EP3532027A1 - Compositions comprising carotenoids and phosphatidylcholine - Google Patents

Compositions comprising carotenoids and phosphatidylcholine

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Publication number
EP3532027A1
EP3532027A1 EP17792173.1A EP17792173A EP3532027A1 EP 3532027 A1 EP3532027 A1 EP 3532027A1 EP 17792173 A EP17792173 A EP 17792173A EP 3532027 A1 EP3532027 A1 EP 3532027A1
Authority
EP
European Patent Office
Prior art keywords
composition
individual
carotenoid
bacterial infection
lycopene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP17792173.1A
Other languages
German (de)
French (fr)
Inventor
Ivan M. PETYAEV
Nailya ZIGANGIROVA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
IP Science Ltd
Original Assignee
IP Science Ltd
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Filing date
Publication date
Application filed by IP Science Ltd filed Critical IP Science Ltd
Publication of EP3532027A1 publication Critical patent/EP3532027A1/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • A61K31/6615—Compounds having two or more esterified phosphorus acid groups, e.g. inositol triphosphate, phytic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/01—Hydrocarbons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/01—Hydrocarbons
    • A61K31/015—Hydrocarbons carbocyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/047—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates having two or more hydroxy groups, e.g. sorbitol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/065—Diphenyl-substituted acyclic alcohols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to new formulations of carotenoids and chaperone molecules.
  • the invention also relates to the use of these formulations for preventing or treating any bacterial infection, especially obligate intracellular bacterial infections such as those of the class Chlamydiae, as well as conditions associated with obligate intracellular bacterial infections.
  • the invention also relates to methods of treating bacterial infections and associated conditions using these formulations.
  • Obligate intracellular bacteria such as those of the class Chlamydiae are responsible for a wide range of diseases in different hosts organs and tissues, including genital and eye inflammatory diseases, arthritis, inflammatory diseases of the respiratory and cardiovascular system. Such intracellular bacteria may also be associated with development of neurodegenerative and behavioral disorders. For example, there is evidence that Chlamydophila pneumoniae may promote differentiation of pre-adipocyte cells into mature fat cells, and be associated with development of metabolic syndrome, fatty liver or non-alcoholic steatohepatitis.
  • Intracellular infection becomes initiated with infectious but metabolically inert elementary bodies which differentiate within the membrane-associated vacuole of the cell into metabolically active but non-infective reticulate bodies.
  • the infective cycle terminates within 48-72 hours by lysis of eukaryotic cells and release of infective progeny to neighboring cells, and subsequent repeat of cellular infections in adjacent epitheliocytes.
  • the whole chlamydial infectious cycle is highly dependent on host cell energy homeostasis and metabolism, since chlamydial species lack crucial enzymes for ATP biosynthesis and are defective in biosynthesis of lipids and many other organic substances.
  • the invention relates to the finding that the high dependence of the obligate intracellular bacterial developmental cycle upon the host's cell metabolism creates an opportunity to control these species by modulating the host cell's metabolic pathway.
  • the inventors have surprising shown that carotenoids target metabolic pathways essential for survival and propagation of obligatory intracellular bacteria. Targeting such metabolic pathways using carotenoids therefore unexpectedly provides an alternative means to prevent or treat such bacterial infections as compared to conventional antibiotics and anti-bacterial drugs.
  • the invention also relates to the finding that lipoprotein metabolism may be compromised when obligate intracellular bacteria such as Chlamydiae hijack the lipid metabolism of the host. Therefore, in order to make carotenoid treatment of such bacteria effective, it is essential to target these molecules to the sites of infection.
  • the formulations of the invention not only target obligate intracellular bacterial infections themselves, but may also be used to treat or prevent their complications and associated conditions such as metabolic syndrome, fatty liver, steatohepatitis, atherosclerosis, cardiovascular pathologies, cerebrovascular, neurodegenerative conditions and/or functional carotenoid deficiency.
  • the invention also relates to the unexpected finding that the formulations of the invention act to boost carotenoid-metabolizing bacteria and thereby inhibit pathogenic bacteria.
  • the invention provides:
  • a composition comprising a carotenoid and phosphatidylcholine (PC) at a weight ratio of at least about 1 : 0.1.
  • PC phosphatidylcholine
  • a method of treating or preventing an obligate intracellular bacterial infection and/or a condition associated with an obligate intracellular bacterial infection in an individual comprising administering a therapeutically or prophylactically effective amount of a composition as defined herein to the individual, and thereby treating or preventing the infection and/or condition.
  • compositions as defined herein in the manufacture of a medicament for treating or preventing an obligate intracellular bacterial infection and/or a condition associated with an intracellular bacterial infection in an individual.
  • a method of reducing the titre of antibodies against an obligate intracellular bacterial infection in an individual comprising administering a therapeutically or prophylactically effective amount of a composition as defined herein to the individual and thereby reducing the titre of antibodies against the bacterial infection.
  • a method of treating or preventing an obligate intracellular bacterial infection and/or a condition associated with an intracellular bacterial infection in an individual comprising administering a therapeutically or prophylactically effective amount of a composition comprising lycopene to the individual and thereby treating or preventing the infection.
  • a composition comprising lycopene for use in a method of treating or preventing an obligate intracellular bacterial infection and/or a condition associated with an intracellular bacterial infection in an individual.
  • composition comprising lycopene in the manufacture of a medicament for the treatment or prevention of an obligate intracellular bacterial infection and/or a condition associated with an intracellular bacterial infection in an individual.
