Patent Grant
7119075
The present invention relates to the field of chemotherapeutics, particularly to their formulation as of oral pharmaceutical compositions containing bioenhancers for increasing bioefficacy of anti-infectives and thereby requiring lower doses and/or decreased frequency of dosing of such anti-infectives while maintaining the therapeutic efficacy of standard doses of such drugs.
A variety of human ailments owe their origin to pathogenic microorgnisms, which include bacteria, virus and fungi. The presence of such pathogenic microorgnisms lead to septicaemia, serious infections of upper and lower respiratory tract, CNS, meningitis, intra-abdominal including peritoneum, genito-urinary tract, skin, and soft tissue, and variety of other infections like systemic mycosis, candidiasis including infections caused by dermatophytes. During the last 100 years, significant progress has been made to combat the diseases caused by such a large family of microbes with innumerable therapeutic agents of diverse chemical and biological nature that have become available as a short and long-term cure. Such antimicrobials include aminoglycosides, penicillins, cephalosporins, macrolides, glycopeptides, fluoroquinolones, tetracyclins, first and second line anti-TB drugs, anti-leprosy, antivirals, polyene, txiazole, and imidazole anti-fungals, combinations like pyrimidine derivatives and trimethoprim and sulphamethoxizole.
While such agents are effective against pathogenic bacteria and fungi and therefore useful in the treatment of disease conditions associated with the presence of such pathogens, there is increasing evidence that use of such agents has certain limitations and led to clinical concern. There are several such factors responsible for such a concern: (a) certain strains of bacteria and fungi become increasingly resistant to one or more of the known anti-infectives and therefore the usual or standard therapeutic doses lead to less beneficial effect, (b) higher doses that are required to combat this cause undesirable side effects and toxicity, and (c) high-cost of treatment and patient-non-compliance. The emergence of drug-resistant pathogenic organisms has also been attributed to uncontrolled antibiotic overuse and under use and even under dosing, irrational frequency of administration. The prolonged and high dose therapy is also a matter of serious concern particularly in pregnant women, geriatrics and children.
While an approach embodying rational use of antibiotics use may help slow the problem of microbial drug resistance, new antimicrobial agents must be discovered to combat those strains that are now resistant to most, if not all, currently available antibiotics. As such, there is a continued interest in the identification of novel antimicrobial agents, which can be used to further supplement the medical practitioner's armamentarium against pathogenic microorganisms
In another approach, two anti-infectives are combined in such a way that the combination produces synergy i.e. one of the anti-infective acts as the potentiator of the other antiinfective. The example of such combination is Trimethoprim-sulfamethoxazole also known as co-trimoxazole or TMP-SMX, which was introduced in 1968 as a broad-spectrum antimicrobial agent. Trimethoprim was specially developed as a potentiator of sulphonamide to act synergistically against bacteria and delay the development of bacterial resistance. The 1:5 ratio of trimethoprim to sulfamethoxazole achieves an approximate 1:20 ratio of peak serum concentrations which is the optimal synergistic ratio of serum concentrations against most susceptible bacteria (Gutman L T, Pediatr Infect Dis 1984;3:349–57, Olin B R, Facts and Comparisons, Inc. 1998; 408b–409d, Cockerill F R, Edson R S, Mayo Clin Proc 1991;66:1260–9)
The combination can also be between one antiinfective agent and another chemical agent which by itself is not antiinfective in nature but when combined with the antiinfective, enhances the effectiveness of this antiinfective. The example of such combination is Amoxicillin+Clavulanic acid, more commonly known as Augmentin. Amoxicillin is an antibiotic of the penicillin type. It is effective against different bacteria such as H. influenzae, N. gonorrhea, E. coli, Pneumococci, Streptococci, and certain strains of Staphylococci. Chemically, it is closely related to penicillin and ampicillin. Addition of clavulanic acid to amoxicillin in Augmentin enhances the effectiveness of this antibiotic against many other bacteria that are ordinarily resistant to amoxicillin. Clavulanic acid is produced by the fermentation of Streptomyces clavuligerus. It is a β-lactam structurally related to the penicillins and possesses the ability to inactivate a wide variety of β-lactamases by blocking the active sites of these enzymes. Clavulanic acid is particularly active against the clinically important plasimd mediated β-lactamases frequently responsible for transferred drug resistance to penicillins and cephalosporins.
