellitus was confirmed by estimating fasting blood glucose levels, body weights; and other biochemical parameters such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine.

Pharmacokinetics of Cefpodoxime Proxetil in Diabetic Rats and Control Rats when administered orally and intravenously: Pharmacokinetic (PK) study was performed in which Cefpodoxime Proxetil was orally administered at 10 and 20 mg/kg dose, and Cefpodoxime sodium was intravenously administered at 10 mg/kg dose in control and diabetic animals. In diabetic rats Cefpodoxime Proxetil was administered after 14th day of Streptozotocin (45 mg/kg; i.p.) administration. The blood samples were collected from the animals by retro-orbital sinus at 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 24 hours. The collected blood samples were kept in bacteriological incubator at 37±2ºC for 15-20 minutes. Blood samples were centrifuged at 6000 rpm at temperature of 18-22ºC for 10 minute and serum was separated. The equal volume of separated serum from three animals were pooled for each time point and stored at -60ºC. Samples were then processed by Acetonitrile precipitation method. Processed samples were then analyzed by HPLC for the estimation of drug in serum. Pharmacokinetic parameters such as maximum concentration (Cmax), time taken to reach maximum concentration (Tmax), Half-life (t1/2), Area under the curve (AUC) and Clearance (Cl) were estimated using WinNonlin software.

Serum samples processing by Acetonitrile precipitation (ACN) method: Serum samples have to be processed by a suitable technique before HPLC analysis. Acetonitrile precipitation addresses the removal of proteins as phospholipids and other contaminants that remain in matrix may cause inconsistency and inaccuracy in detection and quantization of compound of interest as well as reduction in sensitivity and run to run reproducibility of HPLC and failure of columns. Aliquots of 300 µl of serum (either unknown serum samples or blank serum spiked with known amounts of varying concentrations of Cefpodoxime) was taken in a 1.5 ml of eppendorf tube and 10 µl of O-Phosphoric acid was added to it and 1000 µl of acetonitrile was added for precipitation of proteins. The tubes were vortex-mixed for 30 seconds to bring about a complete extraction of drug from proteins. The tubes were then centrifuged at 10000 rpm at 4-6ºC for 3 minutes. The supernatant was collected and analyzed by HPLC.

HPLC method of Analysis: A simple, sensitive, specific, precise, and stability-indicating reverse phase high-performance liquid chromatographic (HPLC-Agilent series-1100) method for determination of Cefpodoxime Proxetil and Cefpodoxime acid was developed and standardized as per the International Conference on Harmonization (ICH) guidelines. A gradient separation was achieved using YMC-pack C-18 HPLC (250 mm × 4.6 mm i.d., 5 µm particle size) column with a flow rate of 0.75 ml/min and a UV detector to monitor the eluent at 235 nm. The mobile phase consisted of 10 mM ammonium formate; pH 3, and acetonitrle in the ratio of 95:5 (v/v). Total run time for each sample analysis was 12 minute. The linear regression analysis data for the calibration plots showed good linear relationship with r2 = 0.9998 in the working concentration range of 20-0.156 µg/ml. The standard drug peaks were best resolved at retention time (tR=8.6). The column was maintained at ambient temperature (50ºC). The mobile phase was vacuum filtered through 0.45 µm nylon membrane filter followed by degassing prior to use. Samples were then injected in the system through sample vials after processing. Data acquisition and integration were performed using Chemstation software.

In situ Single-Pass Intestinal Perfusion Model (SPIP): The in situ single pass intestinal perfusion model in rats was used to determine the absorption characteristics of the drug and to determine regional disposition of drug. The actual fate of the drug after oral ingestion can only be obtained from in situ-absorption studies, as they involve actual process of absorption of drug with presence of blood circulation, mucus layer, and GI secretions. For this, in situ absorption study was performed using SPIP method was adapted from literature. According to Food and Drug Administration (FDA) it is also useful model to classify a compound’s absorption characteristics in the Biopharmaceutics Classification System (BCS).