  • a method of producing a pharmaceutical formulation comprising formulating composition components as defined herein, together with a pharmaceutically acceptable excipient.
  • a method of producing a nutraceutical formulation comprising formulating composition components as defined herein, together with a nutraceutically acceptable excipient.
  • a method of treating or preventing a bacterial infection in an individual comprising administering a therapeutically or prophylactically effective amount of a composition comprising a carotenoid to the individual, wherein the composition increases the activity and/or number of carotenoid-metabolizing bacteria in the individual and thereby treats or prevents the bacterial infection.
  • the invention additionally provides a composition comprising a carotenoid for use in a method of treating or preventing a bacterial infection in an individual, wherein the composition increases the activity and/or number of carotenoid-metabolizing bacteria in the individual.
  • a composition comprising a carotenoid for use in a method of treating or preventing a bacterial infection in an individual, wherein the composition increases the activity and/or number of carotenoid-metabolizing bacteria in the individual.
  • the carotenoid-metabolizing bacteria may be any carotenoid-metabolizing bacteria described herein, such as lactobacilli and/or bifidobacteriacea.
  • the bacterial infection which may be treated or prevented with any of such compositions is any bacterial infection comprising any bacterium or bacteria which are susceptible to inhibition by carotenoid-metabolizing bacteria, e.g. as described in more detail herein.
  • the bacterial infection may be caused by an obligate intracellular bacteria such as Chlamydia, Ehrlichia and/or Rickettsia.
  • any of the carotenoid-comprising compositions defined herein may be used to increase any of the activities described herein of the carotenoid-metabolizing bacteria.
  • FIG. 1 Dose-Dependent inhibition of C. trachomatis growth in B IO.MLM cells in presence of oil-formulated (A) and microencapsulated (B) lycopene.
  • FIG. 1 Inhibition of C. pneumoniae growth in B 10.MLM cells in presence of oil-formulated and microencapsulated lycopene.
  • FIG. 1 Panel A - C. trachomatis cells within a vacuole of the infected macrophages, 48h, ⁇ 17,000.
  • Panel B - Infected microphages + oil form lycopene, 48h, x6,300. Fragments of damaged Chlamydia reticulate body.
  • Panel C - Infected microphages + microencapsulated lycopene, 48h, ⁇ 17,000. Fragmented reticulate body and lysis destroyed Chlamydia bacteria.
  • Figure 4 Reversibility of the apparent decline in lycopene concentration in inflammatory environment, in ex vivo experimental, human blood serum.
  • Figure 5. Box and whisker analysis of serum lycopene levels.
  • Figure 7 Inflammatory oxidative damage (A) and oxidized LDL (B) in patients treated with lycopene.
  • Figure 8 Changes in the gut microbiota after daily ingestion of 7mg GA lycopene for 4 weeks.
  • Panel A analysis of the most powerful probiotics in the gut.
  • Panel B analysis of health negative, potentially pathogenic bacteria. The results demonstrate a significant increase in probiotic bacterial species such as lactobacilli in the gut after administration of the GA lycopene.
  • Figure 9 Changes in the gut microbiota after daily ingestion of 30mg GA lycopene for 4 weeks.
  • Panel A analysis of the most powerful probiotics in the gut.
  • Panel B analysis of health negative, potentially pathogenic bacteria.
  • the results demonstrate a significant increase in levels of probiotic bacterial species such as lactobacilli in the gut after administration of the GA lycopene.
  • a and/or B is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Anywhere herein where a compound is referred to, if a salt of such compound may also be employed, that is also encompassed in the invention, particularly the use of such physiological acceptable salts. Where a given entity is referred to herein for use in a particular method, the method itself is also provided as is use of the entity in the manufacture of a medicament for use in such a method.
  • Carotenoids The invention provides compositions comprising one or more carotenoids.
  • the compositions of the invention may include any carotenoid.
  • Carotenoid compounds are a class of tetraterpenoids which contain long polyene chains.
  • Carotenoids include carotenes, such as beta-carotene, alpha-carotene, zeto-carotene, and lycopene and related molecules, including l -HO-3', 4'-didehydrolycopene, 3, l '-(HO)2 -gamma-carotene, 1, 1'- (HO)2-3, 4, 3', 4'-tetradehydrolycopene, 1 , l'-(HO)2-3, 4-didehydrolycopene.
  • Carotenoids also include xanthophylls such as lutein, meso-zeaxanthin, zeaxanthin and astaxanthin.
  • carotenoid compounds which may be used as described herein include hydrocarbons, such as lycopersene (7,8, 1 1, 12, 15,7', 8', 1 ⁇ , 12', 15'-decahydro-Y,y- carotene), phytofluene, hexahydrolycopene (15-cz ' s-7,8, l 1 , 12,7', 8'-hexahydro-y,Y- carotene), torulene (3',4'-didehydro-P,y-carotene) and a-zeacarotene (7',8'-dihydro-s,y- carotene); alcohols, such as alloxanthin, cynthiaxanthin, pectenoxanthin,
  • cryptomonaxanthin ((3r,3'r)-7,8,7',8'-tetradehydro-P,P-carotene-3,3'-diol), crustaxanthin (P,-carotene-3,4,3',4'-tetrol), gazaniaxanthin ((3r)-5'-cis-P,y-caroten-3-ol), oh- chlorobactene (l ',2'-dihydro-f,Y-caroten-l '-ol), loroxanthin (P,s-carotene-3, 19,3'-triol), lycoxanthin (Y,y-caroten-16-ol), rhodopin (l,2-dihydro-y,Y-caroten-l-ol), rhodopinol (aka warmingol; 13-c/s-l,2-dihydro-y,y-carotene-l ,20-di
  • decaprenoxanthin (2,2'-bis(4-hydroxy-3-methyl-2-butenyl)-e,e-carotene), c.p. 450 (2-[4- hydroxy-3-(hydroxymethyl)-2-butenyl]-2'-(3 -methyl -2-butenyl)-b,b-carotene), c.p.