One of the most notable features that has been associated with the traditional Indian medicine and amply described in Ayurveda is the use of compositions which offer additive, synergistic and potentiating effect of one medicament when used in combination with the other. In Ayurveda there are several natural products, which have been used as an essential ingredient of many formulations used against wide range of diseases. The most prominent of these being ‘Trikatu’ comprising black pepper, long pepper and dry ginger. Detailed and systematic studies have shown that one of the active ingredients of peppers i.e., piperine is a potent bioavailability and/or bioeffacicay enhancer of several drugs and nutrients. The process of obtaining piperine and piperine containing formulations including anti-TB antibiotics with enhanced bioavailability/bioefficacy at lower doses of active drugs has been disclosed in earlier patents (IP 172684,; IP 172690,; IP 176433; U.S. Pat. No. 5,439,891).
The main object of the invention is to provide a oral pharmaceutical compositions containing bioenhancers for increasing bioefficacy of anti-infectives and thereby requiring lower doses and/or decreased frequency of dosing of such anti-infectives while maintaining the therapeutic efficacy of standard doses of such drugs.
The present invention deals with one such combinations, where piperine and other bioenhancers are used as potentiators when combined with various anti-infective agents in vitro using bacteria, viruses and yeast and in vivo using mice and guinea pig infection models. The present invention is aimed to overcome or avoid problems faced in the prior art. The use of products of the present invention offer a low dose regimen that produces enhanced therapeutic action comparable to that of standard dose alone.
Accordingly, the present invention provides a composition useful for enhanced therapeutic effect at reduced doses of the anti infectives against infection caused bya microorganism comprising a mixture of an anti-infective agent and a bioenhancer selected from piperine of formula 1 and 3′,5-Dihydroxy flavone 7-O-β-D-galacturonide-4′-O-β-D-glucopyranoside of formula 2 or a mixture thereof.
In one embodiment of the invention, the anti infective agent is selected from the group consisting of penicillins including semi synthetic, cephalosporins, aminoglycosides, glycopeptides, fluroquinolones, macrolides, tetracyclines, first and second line anti-TB drugs, antileprosy drugs, oxazolidelones, antifungal agents, antiviral agents and pyrimidine derivatives—sulphonamides combination.
In a further embodiment of the invention, the anti-fungal agent is selected from the group consisting of polyenes, imidazoles and triazoles.
In yet another embodiment of the invention, the antiviral agent is selected from the group consisting of Zidovudines, idouridine, acyclovir and ribavarine.
In another embodiment of the invention, the 3′,5-Dihydroxy flavone 7-O-β-D-galacturonide-4′-O-β-D-glucopyranoside is used in pure form or in the form of a HPLC fingerprinted fraction of 3′,5-Dihydroxy flavone 7-O-β-D-galacturonide-4′-O-β-D-glucopyranoside from Cuminum cyminum or a sub fraction.
In another embodiment of the invention, the concentration of the anti infective is two to eight times lesser than when such anti infective is used without the bioenhancer.
In another embodiment of the invention, the composition includes one or more pharmaceutically acceptable additives and excipients.
In another embodiment of the invention, the additives/excipients are selected from the group consisting of nutrients comprising proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, starch-gelatin paste, and/or pharmaceutically acceptable carriers, diluents and solvents.
In another embodiment of the invention, the composition is in oral administration form.
In a further embodiment of the invention, the ratio of the anti-infective to the bioenhancer is in the range of 1:1 to 1:5.
