WO2002022147A1 - Combinaison antibacterienne comprenant un extrait de plante de margousier a feuilles de frene - Google Patents

Combinaison antibacterienne comprenant un extrait de plante de margousier a feuilles de frene Download PDF

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Publication number
WO2002022147A1
WO2002022147A1 PCT/IN2000/000095 IN0000095W WO0222147A1 WO 2002022147 A1 WO2002022147 A1 WO 2002022147A1 IN 0000095 W IN0000095 W IN 0000095W WO 0222147 A1 WO0222147 A1 WO 0222147A1
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Prior art keywords
extract
composition
antibiotic
neem
bacterial
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PCT/IN2000/000095
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English (en)
Inventor
Narasimha Baba Brindavanam
Chandra Kant Khatiyar
Dasalukunte Bhimra Anantanarayana
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Dabur Research Foundation
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Priority claimed from US09/662,809 external-priority patent/US6599541B1/en
Application filed by Dabur Research Foundation filed Critical Dabur Research Foundation
Priority to AU2001235965A priority Critical patent/AU2001235965A1/en
Publication of WO2002022147A1 publication Critical patent/WO2002022147A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/58—Meliaceae (Chinaberry or Mahogany family), e.g. Azadirachta (neem)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • the invention provides a synergistic novel composition which can be used against bacteria that have developed resistance to conventional antibiotics and other chemotherapeutic anti-microbial agents.
  • the invention provides a composition comprising a herbal agent to re-sensitize the resistant bacterial strains and an antibiotic or a chemotherapeutic anti-bacterial drug to which, the bacterial strain is resistant.
  • the invention also provides a process for the preparation of the composition.
  • Bacterial resistance to an antibiotic can be of two kinds (Beam Jr. TR, 1992 on Principles of Anti-infectives Use - chapter-47 in Text Book of Pharmacology, ed. Smith CM and Raynard AM, published by W. B. Saunders Company, 1 st ed., p-812-814.).
  • the first one is called intrinsic resistance. This is a natural phenomenon and hence, all antibiotics do not exhibit activity against all sorts of bacteria but work in a selective manner.
  • the second type is an acquired resistance. In this type, a particular bacterial species appear to be susceptible to an antibiotic initially but develops resistance over a period of time.
  • Ente ob cter species inactivate Erythromycin by producing an esterase.
  • bacteria may alter the target site.
  • Erythromycin normally latches to 50s ribosomal sub-units and inhibits the synthesis of proteins and thereby kills bacteria.
  • certain bacteria are now known to modify the target site.
  • bacteria may reduce the permeability of antibiotic substance by altering the structure of its own cell wall.
  • Amoxycillin is an amino-penicillin and is degraded by ⁇ - lactamase producing bacteria.
  • Clavulanic acid was discovered to inhibit the activity of ⁇ - lactamase but is devoid of antibacterial activity of its own (Reading C & Cole M, 1977 A beta lactamase inhibiting beta lactam from Streptomyces clavuligenus; Antimicrob Agents Chemother, 11, p-852-857, Reading C, Farmer T & Cole, M 1983,_The Beta lactamase stability of Amoxycillin with beta lactamase inhibiting Clavulanic acid, J. Antimicrob Chemother., 11, p-27-32 & Todd PA & Benfield P 1990 Amoxycillin/Clavulanic acid, an up-date of its antibacterial activity, pharmacokinetic properties & Therapeutic use,
  • the neem tree also referred as Azadirachta indica A. juss is known since ages for its healthcare benefits. The tree has been attributed with many therapeutic effects in various Ayurvedic texts and even the common-folk appear to have a fair understanding about its medicinal uses and therefore, they use it often, for such benefits. Usually, the leaves, the seeds and their fixed oil portion and bark of neem are used for their medicinal activities. However, the leaves and bark are preferred for oral use in humans.
  • US Patent 5,372,817 teaches insecticidal compositions obtained from neem oil and neem wax fractions.
  • US patents 5,001,146 and 5,124,346 teach storage stable pesticidal compositions containing Azadirachtin. The extract used in all these compositions is from neem seeds.
  • neem seed oil was found to be effective against standard strains of Bacillus subtilis, Salmonella typhosa and Salmonella paratyphi (Jain PP, Suri RK, Deshmukh SK & Mathur KC 1987, fatty oils from oil seeds of forest origin as antibacterial agents, Ind. For, 113 (4), p-297 - 299).
  • Jain PP Suri RK
  • Deshmukh SK & Mathur KC 1987 fatty oils from oil seeds of forest origin as antibacterial agents, Ind. For, 113 (4), p-297 - 299
  • different fractions of neem leaf extract were found to be devoid of any antibacterial effect (Singh PP, Junnarkar AY, Reddy GS & Singh KV 1987 Azadirachta indica: neuro- psychopharmacological and antimicrobial studies, Fitorick v.58 (4), p-235-238).
  • Neem leaf was also found to intervene the biological systems of certain fungi and was observed to inhibit the synthesis of aflatoxins by Aspergillus parasiticus (Bhatnagar D & McCormick SP (1988): The inhibitory effect of neem (Azadirachta indica) leaf extracts on Aflatoxin Synthesis in Aspergillus parasiticus, JO ACS, 65(7), p-1166-1168).
  • neem can be effectively used to inhibit the growth of certain fungi.
  • the main problem is that these observations do not teach or discuss the effect of neem extracts on bacterial systems, although, there is a stray reference to the antibacterial value of neem in the papers.