  • a composition as described herein may contain a single carotenoid compound or more than one carotenoid compound.
  • a composition as described herein may comprise, one, two, three, four, five, six or more carotenoids, such as any of those numbers of the specific carotenoids specified here.
  • a composition may in one instance comprise one, two or three carotenoids, for instance any of the specific carotenoids specified herein.
  • a composition may comprise one carotenoid, for example where the carotenoids is any of those specified herein.
  • each carotenoid may be, for instance, present in a range of different isomeric forms.
  • the carotenoid compound is lycopene, hence in any of the embodiments described herein where a carotenoid is present, in a preferred instance the carotenoid is lycopene, or where more than one carotenoid is present lycopene may be one of the carotenoids present.
  • Lycopene is an open-chain unsaturated C40 carotenoid of structure I (Chemical Abstracts Service Registry Number 502-65-8).
  • Lycopene occurs naturally in plants such as tomatoes, guava, rosehip, watermelon and pink grapefruit.
  • the lycopene of the invention may comprise one or more different isomers.
  • lycopene may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% , at least 80% , at least 90% , or at least 95% (Z)-isomers, (all-E)-isomers, or cz ' s-isomers, such as 5-cis- or 9-cis- or 13-c/s-isomers, which have improved bioavailability relative to trans isomers.
  • Trans isomers may isomerise into cis forms in vivo, or during storage and processing.
  • the carotenoid compounds of the invention may be natural i .e., obtained from a natural source, for example, extracted from a plant, such as a tomato or melon.
  • a natural source for example, extracted from a plant, such as a tomato or melon.
  • a range of methods for extracting, concentrating and/or purifying carotenoids from plants are known in the art. For example, solvent extraction using ethanol, DMSO, ethyl acetate, hexane, acetone, soya or other vegetable oil, or non-vegetable oils may be employed.
  • a carotenoid compound may be isolated i.e. free or substantially free of other molecules found in its natural source or environment.
  • Carotenoid compounds for use as described herein may be synthetic i.e.
  • lycopene and other carotenoids are known in the art.
  • a three-stage chemical synthesis based on the standard Wittig olefination reaction scheme for carotenoid synthesis may be employed, in which an organic solution of C 15 phosphonium methanesulfonate in dichloromethane (DCM) and an organic solution of Cio dialdehyde in toluene are produced, and the two organic solutions are gradually combined with sodium methoxide solution and undergo a condensation reaction to form crude lycopene.
  • DCM dichloromethane
  • Cio dialdehyde in toluene an organic solution of Cio dialdehyde in toluene
  • the crude lycopene may then be purified using routine techniques, for example by adding glacial acetic acid and deionized water to the mixture, stirring vigorously, allowing the aqueous and organic phases to separate, and extracting the organic phase containing DCM and crude lycopene with water.
  • Methanol is added to the organic phase and the DCM removed via distillation under reduced pressure.
  • the crude methanolic lycopene solution is then be heated and cooled to crystalline slurry that is filtered and washed with methanol.
  • the lycopene crystals may then be recrystallized and dried under heated nitrogen.
  • Synthetic carotenoids, such as lycopene are also available from commercial suppliers (e.g. BASF Corp, NJ USA, DSM Nutritional Products, Basel, CH).
  • Synthetic carotenoids may comprise an increased proportion of cis isomers relative to natural carotenoids.
  • synthetic forms of carotenoids such as lycopene may be up to 25% 5-cis, 1% 9-cis, 1% 13-cis, and 3% other cis isomers
  • natural forms of carotenoids for example lycopene produced by tomatoes, may be 3-5% 5-cis, 0-1% 9-cis, 1% 13-cis, and ⁇ 1% other cis isomers.
  • cis-carotenoids, such as cis-lycopene have increased bioavailability relative to trans-carotenoids, such as trans- lycopene
  • synthetic carotenoids may be preferred in some embodiments.
  • Derivatives of carotenoids as described above may be produced by chemical synthesis analogous to the synthesis described above; by chemical modification of natural carotenoids extracted from plant material or by microbial, yeast, algal, or fungal fermentation.
  • lycopene may be produced by fermentation of the fungus Blakeslea trispora (e.g. LyconatTM, Vitatene SA).
  • the composition may comprise 0.05 to 90% by weight of the carotenoid compound, preferably 0.1% to 10% by weight.
  • the population may be 0.01% or more, 0.05% or more, 0.1% or more, 0.2% or more, 0.5% or more, 1% or more, 10% or more, or 20% or more by weight of carotenoid compound.
  • the population may be up to 90%, up to 80%, up to 70%, up to 60% up to 50%, up to 40%, up to 30%, up to 20% or up to 10% by weight of carotenoid compound.
  • the composition may contain the same or similar amounts of carotenoid compound or the amount of carotenoid compound may vary between particles in the population.
  • Each carotenoid particle in the population may comprise 0.05 to 90% by weight of carotenoid compound.