In yet another embodiment of the invention, the additives have no effect on the antiinfective property of the said composition.
The present invention also provides a process for the preparation of a composition useful for enhanced therapeutic effect at reduced doses of the anti infectives against infection caused bya microorganism comprising a mixture of an anti-infective agent and a bioenhancer selected from piperine of formula 1 and 3′,5-Dihydroxy flavone 7-O-β-D-galacturonide-4′-O-β-D-g of formula 2 or a mixture thereof, said process comprising a physical admixing technique.
In one embodiment of the invention, the physical admixing technique is selected from dialysis, molecular sieving and by membranes.
In another embodiment of the invention, the process of preparation of the bioenhancer comprises use of water, alcohol, combinations of water and alcohol, hydrocarbons, ketones and ethers.
In one embodiment of the invention, the anti infective agent is selected from the group consisting of penicillins including semi synthetic, cephalosporins, aminoglycosides, glycopeptides, fluroquinolones, macrolides, tetracyclines, first and second line anti-TB drugs, antileprosy drugs, oxazolidelones, antifungal agents, antiviral agents and pyrimidine derivatives—sulphonamides combination,
In a further embodiment of the invention, the anti-fungal agent is selected from the group consisting of polyenes, imidazoles and triazoles.
In yet another embodiment of the invention, the antiviral agent is selected from the group consisting of Zidovudines, idouridine, acyclovir and ribavarine.
In another embodiment of the invention, the 3′,5-Dihydroxy flavone 7-O-β-D-galacturonide-4′-O-β-D-glucopyranoside is used in pure form or in the form of a HPLC fingerprinted fraction of 3′,5-Dihydroxy flavone 7-O-β-D-galacturonide-4′-O-β-D-glucopyranoside from Cuminum cyminum or a sub fraction.
In another embodiment of the invention, the concentration of the anti infective is two to eight times lesser than when such anti infective is used without the bioenhancer.
In another embodiment of the invention, the composition includes one or more pharmaceutically acceptable additives and excipients.
In another embodiment of the invention, the additives/excipients are selected from the group consisting of nutrients comprising proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, starch-gelatin paste, and/or pharmaceutically acceptable carriers, diluents and solvents.
In another embodiment of the invention, the composition is in oral administration form.
In a further embodiment of the invention, the ratio of the anti-infective to the bioenhancer is in the range of 1:1 to 1:5.
In yet another embodiment of the invention, the additives have no effect on the anti-infective property of the said composition.
Studies originating from the laboratory of the inventors resulted in conceptualisation of ‘bioenhancers’ wherein such agents, which by themselves are not therapeutic entities but when combined with an active drug lead to the potentiation of the pharmacologic effect of the drug. Such formulations have been found to increase the bioavailability/bioefficacy of a number of drugs even when reduced doses of drugs are present in such formulations. Evidence have been obtained for such classes of drugs which are (a) poorly bioavailable and/or efficacious, (b) require prolonged therapy, and (c) are highly toxic and expensive. For example, Patent Nos. IP 172690, IP 176433 and U.S. Pat. No. 5,744,161 disclose such art. Further studies carried out in the laboratory of the inventors have shown that such bioenhancers are not only capable of increasing bioavailability of a wide variety of therapeutic agents but are also capable of enhancing bioefficacy through a variety of mechanisms underscored in serial nos (a) to (g) below. As a result newer understanding has emerged about the factors involved in decreased cellular concentrations of drugs at which they fail to attain therapeutic levels and the strategies that make it possible to enhance the bioavailability and/or bioefficacy of these active drugs even at lower concentrations compared to standard high dosing. Some of these factors are:
The products of the present invention are novel mechanism based pharmaceutical entities acting through synergism and or additive effect so that drugs contained in the formulation are more bioefficaceous as a result of one or more of the mechanism as revealed above and thereby increasing the sensitivity of the target cell to an anti-infective.