  • Bacteria modify their biological systems in order to protect themselves, from the onslaught of antibiotics. As a result of these modifications, they acquire resistance and survive in their host even when the treatment with an antibiotic is on. Knowing these complex, intrinsic modifications taking place in bacteria - it would be hard to presume that, a mere admixture of two known anti-bacterial substances could do a wonder in case of resistant bacteria. Ideally, it needs an agent/substance, which can intervene these intrinsic biological mechanisms and thereby re-sensitize them to conventional antibiotics. Almost, a similar principle was employed in the discovery of Clavulanic acid - which is presently employed to treat ⁇ lactamase mediated bacterial resistance to amoxycillin.
  • a given substance might affect the biological systems of any organism in many ways. However, such modification in the biological systems should be in a well-specified direction to address selected issues.
  • the prior art lacks methods or even principles of overcoming resistance acquired by bacteria to conventional drug therapy.
  • development of resistance by bacteria to conventional antibiotics is a problem for which no concrete solution is available.
  • the inventors focussed their study on neem extract and its different properties.
  • extract of neem especially the leaves, can be used to address the problem of resistance developed by bacteria to conventional antibiotics.
  • the inventors selected extracts from neem leaves because it is well established fact that, most of Azadirachtin content is deposited in the seeds as compared to other aerial parts of Neem.
  • neem leaf extracts are capable of re-sensitizing bacteria to drugs to which they had developed resistance. This principle has been applied in the invention to provide novel compositions and methods of treatment.
  • the main object of the invention is to provide a composition comprising an extract obtained from aerial parts of the neem tree (Azadirachta indica or Melia azadirach) and an antibiotic or a chemotherapeutic anti-bacterial drug.
  • Another object is to provide a composition containing an extract obtained from the neem tree and an antibiotic or a chemotherapeutic anti-bacterial drug to which the bacteria have developed resistance.
  • Another object is to provide a method to render bacteria susceptible to the drugs to which they had developed resistance.
  • Yet another objective of invention is to provide a process for the preparation of the herbal extract for use in combination with antibiotics.
  • the invention provides a novel composition useful in the treatment of resistant bacterial infections, said composition comprising an extract obtained from Azadiracta indica or Melia azadirachta and an effective amount of an antibiotic or chemotherapeutic antibacterial drug. Further, the invention provides a method for the treatment of resistant bacterial infections. The invention also provides a process for the preparation of the composition of the invention.
  • the invention provides a novel synergistic composition
  • a novel synergistic composition comprising extract obtained from a neem plant, together with an effective amount of an antibiotic or chemotherapeutic antibacterial drug, said composition being useful in the treatment of resistant bacterial infections.
  • the extract is obtained from plants selected from Azadirachta indica or Melia azadirachta.
  • the plant chosen for this invention is Azadirachta indica.
  • the extract is obtained from leaves, bark and tender twigs, or combinations thereof, preferably leaves.
  • the neem extract is obtained using water at its natural pH or pH adjusted from 6.0 to 6.5. The total content of bitter principles in the extract is upto 9%. Further, the extract is obtained using any one of alcohols alone or by combining with water.
  • the amount of extract in the composition is in the range of 50-300mg per unit dose.
  • the extract re-sensitizes the resistant bacteria and renders it susceptible to antibiotics or chemotherapeutic antibacterial drugs.
  • the extract acts in synergy with the antibiotic or chemotherapeutic antibacterial drugs to inhibit bacterial growth and ensure clinical recovery from infection.
  • the extract exhibits selective activity with respect to the antibiotic or chemotherapeutic antibacterial drugs.
  • the antibiotic is a member of the group comprising penicillin derivatives, quinolone derivatives and macrolide antibiotics.
  • the penicillin derivatives comprise ampicillin, amoxycillin and piperacillin.
  • the quinolone derivatives comprise ciprofloxacin and norfloxacin.
  • the macrolide antibiotics comprise erythromycin and roxythromycin.
  • the chemotherapeutic anti-bacterial drug is co-trimoxazole.
  • the concentration of the antibiotic in the composition is the conventional therapeutic dosage regimen. In yet another embodiment, the concentration of chemo-therapeutic anti-bacterial drug is the conventional therapeutic dosage regimen.
  • composition in different pharmaceutical dosage forms suitable for oral administration.
  • the composition is useful for the treatment of infections caused by resistant bacterial strains of gram positive and gram negative classes.
  • the invention also provides a method for using an extract obtained from neem plant in combination with an effective amount of antibiotic or chemotherapeutic antibacterial drug for treatment of resistant bacterial infections.
  • the "effective amount" of the anti-biotic of the chemotherapeutic anti-bacterial drug is the amount that would be necessary to inhibit or control bacterial resistance. This effective amount will vary depending upon the nature of the drug used and the same can be readily determined by a person skilled in the art.
  • the dosage of the neem extract used in the composition or at the time of administration may be in the range of 50-300mg per unit dose.
  • neem extract used in this method is obtained from the neem plants selected from Azadirachta indica or Melia azadirach.
  • the neem extract as said earlier, is obtained from aerial parts selected from leaves, bark and tender twigs or combinations thereof. Especially, the leaves of Azadirachta indica.
  • the extract is used as an adjuvant to conventional drugs to overcome bacterial resistance.
  • the drugs are either antibiotics or chemotherapeutic anti- bacterial drugs.
  • the antibiotic is a member of the group comprising penicillin derivatives, quinolone derivatives and macrolide antibiotics.