  • each carotenoid particle in the population may be 0.05% or more, 0.1% or more, 1% or more, 10% or more, or 20% or more by weight of carotenoid molecules.
  • Each carotenoid particle may be up to 90%, up to 80%, up to 70%, up to 60% up to 50%, up to 40% or up to 30%, up to 90% or more by weight of carotenoid compound.
  • the carotenoid employed is one or more selected from lycopene, meso-zeaxanthin, zeaxanthin, astaxanthin, canthaxanthin and/or lutein.
  • the carotenoid lycopene is employed. Phosphatidylcholine (PC)
  • compositions of the invention further comprise phosphatidylcholine (PC).
  • PC phosphatidylcholine
  • Any phosphatidylcholine may be used in the invention.
  • Phosphatidylcholine is a component of cell membrane bilayers and the main phospholipid circulating in the plasma.
  • Phosphatidylcholine is highly absorbable and supplies choline which is needed to facilitate movement of fats and oils across the cell membrane, and to maintain the cell membranes.
  • Phosphatidylcholine acts as a chaperone molecule for the delivery of carotenoids in the practice of the present invention.
  • the phosphatidylcholine of the invention may be obtained from various sources such as egg yolk or soybeans.
  • the term "phosphatidylcholine” is understood herein to include lecithin, l,2-Diacyl-sn-glycero-3-phosphocholine, choline phosphatide, lecithol, posphatidyl-N-trimethylethanolamine, phospholutein.
  • the phosphatidylcholine is l,2-diacyl-sw-glycero-3-phosphocholine, 10- (perfluorobutyl)decyl phosphatidylcholine, dioleoyl phosphatidylcholine.
  • the phosphatidylcholine of the invention is a commercially available purified form.
  • Phosphatidylcholine is a glycerophosphocholine compound of structure II having O-acyl substituents at both the 1- and 2-positions of the glycerol.
  • composition of the invention comprises a carotenoid
  • the composition of the invention comprises a carotenoid and phosphatidylcholine at weight ratio of at least about 1.00 : 0.14.
  • the composition comprises a carotenoid and phosphatidylcholine at a weight ratio of at least about 1 : 0.2, 1 : 0.4, 1 : 0.6, 1 : 0.8, 1 : 1, 1 : 1.2, 1 : 1.4, 1 : 1.6, 1 : 1.8, 1 : 2, 1 : 3, 1 : 4 , 1 : 5 , 1 : 6 or 1 : 7 or more.
  • the weight ratio of carotenoid to phosphatidylcholine is at least about 1 : about 1.
  • the composition of the invention comprises a carotenoid and phosphatidylcholine at a weight ratio of at least about 1.0 : 1.4. The inventors have shown that, unexpectedly, inclusion of a carotenoid with phosphatidylcholine at a weight ratio of at least about 1 : 1 leads to a significant improvement in anti -bacterial and/or anti-oxidative effects.
  • the weight ratio may be expressed as mg/mg.
  • composition of the invention includes at least about lmg, 2mg,
  • the composition of the invention includes at least about 4mg of carotenoid and at least about lmg, 2mg, 3mg, 4mg, 5mg, 6mg or more of
  • composition of the invention includes at least about 7mg of carotenoid and at least about 7mg of phosphatidylcholine.
  • the composition of the invention comprises at least about 7mg of carotenoid and at least about lOmg of phosphatidylcholine.
  • the carotenoid is lycopene. In preferred embodiments, the carotenoid is lycopene. In preferred
  • the ratio of lycopene to phosphatidylcholine in the composition of the invention includes any of the ratios discussed above.
  • the composition of the invention comprises lycopene and
  • the ratio of lycopene to phosphatidylcholine is at least about 1 : 1 (i.e., at a weight ratio of at least about 1.0 : 1.4).
  • compositions the weight ratio may be expressed as mg/mg.
  • the composition of the invention comprises at least about 7mg of lycopene and at least about lOmg of phosphatidylcholine.
  • the composition of the invention further comprises ascorbic acid (vitamin C).
  • Ascorbic acid is a naturally occurring organic compound with antioxidant properties
  • the inventors have shown that, unexpectedly, the addition of ascorbic acid to the composition of the invention acts to protect and extend the active life of carotenoid.
  • the inventors have also shown that, unexpectedly, the addition of ascorbic acid to the composition of the invention leads to synergistic anti -bacterial and/or anti-oxidative effects.
  • the inventors tested a number of different antioxidants including tocpherol, glutathione, propyl gallate, catalase and superoxide dismutase, only the addition of ascorbic acid led to such effects.
  • the composition of the invention comprises a carotenoid and ascorbic acid at a weight ratio of at least about 1 : about 5.
  • the composition of the invention comprises a carotenoid (such as lycopene) and ascorbic acid at a weight ratio of at least about 1 to about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more.
  • the composition of the invention comprises a carotenoid (such as lycopene), phosphatidylcholine and ascorbic acid at a weight ratio of at least about 1 : about 0.1 : about 5.
  • the composition of the invention comprises a carotenoid (such as lycopene), phosphatidylcholine and ascorbic acid at a weight ratio of at least about 1 : 0.1 : 6, 1 : 0.1 : 7, 1 : 0.1 : 8, 1 : 0.1 : 9, 1 : 0.1 : 10, 1 : 0.1 : 11, 1 : 0.1 : 12, 1 : 0.1 : 13, 1 : 0.1 : 14, 1 : 0.1 : 15 or more.