Rats were fasted 12-18 hours prior to the start of experiment but had free access to water. Rats were weighed and total body weight was recorded for each rat. Rats were anesthetized with i.p. injection of urethane at 1.5 g/kg dose (0.5 ml/200 g of rat). Rats were not reacted to tail pinch before cutting the abdominal cavity to ensure compliance with the Institutional Animal Care and Use Committee (IACUC) protocol. First cut into skin and then abdominal cavity was opened by a middle incision of 3-4 cm and jejunum was located. A segment of jejunum (~10 cm) was cannulated using PE350 tubing. The cannulae were secured with a surgical silk suture. The infusion pump was set at the desired flow rate (0.2 ml/min). Cleared the intestine for about 30 minute using the perfusate buffer (Perfusion buffer (pH=6.8-7.0): Di-Sodium Hydrogen Phosphate (4.47 g), Sodium Dihydrogen Phosphate (9.52 g), and Sodium Chloride (7g) were added in 1000 ml of Mili-Q and adjusted within pH range). Non-cannulated portions of intestine were placed within the abdominal cavity after the cannulation. The rest of the intestinal segment laid flat on the abdominal surface of the rats. The whole area is then covered by a cotton piece wetted with normal saline to keep the intestinal segment moist. For permeability determination, Cefpodoxime was dissolved in perfusion buffer (30 µg/ml) and perfused at the flow rate of 0.2 ml/minute. Phenol red (30 µg/ml) was added to the drug solution as unabsorbable marker to measure Net Water Flux (NWF). A negative NWF indicates loss of fluid from mucosal side (lumen) to serosal side (blood) while a positive NWF indicates secretion of fluid into the segment. The drug solution was infused into the segment for 30 minutes to achieve steady state concentration. And then outlet perfusate was collected at 0, 5, 10, 15, 30, 45, 60, 90, and 120 minutes interval. At the end of 120 minutes, the length and diameter of cannulated segment was measured. Rats were euthanized by cervical dislocation technique. Collected perfusate samples were prepared for further measurements. Samples were centrifuged at 10000 rpm for 3 minutes at 6-8ºC to pellet any proteins, cells, or cellular debris before samples were injected in HPLC. Same procedure was followed for all drugs like: std. drug i.e. Propranolol, and Cimetidine. All the samples were analyzed on HPLC from where concentration can be estimated and permeability coefficient can be calculated by using following formula:

Peff(cm/s ) = [ -Q ln(Cout / Cin)]/ 2pRL
wherein,
Q is flow rate (0.2 ml/min);
Cin and Cout are the inlet and outlet perfusate concentrations;
L is length (approximately 10 cm); and
R is radius (for duodenum, jejunum and ileum =0.18 cm).

The intestinal Net Water Flux was calculated using following formula:-
NWF= (1-[Ph. Red out / Ph. Red in]*Q in)/L
wherein, Ph. Red out and Ph. Red in are the inlet and outlet concentrations.

Serum protein binding estimation of Cefpodoxime: Binding of the drug with serum proteins were also estimated to get an idea for total protein binding of drug and its free concentration available in serum of diabetic rats at 5µg/ml and 10µg/ml concentration as compared to control rats. The overnight fasted rats were taken. Blank (untreated rats) blood of normal and diabetic wistar rats was withdrawn separately by retro-orbital bleeding technique. After collection of blood rats were cervically dislocated and blood was incubated in microbiological incubator for 30 minutes at 37ºC for serum separation. Serum was separated by centrifusing at 5000 rpm for 15 minutes. Stock solution of 1 mg/ml of Cefpodoxime was prepared and required concentration were added to equal volume of serum in tubes in duplicate manner to estimate better protein binding. Samples were then centrifuged in ultracentrifuge at 300000 rpm for 3:45 hours at 0-4ºC. Supernatant was collected from each tube individually and analyzed directly on HPLC. Method for HPLC analysis was same as for pharmacokinetic studies. Respective concentrations were estimated by HPLC analysis and protein binding was calculated by the following formula:

% Binding = Actual conc. of drug – Free drug conc.
Actual conc. of drug

Table 1 gives the details of the physiological and biochemical examinations in control and Streptozotocin induced diabetic rats. Body weight, blood glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine were measured on day 14th after Streptozotocin administration. As can be seen from the data in the Table 1, there was 4 fold increase in the blood glucose level in diabetic rats compared to control. It was also observed that there was decrease in body weight and increase in AST, ALT, BUN and creatinine levels in diabetic rats compared with controls.