  • the chemotherapeutic anti-bacterial drug is co-trimoxazole.
  • the invention provides a method for treatment of resistant bacterial infections, said method comprising the steps of oral administration of an extract obtained from the neem plant to a subject in need thereof, in combination with an effective amount of an antibiotic or chemotherapeutic anti-bacterial drug to which the bacteria has developed resistance.
  • the focus of the invention is not to modify the drug therapy or the dosage regimen of the conventional antibiotic or chemotherapeutic antibacterial drug treatment, but to effectively use neem extract and thereby facilitate eradication of drug resistant infections.
  • the extract from the neem plant and the antibiotics or chemotherapeutic antibacterial drugs are administered together as a combination or separately.
  • the extract is obtained from neem plant selected from Azadirachta indica and Melia azadirach.
  • the extract from neem plant is administered before or after administration of the antibiotic chemotherapeutic anti-bacterial drug, with a gap of not more than 30 minutes.
  • the extract contains total bitter principles of about 9% w/w.
  • the amount of extract administered ranges between 100 to 600 mg per day in two or three divided doses.
  • the antibiotic is a member of the group comprising penicillin derivatives, quinolone derivatives and macrolide antibiotics.
  • the penicillin derivatives comprise ampicillin, amoxycillin and piperacillin.
  • the quinolone derivatives comprise ciprofloxacin and norfloxacin.
  • the macrolide antibiotics comprise erythromycin and roxythromycin.
  • the chemotherapeutic anti-bacterial drug is co-trimoxazole.
  • the antibiotic or the chemotherapeutic anti-bacterial drug administered is the antibiotic or chemotherapeutic anti-bacterial drug to which the bacteria has acquired resistance.
  • the extract re-sensitizes the resistant bacteria and renders it susceptible to antibiotics and chemotherapeutic antibacterial drugs.
  • the extract acts in synergy with the antibiotic or chemotherapeutic antibacterial drugs to inhibit the bacterial growth and ensure clinical recovery from infection. Further, the extract exhibits selective activity with respect to the antibiotic or chemotherapeutic antibacterial drugs used.
  • the concentration of antibiotic or chemotherapeutic anti-bacterial drug administered is the conventional therapeutic dosage regimen.
  • the administration of neem extract is continued during the entire period of antibiotic therapy.
  • the invention provides a process for the preparation of the composition of the invention, said process comprising the steps of: a) preparing dry coarse powder of bark, dry tender twigs and leaves of
  • Azadirachta indica ox Melia azadirach b) preparing an aqueous extract wherein the pH of water is adjusted between 6 to 6.5 by addition of acids, c) alternatively, preparing an extract using one of alcohols or its blend with water, d) refluxing the extract by conventional methods to obtain a clear liquid, and e) concentrating and drying the extract to obtain a fine powder.
  • the acids are selected from any of the carboxylic acids, mineral acids or a combination thereof.
  • the alcohols comprise ethyl alcohol, methyl alcohol, isopropyl alcohol.
  • the extraction is carried-out using a blend of one of the alcohols and water.
  • extract is obtained by a reflux technique or open pan boiling technique, or any other conventional method.
  • the extract is concentrated by open-pan boiling technique or under vacuum or using falling film evaporators or any other conventional method. Finally, the extract is dried by tray or spray drying technique or under vacuum.
  • the focus of the invention is to use extracts obtained from the plants Azadirachta indica and Melia azadirachta (neem extract) in combination with antibiotics to which the bacterium causing the infection has developed resistance.
  • the neem extract is capable of being used/administered independent (before or after) of the antibiotic in question, or the neem extract can be administered in combination or concurrent with the antibiotic as a single composition, suitable for oral administration.
  • concurrent administration of the neem extract and the antibiotic refers to administration such that the two agents can exert their advantageous effect in combination in the subject.
  • the inventors have observed that such administration of the neem extract renders the bacteria susceptible to the same drug (antibiotic or chemotherapeutic antibacterial drug) to which it had acquired resistance.
  • the inventors also observed during their investigation that, in cases where the neem extract is administered independent of the antibiotic to the subject, the interval between administration of the neem extract and the antibiotic should not exceed 30 minutes. The said period of 30 minutes is not critical, but is recommended for best results.
  • the inventors have tested the efficacy of the combination of the neem extract with antibiotics in several bacterial cultures as exemplified by the examples provided herein below and the results of their clinical studies, indicate that the combination of neem extract with a particular antibiotic renders the bacterium in question susceptible to the antibiotic or the chemotherapeutic anti-bacterial drug.
  • the invention as discussed hereinbelow is yet another route to solve the problem of "acquired resistance" of bacteria. Since the neem extract sensitizes the bacteria to the antibiotics to which it has acquired resistance when the neem extract is administered in combination with the drug, it is the neem extract is said to have synergistic effect.
  • Phase (c) Elaborate investigations to confirm the observations made in Phase (b).
  • Azadirachta indica or Melia azadirachta both belonging to family Meliaceae
  • Azadirachta indica were used to obtain the herbal extract employed in this invention.
  • the present invention seeks to employ neem extract for a defined therapeutic application and considers the aqueous extract to be a safer option for human consumption - as opposed extracts obtained using organic solvents. At the same time, it seeks to impart a reliable shelf life to such aqueous extracts.