  • the ratio of carotenoid to phosphatidylcholine to ascorbic acid in the composition of the invention is at least about 1 : 1 : 5, 1 : 1 : 6, 1 : 1 : 7, 1 : 1 : 8, 1 : 1 : 9, 1 : 1 : 10, 1 : 1 : 11, 1 : 1 : 12, 1 : 1 : 13, 1 : 1 : 14, 1 : 1 : 15 or more.
  • the carotenoid is lycopene, lutein-meso-zeaxanthin, zeaxanthin, astaxanthin, or any combination thereof.
  • the carotenoid is lycopene.
  • the composition of the invention includes at least about 4mg, 5mg, 6mg, 7mg or more of carotenoid, and so at least about 0.4mg, 0.5mg, 0.6mg, 0.7mg or more of phosphatidylcholine respectively and at least about 20 mg, 25mg, 30mg, 35mg or more ascorbic acid respectively.
  • the composition of the invention includes at least about 7mg of carotenoid, at least about lOmg of
  • phosphatidylcholine and at least about 20mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, 70mg or more ascorbic acid.
  • the carotenoid is lycopene. In preferred embodiments, the carotenoid is lycopene. In preferred
  • the ratio of lycopene to phosphatidylcholine to ascorbic acid in the composition of the invention includes any of the ratios discussed above.
  • the composition of the invention comprises lycopene, phosphatidylcholine and ascorbic acid at weight ratio of at least about 1 : 1 : 7.
  • the composition of the invention may comprise at least 7mg or more lycopene, at least lOmg or more phosphatidylcholine and at least 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg or more ascorbic acid.
  • compositions described herein further comprising ascorbic acid the weight ratio may be expressed as mg/mg, or mg/mg/mg.
  • a composition comprising a carotenoid and phosphatidylcholine (PC) at a weight ratio of at least about 1 : 0.14, preferably wherein the weight ratio is mg/mg.
  • PC phosphatidylcholine
  • a composition comprising lycopene and phosphatidylcholine (PC) at a weight ratio of at least about 1 : 0.14, preferably wherein the weight ratio is mg/mg.
  • PC phosphatidylcholine
  • a composition comprising about 7mg lycopene and about lmg
  • PC phosphatidylcholine
  • a composition comprising a carotenoid, phosphatidylcholine (PC) and ascorbic acid at a weight ratio of at least about 1 : 0.14 : 7.1, preferably wherein the weight ratio is mg/mg.
  • PC phosphatidylcholine
  • a composition comprising lycopene, phosphatidylcholine (PC) and ascorbic acid at a weight ratio of at least about 1 : 0.14 : 7.1, preferably wherein the weight ratio is mg/mg.
  • a composition comprising about 7mg lycopene, about lmg phosphatidylcholine (PC) and about 50mg ascorbic acid.
  • a composition comprising a carotenoid and phosphatidylcholine (PC) at a weight ratio of at least about 1 : 1.4, preferably wherein the weight ratio is mg/mg.
  • PC phosphatidylcholine
  • a composition comprising lycopene and phosphatidylcholine (PC) at a weight ratio of at least about 1 : 1.4, preferably wherein the weight ratio is mg/mg.
  • a composition comprising about 7mg lycopene and about lOmg
  • PC phosphatidylcholine
  • a composition comprising a carotenoid, phosphatidylcholine (PC) and ascorbic acid at a weight ratio of at least about 1 : 1.4 : 7.1, preferably wherein the weight ratio is mg/mg.
  • a composition comprising lycopene, phosphatidylcholine (PC) and ascorbic acid at a weight ratio of at least about 1 : 1.4 : 7.1, preferably wherein the weight ratio is mg/mg.
  • a composition comprising about 7mg lycopene, about lOmg
  • PC phosphatidylcholine
  • compositions of the invention may typically comprise, for instance, one or more pharmaceutically or nutraceutically acceptable carriers, excipients, buffers, adjuvants, stabilizers, or other materials, as described herein.
  • pharmaceutically acceptable typically pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a subject e.g., human
  • Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.
  • GRAS Generally Recognized as Safe
  • formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy, food science or nutrition. Such methods include the step of incorporating a carrier which may constitute one or more accessory ingredients.
  • Formulations may be in the form of food products, beverages, liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, lozenges, granules, powders, capsules, cachets, pills, ampoules, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols.
  • a composition of the invention may be preferably in a form which is suitable for administration orally for delivery via the gastro-intestinal tract.
  • Formulations suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.
  • a formulation of the invention may be provided in a capsule, hence the present invention provides a capsule comprising a composition of the invention.
  • Formulations of the invention will, in particular, be suitable for oral administration. Oral administration is the most preferred route of administration for the invention.
  • a tablet may be made by conventional means, e.g. , compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g., povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica); disintegrants (e.g., sodium starch glycolate, cross- linked povidone, cross-linked sodium carboxymethyl cellulose); surface-active or dispersing or wetting agents (e.g., sodium lauryl sulfate); and preservatives (e.g., methyl p-hydroxybenzoate, propy
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile.
  • Compositions for oral administration may further comprise sweeteners, texture modifiers, colorings and flavorings.
  • compositions or “nutritional supplements.”
  • any of the compositions described herein may be provided as a nutritional composition or supplement.
  • a composition of the invention may be a "nutraceutical” and that term may include: food products, foodstuffs, dietary supplements, nutritional supplements or a supplement composition for a food product or a foodstuff.