Table 1. Effect of Streptozotocin after 14 days of administration on plasma glucose, body weight, ALT, AST, BUN and creatinine levels of rats.
Sr. Parameters Control Diabetes
1 Body weight (g) 260.6 ± 1.62 183.4 ± 2.40***
2 Blood glucose (mg/dl) 91.59 ± 2.44 430.7 ± 19.20***
3 AST (µg/l) 151.62 ± 5.59 297.55 ± 24.40***
4 ALT (µg/l) 51.28 ± 1.96 78.56 ± 5.60***
5 BUN (mg/dl) 13.44 ± 0.78 32.09 ± 1.86***
6 Creatinine (mg/dl) 0.105 ± 0.017 0.198 ± 0.03*
Values are expressed as mean ± SEM (n=6), ***p<0.001, **p<0.01 and *p<0.05 when compared to control.

Figures 1 A and 1 B gives the serum concentration versus time profile after the oral administration of Cefpodoxime Proxetil (10 and 20 mg/kg) in diabetic and control rats. Table 2 and Table 3 enlists the corresponding estimated pharmacokinetic parameters after the oral administration of Cefpodoxime Proxetil (10 and 20 mg/kg) in diabetic and control rats. Similarly, Figure 1 C gives the serum concentration versus time profile after the intravenous administration of Cefpodoxime Sodium (10 mg/kg) in diabetic and control rats, and Table 4 gives the corresponding pharmacokinetic parameters. It was observed that the mean AUC and Cmax of diabetic rats were decreased significantly in comparison to control after administering 10 and 20 mg/kg oral dose of Cefpodoxime Proxetil, and 10 mg/kg intravenous administration of Cefpodoxime Sodium. The reduced concentrations and AUC of Cefpodoxime in diabetic rats reduced the bioavailability in diabetic rats. Thus results indicate that diabetes is affecting the pharmackinetics of Cefpodoxime.

To investigate whether lower exposure of Cefpodoxime in serum of diabetic rats resulted from an increase in systemic clearance, pharmacokinetic of Cefpodoxime was studied in diabetic and control rats following intravenous administration of Cefpodoxime sodium (10mg/kg). The results indicated that diabetic condition increased systemic clearance of Cefpodoxime (Table 4), and hence lowered bioavailability of Cefpodoxime. Reduction in AUC and Cmax could have been due to significantly faster clearance of Cefpodoxime.
Table 2. Pharmacokinetic parameters at the dose of 10 mg/kg oral administration of Cefpodoxime Proxetil.
Groups Treatment AUC (µg .h/ml) C max (µg/ml) t1/2 (h)
Control Cefpodoxime Proxetil 14.5 ± 0.320 6.10 ± 0.175 1.18 ± 0.27
Diabetic Streptozotocin +
Cefpodoxime Proxetil 8.98 ± 1.41*** 4.80 ± 0.200*** 1.37 ± 0.38
Values are expressed as mean ± SEM (n=6), ***p<0.001, **p<0.01 and *p<0.05 when compared to control.

Table 3. Pharmacokinetic parameters at the dose of 20 mg/kg oral administration of Cefpodoxime Proxetil.
Groups Treatment AUC (µg .h/ml) C max (µg/ml) t1/2 (h)
Control Cefpodoxime proxetil 23.0 ± 0.535 13.0 ± 0.320 1.05 ± 0.27
Diabetic Streptozotocin +
Cefpodoxime proxetil 18.8 ± 0.815*** 8.99 ± 0.680*** 1.22 ± 0.27
Values are expressed as mean ± SEM (n=6), ***p<0.001, **p<0.01 and *p<0.05 when compared to control.

Table 4. Pharmacokinetic parameters at the dose of 10 mg/kg intravenous administration of Cefpodoxime Proxetil.
Groups Treatment AUC
(µg.h/ml) C max
(µg/ml) t1/2
(h) Cl
(l/h/kg)
Control Cefpodoxime proxetil 28.46 ± 0.983 59.96 ± 9.90 0.80 ± 0.15 0.35 ± 0.03
Diabetic Streptozotocin +
Cefpodoxime proxetil 10.16 ± 0.13*** 21.80 ± 0.71** 1.2 ± 0.57 0.97 ± 0.03
Values are expressed as mean ± SEM (n=6), ***p<0.001, **p<0.01 and *p<0.05 when compared to control.