  • the extraction procedure was standardized by assessing various options. After few experiments, the following solvent options were found to be useful for the proposed purpose:
  • the extraction may be carried-out using any one of the polar solvents like ethyl, methyl, isopropyl alcohols
  • Stage-I Extraction Phase: For extraction purposes, the pH of water was first modified to 6 - 6.5. This step is achieved by addition of any one of the carboxylic acids like Citric acid, Phosphoric acid, Oxalic acid and acetic acid or mineral acids like hydrochloric acid or sulfuric acid or an admixture of any of these carboxylic and mineral acids to water. Dried parts of Neem like Leaves, bark, flowers and preferably, the Neem Leaves were extracted using water having weak acidity - as obtained from the above procedure. Alternatively, the extraction can be carried-out, using any of the alcohol ethyl, methyl, isopropyl alone or in combination with water.
  • carboxylic acids like Citric acid, Phosphoric acid, Oxalic acid and acetic acid or mineral acids like hydrochloric acid or sulfuric acid or an admixture of any of these carboxylic and mineral acids.
  • Dried parts of Neem like Leaves, bark, flowers and preferably, the Neem Leaves were extracted
  • Stage-II Filtration Phase: The extract is then filtered using suitable filtration media like, muslin cloth or filtration pads made of cellulose or a filter bed created by adsorbing media to obtain a clear solution.
  • suitable filtration media like, muslin cloth or filtration pads made of cellulose or a filter bed created by adsorbing media to obtain a clear solution.
  • Stage-Ill The Concentration Phase: The filtrate obtained from Stage-II was subjected to concentration by distillation or by an evaporation technique employed directly or under vacuum.
  • Stage-IV The drying Phase: To obtain the soluble solid constituents form the extraction - the concentrated extract was dried by conventional tray drying method or by spray-drying technique.
  • the extract described above contains a group of substances termed as Total Bitters and their quantitative value may go upto 9% (depending upon the natural variations in the raw material used for the purpose) in the final extract.
  • Literature suggests that, these compounds comprise limonoids and protolimonoids.
  • the extract was also characterized by qualitative presence of nimbin, and nimbidin in combination or in isolation with nimbidin.
  • the specific character of the aforesaid extract is also that, it contains nimbin and nimbidin only in traces and generally, not amenable to quantification. This very specific feature of the extract renders it safe for human use.
  • the product thus, obtained is labeled as Neem Extract for further experiments and hereinafter referred to as NE.
  • Phase (b) With the neem extract obtained by the method described herein above, further investigations on limited number of bacterial species isolated from wider clinical samples were used to ascertain the sensitization role of NE against a wider range of antibiotics. Once, the spectrum of its sensitization activity was identified, more focused investigations were done on a larger number of culture isolates representing a limited number of bacterial species and a limited number of antibiotic substances.
  • the clinical samples referred for routine culture and sensitivity tests were monitored.
  • the cultures of all bacterial species/strains exhibiting resistance to commonly used antibiotics - were preserved for the purpose of investigations.
  • the resistant bacterial cultures used in the investigation included ⁇ -haemolytic streptococci, Coi ⁇ naebacterium diphetheriae, Gram Negative enteric bacilli, Staphylococcus aureus, Escherichia coli, Klebseilla pneumoniae, Proteus vulgaris, Enterobacter species, Salmonella senftenberg etc.
  • MBC Minimum Bactericidal Concentration
  • Ciprofloxacin ( ⁇ g/ml)
  • Co-trimoxazole (Sulphamethoxazole/ Trimethoprim/ ⁇ g/ ⁇ g per ml) 0.5/9.5, 1.0/19, 4.0/76, 8.0/152, 16/304, 32/608
  • Erythromycin (used only for ⁇ -haemolytic streptococci and Staphylococcus aureus) in ⁇ g /ml 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256
  • a minimum inhibitory concentration is the lowest level of antibiotic needed to cause an inhibition to bacterial growth in culture medium whereas, the minimum bactericidal concentration is the lowest concentration of antibiotic to achieve a 100% killing of bacteria.
  • these two values are the practical indices of intensity of bacterial resistance to a given antibiotic.
  • a break-even point of any antibiotic indicates the attainable blood levels after an administration of full dose, in human/animal body.
  • this point indicates the practical dosage of administering any given antibiotic effectively and beyond this point, even if an antibiotic is found to act effectively in experimental situation - such dose cannot be administered in a clinical situation. Therefore, an effective antibiotic should exhibit activity against given bacterial cultures at this concentration otherwise; it becomes a useless compound for that particular infection.
  • NE is expected to sensitize the resistant bacteria to a given antibiotic and make it, susceptible to antibiotic at a break-even point concentration in the culture medium.
  • each antibiotic is taken at its breakpoint concentration, (the highest serum concentration of that particular antibiotic reached after the maximum therapeutic dose in-vivo).
  • the break points for various antibiotics are as follows: 1. Ampicillin/ Amoxacillin 16 ⁇ g/ml
  • Neem Extract Stock solution of neem extract (1 mg/ml) was prepared, filtered and autoclaved. It was used in 4 concentrations - 1000 ⁇ g/ml; 500 ⁇ g/ml; 250 ⁇ g/ml; 125 ⁇ g/ml. A set of four tubes with variable concentrations of the extract is prepared and to each of these tubes, the break point concentration of antibiotics is added. Now, these sets are inoculated with the study isolate in the same manner as per the broth dilution procedure. A set of six controls is inoculated with each isolate - in parallel with the main experiment. 1. Only medium
  • NE does not possess any inherent anti- bacterial activity. However, it sensitizes su ⁇ risingly, a resistant bacterial strain and renders it susceptible to well-known antibiotic drugs.