  • composition of the invention may be provided in an enteric soft capsule shell.
  • the shell of a capsule may be, for instance, made of naturally occurring ingredients.
  • the composition of the invention may be taken by an individual after a meal.
  • a composition of the invention may be, for instance, given on a daily basis, for examples after meals, or for instance at any appropriate intervals such as at weekly, fortnightly or monthly intervals.
  • a composition of the invention may be one that does not need to be prescribed by a doctor to be administered.
  • a composition of the invention is a supplement. It may be that the composition is one sold as an over the counter medicine. It may be that the composition is a nutraceutical.
  • a composition of the invention is not one that requires regulatory approval prior to marketing. However, the invention may be also applied to pharmaceutical products, such as those that have to be prescribed.
  • a composition of the invention may be one with an active agent such as that the composition requires regulatory approval.
  • the term "effective amount” refers to a quantity sufficient to achieve a desired effect and in particular a desired therapeutic and/or prophylactic effect.
  • a “therapeutically effective amount” may be, for instance, the amount needed to reduce or eliminate the presence, frequency, or severity of one or more signs, or symptoms of the conditions mentioned herein.
  • the amount of a formulation administered to the subject will depend on the type, degree, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled person will be able to determine appropriate dosages depending on these and other factors.
  • an oral pharmaceutical dosage form comprising any of the compositions described herein, particular a capsule comprising one of the compositions described herein, particularly a capsule provided a daily dose of the composition as described herein.
  • a composition of the invention may be administered.
  • the carotenoid may be administered at an amount from about lmg to about 50 mg, preferably from about 1 to about 25 mg, such as from about 1 to about 15 mg, such as from about 1 to about 10 mg.
  • a composition provides at least about 4 mg of carotenoid.
  • a composition of the invention may be in unit dose form, and so provide the recommended daily amount of the carotenoid.
  • the invention provides a method of producing a pharmaceutical formulation comprising formulating composition components as defined herein, together with a pharmaceutically acceptable excipient. In certain embodiments, the invention provides a method of producing a nutraceutical formulation comprising formulating composition components as defined herein, together with a nutraceutically acceptable excipient.
  • the invention provides a method of treating or preventing an obligate intracellular bacterial infection in an individual, comprising administering a
  • the invention provides a method of treating or preventing any bacterial infection wherein the bacteria cannot reproduce outside it' s host cell, meaning that it's reproduction is entirely reliant on intracellular resources.
  • the bacterial infection is caused by Chlamydiae, Rickettsia, Ehrlichia and/or Coxiella.
  • the composition may act to reduce or inhibit the formation of infective progeny of the obligate intracellular bacteria in the individual.
  • the composition may act to reduce or inhibit lysis of eukaryotic cells and the release of infective progeny to neighboring cells.
  • the composition may therefore act to prevent the subsequent repeat of cellular infections in adjacent epitheliocytes.
  • the composition acts to reduce or inhibit the release of infective progeny into the bloodstream of the individual.
  • the invention therefore provides a method of inhibiting the formation of infective progeny of obligate intracellular bacteria in an individual, the method comprising administering a therapeutically or prophylactically effective amount of a composition of the invention, and thereby reducing or inhibiting the formation of infective progeny of obligate intracellular bacteria in the individual.
  • he invention also provides a method of inhibiting the release of infective progeny of obligate intracellular bacteria into the bloodstream of an individual, the method comprising administering a therapeutically or prophylactically effective amount of a composition of the invention, and thereby reducing or inhibiting the release of infective progeny into the bloodstream of the individual.
  • reducing or inhibiting the formation of infective progeny means reducing or inhibiting the formation or release of infective progeny in the individual as compared to one or more control individual(s).
  • reducing or inhibiting the formation or release of infective progeny is understood to mean a statistically significant reduction or inhibition in the formation or release of infective progeny in the individual as compared to one or more control individual(s).
  • the number of infective progeny being formed and/or released in the individual may be reduced by at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more as compared to one or more control individual(s).
  • the control individual may be same individual, prior to or at an earlier stage of treatment with the composition of the invention.
  • the control individual may be a different individual, i.e., receiving a control composition that includes a carotenoid but does not include PC and/or ascorbic acid.
  • any standard method may be used to determine the number of infective progeny being produced or released in the individual, such as those described in the Examples. For instance, in clinical cases where the obligate intracellular bacteria are not present in the circulating blood and collection of tissue biopsy of the infected tissues is limited or not practical, the reduction or inhibition of the formation or release of infective progeny can be determined by the standard diagnostic practice of measuring titers of specific antibodies against the obligate intracellular bacteria. As discussed further below, the invention therefore further provides a method of measuring the titers of antibodies against the obligate intracellular bacteria in a sample taken from the individual, as compared to one or more control samples.
  • the control sample may be from the same individual, prior to or at an earlier stage of treatment with the composition of the invention. Alternatively, the control sample may be from a different individual receiving a control composition that includes a carotenoid but does not include PC and/or ascorbic acid.
  • the sample may be from any tissue or bodily fluid.
  • the sample typically comprises a body fluid and/or cells of the individual and may, for example, be obtained using a needle.
  • the sample may be, or be derived from, plasma, serum or whole blood from the individual.
  • the sample is typically processed prior to being assayed, for example by centrifugation or by passage through a membrane that filters out unwanted molecules or cells, such as red blood cells.
  • the sample may be measured immediately upon being taken.
  • the sample may also be stored prior to assay, preferably below -70°C.