Table 5 gives the details of the effective permeability of Cefpodoxime. The effective permeability was determined by In-situ single pass intestinal perfusion model (SPIP). No significant change in P eff value of Cefpodoxime was observed in diabetic and control rats. Table 6 details the protein binding behavior of Cefpodoxime in diabetic and control rats. No significant change in protein binding was observed in diabetic rat for binding of drug to the serum proteins.

Table 5. Effect of Diabetes Mellitus on the effective permeability (Peff) of Cefpodoxime Proxetil (30mcg/ml).
S.No. Drugs Peff (×10-5cm sec)
1 Propranolol 2.717 ± 0.5096
2 Cimetidine 0.0922 ± 0.3983
3 Cefpodoxime (control) 0.055 ± 1.38
4 Cefpodoxime (diabetic) 0.0572 ± 1.31
Values are expressed as mean ± SEM (n=3)

Table 6. Effect of Diabetes mellitus on serum protein binding behavior of Cefpodoxime by ultracentrifugation method.
Groups Protein binding at 5 µg/ml (%) Protein binding at 10 µg/ml (%)
Control 69.76 ± 1.14 67.65 ± 0.07
Diabetic 67.65 ± 0.412 68.92 ± 1.12
Values are expressed as mean ± SEM (n=6)

The results of Tables 1-4 and Figures 1-3 clearly show the alterations in pharmacokinetic parameters of Cefpodoxime upon administration in a diabetic subject. Thus, the alteration of these pharmacokinetic parameters helps in designing better dosage regimen for treatment of bacterial infections in diabetic patients.


(A) (B)

(C)
Figure 1. Serum concentration-Time profile of Cefpodoxime Proxetil after intragastric administration of 10 mg/kg (A), 20 mg/kg (B) and Cefpodoxine Sodium after intravenous administration of 10 mg/kg (C). (Values as expressed as mean ± SEM (n=32), ***p<0.001 when compared to control).
,CLAIMS:1. A method of treating bacterial infections in a diabetic subject, said method comprising administrating high dose of Cephalosporin antibacterial agent or pharmaceutical acceptable derivative thereof, wherein said Cephalosporin exhibits altered pharmacokinetic parameters in a diabetic subject.

2. A method according to Claim 1, wherein bacterial infections are caused by bacteria producing one or more beta-lactamase enzymes.

3. A method according to Claim 1, for treatment of bacterial infections selected from the group consisting of skin and soft tissue infections, septicemic infections, febrile neutropenia, urinary tract infection, intraabdominal infections, respiratory tract infections, pneumonia (nosocomial), bacteremia meningitis or surgical infections.

4. A method according to Claim 1, wherein Cephalosporin antibacterial agent is selected from the group consisting of Cefixime, Ceftriaxone, Cefcapene, Cefdaloxine, Cefdinir, Cefditoren, Cefetamet, Cefmenoxime, Cefodizime, Cefotaxime, Cefpimizole, Cefpiramide, Cefpodoxime, Cefsulodin, Cefteram, Ceftibuten, Ceftiolene, Ceftizoxime, Flomoxef, Latamoxef or pharmaceutically acceptable salts thereof.

5. A method of treating bacterial infections in a diabetic subject, said method comprising administrating high dose of Cefpodoxime or pharmaceutical acceptable derivative thereof, wherein said Cefpodoxime exhibits altered pharmacokinetic parameters in a diabetic subject.

6. A method according to Claim 5, wherein Cefpodoxime exhibits reduction in dose proportional pharmacokinetic parameters selected from the group consisting of Cmax and AUC, in a diabetic subject.

7. A method according to Claim 5, wherein Cefpodoxime exhibits increase in Clearance in a diabetic subject.

8. A method of treating bacterial infections in a diabetic subject, said method comprising high dose of Cefpodoxime or pharmaceutical acceptable derivative thereof, wherein said dose is in an amount 20-50% greater than that administered in non-diabetic subject.

9. A method according to Claim 8, wherein the dose of Cefpodoxime is in range of 240 mg/day to 1200 mg/day.

10. A pharmaceutical composition comprising high dose of Cephalosporin antibacterial agent for treatment of bacterial infections in a diabetic subject.