  • NE did not inhibit the growth of any of these 206 cultures and there was confluent growth of each of the resistant bacteria even in a plate containing highest concentration of NE.
  • the culture medium (Mueller Hinton Agar) was melted and distributed into a number of test tubes, ii) The stock solution of NE was dispensed into these test tubes in four different concentrations viz. 125 ⁇ g/mL, 250 ⁇ g/mL, 500 ⁇ g/mL, and 1000 ⁇ g/mL. iii) In the same medium, one of the antibiotics under investigation was also added so as to provide a variable concentration ( at break-even point concentration) as described below:
  • Ciprofloxacin 4 ⁇ g/mL
  • neem extract did not show any sensitization effect on bacteria resistant to aminoglycoside group of antibiotics (for example gentamicin, amikacin etc) or those resistant to cephalosporin group of antibiotics (such as Cephelexin, Cefotaxime etc) or to tetracycline group of antibiotics. Based on these negative observations, the inventors concluded that NE might not work in synergy with these antibiotics.
  • antibiotics for example gentamicin, amikacin etc
  • cephalosporin group of antibiotics such as Cephelexin, Cefotaxime etc
  • Escherichia coli was isolated from a wound infection and this culture was found to be resistant to Ciprofloxacin by a standard disc diffusion technique in routine culture and sensitivity test.
  • the minimum inhibitory concentration (MIC) was 8 ⁇ g/ml and minimum bactericidal concentration (MBC) goes to be 64 ⁇ g/ml concentration.
  • MMC minimum bactericidal concentration
  • the break-even point for this antibiotic is 4 ⁇ g/ml concentration.
  • 4 ⁇ g/ml of Ciprofloxacin was combined with four different concentrations of NE i.e. 125 ⁇ g /ml, 250 ⁇ g/ml, 500 ⁇ g/ml and 1000 ⁇ g/ml respectively.
  • Ciprofloxacin combined with NE could attain a 100% bactericidal effect at its break point concentration.
  • NE and Ciprofloxacin were added individually in control tubes and the bacterial growth was observed to be unhindered in both the controls.
  • the NE contributes to reversal of resistance and at the same time, it is not capable of inhibiting/killing a resistant bacteria on its own.
  • Escherishia coli was isolated from endotracheal catheter tip culture and this culture was found to be resistant to Ciprofloxacin by a standard disc diffusion technique in routine culture and sensitivity test.
  • the minimum inhibitory concentration (MIC) was 32 ⁇ g/ml and minimum bactericidal concentration (MBC) was noted to be > 128 ⁇ g/ml concentration.
  • MMC minimum bactericidal concentration
  • the break-even point for this antibiotic is 4 ⁇ g/ml concentration.
  • 4 ⁇ g/ml of Ciprofloxacin was combined with four different concentrations of NE i.e. 125 ⁇ g /ml, 250 ⁇ g/ml, 500 ⁇ g/ml and 1000 ⁇ g/ml respectively.
  • Ciprofloxacin combined with NE could attain a 40% bactericidal effect at its break point concentration.
  • NE and Ciprofloxacin were added individually in control tubes and the bacterial growth was observed to be unhindered in both the controls. These observations imply that the NE contributes to reversal of resistance but is incapable of inhibiting/killing resistant bacteria on its own.
  • Ciprofloxacin achieved a 99% killing (in 4 ⁇ g/ml concentration) when used in combination with NE in 4 different concentrations.
  • the results were reproducible in all the concentrations i.e. 1000 ⁇ g/ml, 500 ⁇ g/ml, 250 ⁇ g/ml and 125 ⁇ g/ml.
  • a strain of Klebsiella pneumoniae was isolated from a wound swab culture. This isolate was observed to be resistant to Ciprofloxacin by disc diffusion technique. The MIC and MBC of Ciprofloxacin for this strain were observed to be 32 ⁇ g/ml and >128 ⁇ g/ml respectively as against 4 ⁇ g/ml of break-even point concentration. However, when Ciprofloxacin was added to NE, a 90% killing was achieved in the culture tubes. This effect was found to be uniform at all four concentrations of the test compound used in this study. NE does not exhibit any degree of bactericidal effect per-se against this resistant strain also as observed in the control tubes.
  • the above organism was also found to be resistant to Co-trimoxazole.
  • the known break even point for this chemotherapeutic combination is 4/76 ⁇ g/ ⁇ g/ml.
  • the MIC and MBC were identified to be 8/152 ⁇ g/ ⁇ g/ml, 16/304 ⁇ g/ ⁇ g/ml respectively.
  • Co-trimoxazole became effective in its breakeven concentrations i.e. 4/76 ⁇ g/ ⁇ g ml when used in combination with NE and was able to achieve a 90% killing in the culture tubes.
  • Such reversal of resistance to Co-trimoxazole was found to be uniform in all the four concentrations of the NE used in the study.
  • Klebsiella pneumoniae was isolated from Endotracheal Catheter Tip culture and this culture was found to be resistant to Ciprofloxacin by a standard disc diffusion technique in routine culture and sensitivity test.
  • the minimum inhibitory concentration (MIC) was 32 ⁇ g/ml and minimum bactericidal concentration (MBC) goes to >128 ⁇ g/ml concentration.
  • MMC minimum bactericidal concentration
  • the break-even point for this antibiotic is 4 ⁇ g/ml concentration.
  • 4 ⁇ g/ml of Ciprofloxacin was combined with four different concentrations of NE i.e. 125 ⁇ g /ml, 250 ⁇ g/ml, 500 ⁇ g/ml and 1000 ⁇ g/ml respectively.