The ‘drug’ in the present invention refers to a chemical entity capable of affecting organism's patho-physiology and used for the treatment or prevention of disease. Drugs include a number of classes of compounds, but not limited to aminoglycoside, penicillins, cephalosporins and other β-lactam agents, macrolides, glycopeptides, fluoroquinolones, tetracyclines, first and second line anti-TB drugs, anti-leprosy, antivirals, polyene, triazole, and imidazoles and combinations like pyrinidines, sulphamethoxazole. Drugs may be a pro-drug, activated or metabolised form, consisting of charged, uncharged, hydrophilic, hydrophobic or zwitter-ion species which make their entry by simple diffusion, carrier mediated transport dependent and not dependent on energy requirements, through ion and/or voltage gated channels.
The ‘bioenhancer’ refers to piperine (formula 1) or other such molecules, characterised fractions and/or extracts as a chemical entity. The process of obtaining piperine as more than 90% pure chemicaly characterized form has been disclosed in IP 172689, IP 172690, IP 176433, U.S. Pat. No. 5,439,891 and a co-pending U.S. patent application Ser. No. 60/306917/2001. The processes for preparation of a characterised fraction (HPLC profile enclosed) and a pure chemically characterised molecule (
The checkerboard method:
This is the most frequently used method to access the antimicrobial combinations in vitro. The term “checkerboard” refers to the pattern (of tubes or microtiter plate wells) formed by multiple dilutions of two drugs being tested (Eliopoulos G M, Moellering R C. Antimicrobila Combinations. In: Antibiotics in Laboratory Medicine: USA: Williams & Wilkins). In the present study the checkerboard consisted of columns in which each tube (or well) contains the same amount of the standard drug (antibacterial/antifungal/anti-TB/antiviral) being diluted along the x-axis and rows in which each tube (or well) contains the same amount of the bioenhancer being diluted on the y-axis (
Agar Method:
In this method the agar (Mueller Hinton agar, Middlebrook 7H10 agar) was autoclaved and allowed to cool to 50° C. to 55° C. The combination of the standard drug and the bioenhancer was added to the agar. Serial two fold dilutions of each of standard drug and the bioenhancer were prepared in appropriate solvents. In order to maintain the desired concentrations of both agar and drugs, and to rule out the effect of solvent, the volume of solvent (containing standard drug or bioenhancer) added to agar was kept small (i.e ≦5% of the total volume). After the agar plates have been poured and allowed to dry, the bacteria to be tested were applied to the surface of agar with a replicating device designed to deliver a standard inoculum (approx 104 cfu|spot). The plates were incubated at 37° C. for 24 hrs (3 weeks in case of Mycobacterium tuberculosis)
Broth Method:
The above-mentioned checkerboard was also performed with liquid media in a microtiter plate format. This method was used to study the combination of antibacterial/antifingal/antiviral drugs with bioenhancer.