  • the titer of antibodies in the one or more sample(s) may be measured by any standard technique.
  • ELISA assays may be used to measure the antibodies against the obligate intracellular bacteria.
  • the antibodies are against a surface protein of the obligate intracellular bacteria.
  • the antibodies are against Chlamydophilia. More typically, the antibodies are anti -Chlamydia IgG or IgA.
  • Obligate intracellular bacterial infections such as Chlamydia and/or conditions associated with obligate intracellular bacterial infections are associated with depressed tissue oxygenation and/or impaired flow-mediated dilation in the infected individual.
  • the administration of a therapeutically or prophylactically effective amount of a composition of the invention may increase depressed tissue oxygenation and/or impaired flow-mediated dilation in the individual, thereby restoring such parameters to their physiological norm.
  • tissue oxygenation and/or impaired flow- mediated dilation means increasing depressed tissue oxygenation and/or increasing impaired flow-mediated dilation in the individual as compared to one or more control individual(s).
  • tissue oxygenation and/or flow-mediated dilation is understood to mean a statistically significant increase in tissue oxygenation and/or flow-mediated dilation in the individual as compared to one or more control individual(s).
  • tissue oxygenation and/or flow-mediated dilation may be increased by at least about 5%, 10%, 20%, 30%, 40% , 50%, 60% or more as compared to one or more control individuals.
  • the control individual may be same individual, prior to or at an earlier stage of treatment with the composition as defined herein.
  • control individual may be a different individual, i.e., receiving a control composition that includes a carotenoid such as lycopene but does not include PC and/or ascorbic acid.
  • a control composition that includes a carotenoid such as lycopene but does not include PC and/or ascorbic acid.
  • Obligate intracellular bacterial infections such as Chlamydia and/or conditions associated with obligate intracellular bacterial infections are also associated with tissue hypoxia, impaired microcirculation, pre-tension and/or hypertension in the infected individual.
  • the administration of a therapeutically or prophylactically effective amount of a composition of the invention may reduce or inhibit tissue hypoxia, impaired microcirculation, pre-tension and/or hypertension, thereby restoring such parameters to their physiological norm.
  • Spectroscopy Laser Doppler Flowmetry, transcutaneous oxygen monitoring, magnetic resonance imaging and/or angiography may be used to measure such parameters.
  • measurements are made from a sample taken from the individual, as compared to one or more control samples. Any sample may be used for such measurements, such as those described herein.
  • the bacterial infection is caused by Chlamydiae.
  • the bacterial infection is caused by Chlamydophilia, typically Chlamydophila psittaci, Chlamydophila trachomatis or Chlamydophila pneumoniae.
  • the composition used to treat or prevent such Chlamydial bacterial infections comprises lycopene and phosphatidylcholine (PC) at a weight ratio of at least 1 : 1 , preferably mg/mg.
  • the composition used to treat or prevent such Chlamydial infections further comprises ascorbic acid, typically at a weight ratio of at least 1 : 1 : 7, preferably mg/mg/mg.
  • the invention also provides a method of treating or preventing any condition associated with an obligate intracellular bacterial infection in an individual, comprising administering a therapeutically or prophylactically effective amount of a composition of the invention, and thereby treating or preventing the condition.
  • the composition is selected from a metabolic syndrome, fatty liver, steatohepatitis, coronary heart disease (CHD), coronary vascular disease (CVD), atherosclerosis, cardiovascular pathology, cerebrovascular pathology and/or a neurodegenerative condition.
  • the individual that is treated is not infected with an obligate intracellular bacteria and/or the individual does not display any symptoms of the bacterial infection.
  • the individual that is treated is infected with an obligate intracellular bacteria and/or the individual displays symptoms of the bacterial infection.
  • an individual who is infected with an obligate intracellular bacteria and/or who displays symptoms of the bacterial infection has increased serum titer of antibodies against the bacterial infection, as compared to one or more control serum samples.
  • composition used to treat the condition associated with an obligate intracellular bacterial infection comprises lycopene and
  • composition typically at a weight ratio of at least about 1 : 1, preferably mg/mg.
  • composition further comprises ascorbic acid, typically at a weight ratio of at least about 1 : 1 : 7, preferably mg/mg/mg.
  • the invention also provides a method of treating or preventing a functional carotenoid deficiency in an individual, comprising administering a therapeutically or prophylactically effective amount of a composition of the invention, and thereby treating or preventing the functional carotenoid deficiency.
  • Any individual may have a functional carotenoid deficiency.
  • lipoprotein metabolism may be compromised by ageing, and individuals older than approximately 50 years may lose the ability to assemble new lipoproteins. This may lead to functional carotenoid deficiency, leading to age-associated subclinical inflammation and oxidation, especially in cells such as enterocytes and hepatocytes and associated tissues where these processes occur.
  • the invention therefore provides a method of increasing the bioavailability of carotenoids by administering a therapeutically or prophylactic effective amount of a composition of the invention.
  • the individual having a functional carotenoid deficiency is at least approximately 50, at least approximately 55, at least approximately 60, at least approximately 65, at least approximately 70 years in age or more.
  • the individual having a functional carotenoid deficiency is not infected with an obligate intracellular bacteria and/or the individual does not display any symptoms of the bacterial infection.
  • the individual having a functional carotenoid deficiency is infected with an obligate intracellular bacteria and/or the individual displays symptoms of the bacterial infection.