  • Ciprofloxacin combined with NE could attain a 90% bactericidal effect at its break-even point concentration.
  • NE and Ciprofloxacin were added individually in control tubes and the bacterial growth was observed to be unhindered in both the controls.
  • Klebsiella pneumoniae was isolated from Endotracheal Catheter tip culture and this culture was found to be resistant to Ciprofloxacin by a standard disc diffusion technique in routine culture and sensitivity test.
  • the minimum inhibitory concentration (MIC) was 32 ⁇ g/ml and minimum bactericidal concentration (MBC) was noted to be > 128 ⁇ g/ml.
  • MMC minimum bactericidal concentration
  • the break-even point for this antibiotic is 4 ⁇ g/ml concentration.
  • 4 ⁇ g/ml of Ciprofloxacin was combined with four different concentrations of NE i.e. 125 ⁇ g /ml, 250 ⁇ g/ml, 500 ⁇ g/ml and 1000 ⁇ g/ml respectively.
  • Ciprofloxacin combined with NE could attain 80% bactericidal effect at its break point concentration.
  • NE and Ciprofloxacin were added individually in control tubes and the bacterial growth was observed to be unhindered in both the controls. These observations imply that the NE contributes to reversal of resistance but is not effective in inhibiting/killing resistant bacteria on its own.
  • Example-7 Klebsiella pneumoniae was isolated from endotracheal catheter tip culture and this culture was found to be resistant to Ciprofloxacin by a standard disc diffusion technique in routine culture and sensitivity test.
  • the minimum inhibitory concentration (MIC) was 32 ⁇ g/ml and minimum bactericidal concentration (MBC) was noted to be > 128 ⁇ g/ml concentration.
  • MMC minimum bactericidal concentration
  • the break- even point for this antibiotic is 4 ⁇ g/ml concentration.
  • 4 ⁇ g/ml of Ciprofloxacin was combined with four different concentrations of NE i.e. 125 ⁇ g /ml, 250 ⁇ g/ml, 500 ⁇ g/ml and 1000 ⁇ g/ml respectively.
  • Ciprofloxacin combined with NE could attain a 50% bactericidal effect at its break point concentration.
  • NE and Ciprofloxacin were added individually in control tubes and the bacterial growth was observed to be unhindered in both the controls.
  • Example - 8 ⁇ -haemolytic streptococci were isolated from a throat swab culture and this culture was found to be resistant to Erythromycin by a standard disc diffusion technique in routine culture and sensitivity test.
  • the minimum inhibitory concentration (MIC) was 64 ⁇ g/ml and minimum bactericidal concentration (MBC) goes to > 128 ⁇ g/ml concentration.
  • MMC minimum bactericidal concentration
  • the break-even point for this antibiotic is 4 ⁇ g/ml concentration.
  • 4 ⁇ g/ml of Erythromycin was combined with four different concentrations of NE i.e.
  • Example - 9 ⁇ -haemolytic streptococci were isolated from a sputum culture and this culture was found to be resistant to Erythromycin by a standard disc diffusion technique in routine culture and sensitivity test.
  • MIC minimum inhibitory concentration
  • MMC minimum bactericidal concentration
  • the break-even point for this antibiotic is 4 ⁇ g/ml concentration.
  • Example - 10 Enterobacter spp. was isolated from Endotracheal Catheter Tip culture and this culture was found to be resistant to Ciprofloxacin by a standard disc diffusion technique in routine culture and sensitivity test.
  • the minimum inhibitory concentration (MIC) was 32 ⁇ g/ml and minimum bactericidal concentration (MBC) goes to >128 ⁇ g/ml concentration.
  • MMC minimum bactericidal concentration
  • the break-even point for this antibiotic is 4 ⁇ g/ml concentration.
  • 4 ⁇ g/ml of Ciprofloxacin was combined with four different concentrations of NE i.e. 125 ⁇ g /ml, 250 ⁇ g/ml, 500 ⁇ g/ml and 1000 ⁇ g/ml respectively.
  • Ciprofloxacin combined with NE could attain 80% bactericidal effect at its break point concentration.
  • NE and Ciprofloxacin were added individually in control tubes and the bacterial growth was observed to be unhindered in both the controls. These observations imply that the NE contributes to reversal of resistance but is incapable of inhibiting/killing resistant bacteria on its own.
  • Example - 11 Enterobacter spp. was isolated from Endotracheal catheter tip culture and this culture was found to be resistant to Ciprofloxacin by a standard disc diffusion technique in routine culture and sensitivity test.
  • the minimum inhibitory concentration (MIC) was 32 ⁇ g/ml and minimum bactericidal concentration (MBC) was noted to be >128 ⁇ g/ml concentration.
  • MMC minimum bactericidal concentration
  • the break-even point for this antibiotic is 4 ⁇ g/ml concentration.
  • 4 ⁇ g/ml of Ciprofloxacin was combined with four different concentrations of NE i.e. 125 ⁇ g /ml, 250 ⁇ g/ml, 500 ⁇ g/ml and 1000 ⁇ g/ml respectively
  • Ciprofloxacin combined with NE could attain a 50% bactericidal effect at its break point concentration.
  • NE and Ciprofloxacin were added individually in control tubes and the bacterial growth was observed to be unhindered in both the controls.