All bioenhancers were evaluated for their own inhibitory effect if any, at a concentration range of 500 μg/ml to 0.2 μg/ml. (Table 1, 2, & 3)
Staphylococcus aureus ATCC
Staphylococcus aureus
Staphylococcus epidermidis
Staphylococcus hemolyticus
Bacillus cereus RRL 02
Escherichia coli ATCC 25922
Salmonella typhi AIIMS 2
Klebsiella pneumoniae
Pseudomonas aeruginosa
Candida albicans
Candida parapsilosis
Candida glabrata
Candida tropicalis ATCC 750
Candida krusei ATCC 6258
Aspergillus flavus
Aspergillus fumigatus SI-1
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium avium ATCC
Mycobacterium intracelluare
Staphylococcus aureus ATCC
Staphylococcus aureus
Staphylococcus epidermidis
Staphylococcus. hemolyticus
Bacillus cereus RRL 02
Escherichia coli ATCC 25922
Salmonella typhi AIIMS 2
Klebsiella pneumoniae ATCC
Pseudomonas aeruginosa
Candida albicans
Candida parapsilosis
Candida glabrata
Candida tropicalis ATCC 750
Candida krusei ATCC 6258
Aspergillus flavus
Aspergillus fumigatus SI-1
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium avium ATCC
Mycobacterium intracelluare
Cuminum cyminum μg/ml
Staphylococcus aureus ATCC
Staphylococcus aureus
Staphylococcus epidermidis
Staphylococcus, hemolyticus
Bacillus cereus RRL 02
Escherichia coli ATCC 25922
Salmonella typhi AIIMS 2
Klebsiella pneumoniae ATCC
Pseudomonas aeruginosa
Candida albicans
Candida parapsilosis
Candida glabrata
Candida tropicalis ATCC 750
Candida krusei ATCC 6258
Aspergillus flavus
Aspergillus fumigatus SI-1
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium avium ATCC
Mycobacterium intracelluare
The following examples are intended to demonstrate some of the preferred embodiments but in no way be construed so as to limit the scope of the invention. Any person skilled in the art can design more formulations, which may be considered as part of the present invention.
This was done by the process claimed in Indian Patents 1726891 and IP 172690 and U.S. Pat. No. 5,439,891 and U.S. application Ser. No, 60/306917/2001, which is incorporated herein by reference.
Minimum Inhibitory Concentration (MIC) of rifampicin alone and in combination with piperine was performed against Mycobacterial species, using method described in the study design.
Two-fold reduction in MIC of rifampicin was observed in combination with piperine and fraction of Cuminum cyminum (Table 4-a, 4-b)
The study was conducted to see the in vivo response of rifampicin in combination with piperine. The Swiss albino mice were infected intravenously with M.tuberculosis H37Rv (106 CFU/mouse). The infected mice were divided in groups and each group consisted of 6 mice.
The treatment started 24 hrs post infection and continued for 4 weeks in once a day for 5 days in a week dosing schedule. The mice were sacrificed after 4 weeks and the CFU was enumerated from the lungs and the spleen. Rifampicin alone at 20 mg/kg was able to bring about 2-log deduction in log 10 CFU. Same effect was observed with rifampicin at 10 mg/kg when given in combination with pipette at 20 mg/kg. Whereas fraction of Cuminum cyminum was more effective as it yielded the same reduction in log 10 CFU at 5 mg/kg dose of rifampicin. (FIGS. 4-a, 4-b)
M tuberculosis
M tuberculosis
M tuberculosis
M tuberculosis
M avium
M intracellure
M tuberculosis
M tuberculosis
M tuberculosis
M tuberculosis
M avium
M intracellure
Minimum Inhibitory Concentration (MIC) of ciprofloxacin alone and in combination with piperine was performed against bacterial species, using method described in the study design. Two to more than eight fold reductions in MIC of ciprofloxacin was observed in combination with piperine. (Table-5)
S aureus ATCC
S epidermidis
S hemolyticus
Minimum Inhibitory Concentration (MIC) of fluconazole alone and in combination with piperine was performed against fungal species, using method described in the study design Two to eight fold reductions in MIC of fluconazole was observed in combination with piperine. (Table-6)
Candida albicans
Candida albicans
Candida
parapsilosis
Candida glabrata
M. tuberculosis
M. tuberculosis
M. tuberculosis
M. tuberculosis
M. tuberculosis
M. tuberculosis
| Number | Name | Date | Kind |
|---|---|---|---|
| 5439891 | Kapil et al. | Aug 1995 | A |
| 5665386 | Benet et al. | Sep 1997 | A |
| 5744161 | Majeed et al. | Apr 1998 | A |
| 6841174 | Shalaby et al. | Jan 2005 | B1 |
| Number | Date | Country |
|---|---|---|
| 9714319 | Apr 1997 | WO |
| 2004009061 | Jan 2004 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20040198672 A1 | Oct 2004 | US |