  • Example-12 A strain of Staphylococcus aureus was isolated from a non-healing burn wound swab culture. This strain was found to be resistant to Ciprofloxacin by the disc diffusion technique. In subsequent test, the MIC was noted to be 32 ⁇ g ml and MBC was noted to be > 128 ⁇ g/ml as against 4 ⁇ g/ml of break even point. Contrary to this, it was observed that Ciprofloxacin achieved an 80% killing (in 4 ⁇ g/ml concentration) when used in combination with 250 ⁇ g/ml and 125 ⁇ g/ml NE.
  • a strain of Salmonella senftenberg was isolated from a burn wound swab culture. This isolate was observed to be resistant to Ciprofloxacin by disc diffusion technique. The MIC and MBC of Ciprofloxacin for this strain were observed to be 32 ⁇ g ml and >128 ⁇ g/ml respectively as against 4 ⁇ g/ml of Break even point concentration. However, when Ciprofloxacin was added to 1000 ⁇ g/ml NE, a 50% killing was achieved in the Culture tubes. With 500 ⁇ g/ml NE, this effect was somewhat low and at other concentrations this effect was not found. NE does not exhibit any degree of bactericidal effect per-se against this resistant strain also as observed in the control tubes.
  • the above organism was also found to be resistant to Co-trimoxazole.
  • the break- even point for this chemotherapeutic combination is 4/76 ⁇ g/ml.
  • the MIC and MBC were identified to be 8/152 ⁇ g/ ⁇ g/ml and 16/304 ⁇ g/ ⁇ g/ml respectively.
  • Co-trimoxazole became effective to some extent when used in combination with different concentration of NE and was able to achieve a 60% killing in the culture tubes.
  • Such reversal of resistance to Co-trimoxazole was found to be uniform in all the four concentrations of the test compounds used in the study.
  • Example - 14 During confirmatory investigations on NE, 102 cultures of Staphylococcus aureus were employed to ascertain its sensitization effect. These cultures were obtained various clinical samples, such as sputum, non-healing infected wounds, throat swabs etc. All these cultures were confirmed to be resistant to ampicillin, Cephalexin, Ciprofloxacin, Erythromycin, Gentamicin.
  • +++ Shows the growth of organism and — show the inhibition of growth of organism.
  • Staphylococcus aureus cultures were sensitized significantly entailing in their increased susceptibility. As a result, out of 102 cultures, NE + Ampicillin could inhibit the growth of
  • NE could sensitize 50% of clinical strains to erythromycin and this antibiotic represents the class of macrolide antibiotics.
  • Example - 15 In case of other two antibiotics, its sensitization effect is not so marked and NE could sensitize only a small number of resistant strains employed in the investigation.
  • Example - 15 In case of other two antibiotics, its sensitization effect is not so marked and NE could sensitize only a small number of resistant strains employed in the investigation.
  • sensitization effect of NE was evaluated on resistant isolates of Klebsiella pneumoniae. A total of 41 strains were tested for this organism and all of them were initially resistant to Ampicillin and Ciprofloxacin. NE also, did not show any inhibitory effect per-se - against these 41 isolates of Klebsiella pneumoniae.
  • the following table shows the additive/synergistic/sensitization effect of NE as evidenced in this example:
  • +++ Shows the growth of organism and — show the inhibition of growth of organism. In this particular organism also, NE could sensitize the resistant organism only to
  • Piperacillin represents penicillin derivative class of broad-spectrum antibiotics like
  • +++ Shows the growth of organism and — show the inhibition of growth of organism In this organism, the test substance has shown significant sensitization to Ciprofloxacin and in case of Ampicillin the activity is somewhat, low.
  • the samples of NE were prepared in form of film coated tablets (containing 100 of NE per tablet). Patients suffering from respiratory and/or surgical infections were enrolled into the study after a due consideration to inclusion and exclusion criteria:
  • Ampicillin/amoxycillin (representing penicillin derivatives) - the following therapeutic regime was instituted: 1. One of the above antibiotic, (to which the bacterial resistance is established) in a standard dosage regimen.
  • the above treatment schedule was continued for 1 - 3 weeks, depending upon the chronicity of infection. Subjective response to treatment was elicited once in every week. As and when a significant relief was noted, the culture and sensitivity test was repeated, to correlate the clinical response to bacteriological response.
  • Grade - II Response Cases cleared from subjective symptoms and when the culture was found to be sterile after 48 hours incubation - were considered as Complete response. These cases were labeled as Grade -II response.
  • Grade - 1 Response Cases with a distinct subjective response but the culture test was positive for bacterial infection.
  • Grade - 0 Response Cases with no clinical or bacteriological response - after addition of NE to the antibiotic.
  • Results A total of 24 patients were enrolled into the study and two cases didn't go for follow-up investigations and hence, were treated as dropout cases. Out-of 22 evaluable cases, there were 15 cases of surgical infections and 7 cases of respiratory infection. All these patients received some or the other antibiotic therapy for a minimum of 15 days before enrollment into the study and therefore, were clinically considered to be of resistant infections.
  • Table-II Showing the Age distribution in the Study Population.
  • Table - III shows the infecting organism isolated during cultures of clinical specimen.
  • Bacterial resistance patterns to the selected three antibiotic substances, at the time of enrollment.
  • Staphylococcus aureus was found in 9 cases, mixed infections in 5 cases and one case had Gram + ve cocci as infecting organism.
  • Staphylococcus aureus was found in 9 cases, mixed infections in 5 cases and one case had Gram + ve cocci as infecting organism.
  • Staph. aureus predominated with
  • Ciprofloxacin resistance & Role of Neem Extract The resistance patterns suggest that, 8 isolates of Staphylococcus aureus were resistant to Ciprofloxacin. Of these 8 cases, NE - DR could sensitize the infecting organism in 7 cases. In one case - there was no clinical or bacteriological response to the addition of test drug. These observations indicate that, in a majority of cases, DRF - 11198 was able to sensitize Staphylococcus aureus and to subscribe to the clinical recovery with Ciprofloxacin.
  • Ciprofloxacin could be reverted by test substance- in 9 cases. A partial remission could be achieved one case and there was no practical effect of the test drug in Two cases.
  • Staphylococcus aureus which were resistant to Ampicillin / amoxycillin, involved in surgical infections. Both these isolates were sensitized to, and the cases responded to treatment with Ampicillin. Similarly, out of 3 mixed infections resistant to Ampicillin, 2 cases responded completely and there was a partial remission in one case. Apart from these five cultures, there was one case diagnosed be of Gram + ve cocci and this case did not respond to therapy with NE in any manner.
  • Roxythromycin is the latest introduction in this category and with its introduction, the use of earlier drug, erythromycin is not much in clinical use. However, bacteria developed resistance to this drug as well. In the present study, 4 strains of Staphylococcus aureus were isolated. NE could sensitize 2 of these strains and there was complete clinical and bacteriological response to Roxythromycin. There was one case with a partial remission and one did not respond to its addition.

Abstract

L'invention concerne une nouvelle composition pouvant être utilisée dans le traitement des infections bactériennes résistantes. Ladite composition comprend un extrait obtenu à partir d'Azadiracta indica ou Melia azadirachta et une quantité efficace d'un médicament antibiotique ou antibactérien chimiothérapeutique. L'invention a pour objet un procédé permettant de traiter les infections bactériennes résistantes ainsi qu'un procédé pour la préparation de la nouvelle composition.
PCT/IN2000/000095 2000-09-15 2000-10-04 Combinaison antibacterienne comprenant un extrait de plante de margousier a feuilles de frene WO2002022147A1 (fr)

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US09/662,809 US6599541B1 (en) 1999-09-17 2000-09-15 Composition for treatment of drug resistant bacterial infections and a method of treating drug resistant bacterial infections

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EP1667528A1 (fr) * 2003-09-22 2006-06-14 Genyous Biomed International Inc. Compositions d'hippophae rhamnoides pour therapie anticancereuse
WO2008004244A1 (fr) * 2006-07-05 2008-01-10 Hafiz Anwarul Aziz Engrais sans produits chimiques
JP2008509133A (ja) * 2004-08-03 2008-03-27 ブリッジワークス・アクチエンゲゼルシャフト 治癒効果、忌避効果、および生物致死性を有する天然物質を含む外傷の処置および治療のための組成物
US8173177B2 (en) 2003-09-08 2012-05-08 Genyous Biomed International Inc. Compositions of botanical extracts for cancer therapy
US20130243702A1 (en) * 2010-11-04 2013-09-19 Divya Ravindran Mouthwash Composition for Managing Oral Mucositis, Process and Methods Thereof
KR101818146B1 (ko) * 2016-01-29 2018-01-12 제이앤팜유한책임회사 화이트윌로우바크(White Willow Bark), 북미사시나무껍질(Aspen Bark), 님잎(Azadirachta Indica Leaf), 청호(Atremisia Annua) 추출물로 이루어지는 천연방부제 조성물
WO2022149619A1 (fr) * 2020-12-11 2022-07-14 Bigham Herman Lee Jr Composition pour aérosol contre les virus de la covid19/grippe et procédés de lutte contre la contamination par les virus et les rhumes et contre leur propagation

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8173177B2 (en) 2003-09-08 2012-05-08 Genyous Biomed International Inc. Compositions of botanical extracts for cancer therapy
EP1667528A1 (fr) * 2003-09-22 2006-06-14 Genyous Biomed International Inc. Compositions d'hippophae rhamnoides pour therapie anticancereuse
EP1667528A4 (fr) * 2003-09-22 2007-10-24 Genyous Biomed Internat Inc Compositions d'hippophae rhamnoides pour therapie anticancereuse
JP2008509133A (ja) * 2004-08-03 2008-03-27 ブリッジワークス・アクチエンゲゼルシャフト 治癒効果、忌避効果、および生物致死性を有する天然物質を含む外傷の処置および治療のための組成物
JP4851450B2 (ja) * 2004-08-03 2012-01-11 ファイトシューティカルズ・リミテッド 治癒効果、忌避効果、および生物致死性を有する天然物質を含む外傷の処置および治療のための組成物
WO2008004244A1 (fr) * 2006-07-05 2008-01-10 Hafiz Anwarul Aziz Engrais sans produits chimiques
US20130243702A1 (en) * 2010-11-04 2013-09-19 Divya Ravindran Mouthwash Composition for Managing Oral Mucositis, Process and Methods Thereof
KR101818146B1 (ko) * 2016-01-29 2018-01-12 제이앤팜유한책임회사 화이트윌로우바크(White Willow Bark), 북미사시나무껍질(Aspen Bark), 님잎(Azadirachta Indica Leaf), 청호(Atremisia Annua) 추출물로 이루어지는 천연방부제 조성물
WO2022149619A1 (fr) * 2020-12-11 2022-07-14 Bigham Herman Lee Jr Composition pour aérosol contre les virus de la covid19/grippe et procédés de lutte contre la contamination par les virus et les rhumes et contre leur propagation

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