  • an individual who is infected with an obligate intracellular bacteria and/or who displays symptoms of the bacterial infection has increased serum titer of antibodies against the bacterial infection, as compared to one or more control serum samples.
  • the composition used to treat functional carotenoid deficiency comprises lycopene and phosphatidylcholine (PC), typically at a weight ratio of at least about 1 : 1, preferably mg/mg.
  • the composition further comprises ascorbic acid, typically at a weight ratio of at least about 1 : 1 : 7, preferably mg/mg/mg.
  • compositions of the invention may be administered in the methods described herein.
  • the compositions of the invention are employed as supplements, such as nutritional supplements or nutraceuticals.
  • the individual may be a healthy individual.
  • compositions of the invention may be used prophylactically, to help prevent or reduce the risk of developing an intracellular bacterial infection and/or a condition associated with an intracellular bacterial infection, such as any of those described above.
  • Any of the methods discussed herein may be used to prevent, or delay the onset of, a condition, such as the conditions specified or to treat the condition once it has arisen in an individual.
  • treatment may include, for instance, elimination of a condition or reducing the severity of the condition. It may, for instance, involve elimination or reduction of a symptom or symptoms of the condition. Treatment may include bringing about regression of a disorder.
  • the effect of administering a carotenoid (such as lycopene) and ascorbic acid is a synergistic effect than if the same amount of a composition was administered by adding a carotenoid (such as lycopene) or ascorbic acid individually with PC.
  • administration of lycopene in conjunction with ascorbic acid agent leads to an improved effect in preventing or treating the obligate intracellular bacterial infection and/or associated conditions, and so is greater than when lycopene is administered individually with PC.
  • the invention may be applied to any suitable individual.
  • the individual may be an individual organism, a vertebrate, a mammal, or a human.
  • the invention is applied to a human.
  • the invention may, for instance, also be applied to non-human animals, such a pets or commercial animals.
  • Such animals include, for instance, dogs, cats, cattle, pigs and sheep.
  • the individual may be elderly, for instance over 50, 55, 60, 65, 70, 75 or 80 years of age.
  • the subject may be male or female. In some instances, the subject is pregnant.
  • the invention further provides a method of reducing the titer of antibodies against a bacterial infection in an individual, comprising administering a therapeutically or prophylactically effective amount of a composition of the invention.
  • reducing is understood herein to mean, for example, a statistically significant reduction in the titer of antibodies against a bacterial infection in a sample from an individual as compared to a control sample.
  • the invention provides a method of reducing the titer of antibodies by at least about 45%, 50%, 55%, 60% or more as compared to one or more control samples.
  • the control sample may be from the same individual, prior to or at an earlier stage of treatment with the composition of the invention. Alternatively, the control sample may be from a different individual receiving a control composition that includes a carotenoid but does not include PC and/or ascorbic acid.
  • the sample may be from any tissue or bodily fluid.
  • the sample typically comprises a body fluid and/or cells of the individual and may, for example, be obtained using a needle.
  • the sample may be, or be derived from, plasma, serum or whole blood from the individual.
  • the sample is typically processed prior to being assayed, for example by centrifugation or by passage through a membrane that filters out unwanted molecules or cells, such as red blood cells.
  • the sample may be measured immediately upon being taken.
  • the sample may also be typically stored prior to assay, preferably below -70°C.
  • any method may be used to determine whether or not the titer of antibodies is reduced in the individual.
  • standard ELISA assays may be used to measure the titer of the antibodies against the bacterial infection.
  • the antibodies are against a surface protein of the obligate intracellular bacteria.
  • the bacterial infection is caused by Chlamydophilia. More typically, the antibodies are anti-Chlamydia IgG or IgA.
  • C. trachomatis was initially propagated in McCoy cells and purified by Renografin gradient centrifugation as previously described [1]. Chlamydial titers were determined by infecting McCoy cells with 10-fold dilutions of thawed stock suspension. Purified elementary bodies (EB) of known titer were suspended in sucrose-phosphate- glutamic acid buffer (SPG) and used as inoculums for B IO.MLM cells. Cells were grown in 24 well plates until a confluence rate of 80% was reached. BIO.MLM plates were infected with C.
  • SPG sucrose-phosphate- glutamic acid buffer
  • trachomatis at multiplicity of infection MOI
  • MOI multiplicity of infection
  • B IO.MLM cells were harvested for reinfection after 42 h of cultivation, as described before [22].
  • Serial dilutions of lysates were inoculated onto the McCoy cells.
  • Infected McCoy cells were grown for 42 h on coverslips in 24-well plates, fixed with methanol and visualized with C. trachomatis species-specific monoclonal antibody (Bio- Rad).
  • the semi -quantitative analysis was based on the counting of infected cells in 20 random visual fields at magnification of x200 and calculating the mean number of inclusion-forming units (IFU) per ml of the specimen. Every experiment was repeated three times.
  • Lycopene toxicity verification Lycopene toxicity was controlled in MTT test in 24 hours after lycopene addition using 96 well dishes.
  • B10.MLM cells grown on coverslips were incubated with Lycopene for 24, 30 and 42 hours. Then cells were washed with PBS twice, fixed with 3% formaldehyde/ 0.025% glutaraldehyde at room temperature for 20 min and stained with BODIPY 493/503 (Molecular Probes, Invitrogen Life Technologies, Carlsbad, California, USA) according to manufacturer' s instructions. Cells were visualized using a Nikon Eclipse 50i fluorescence microscope at x lOOO magnification.
  • TEM Transmission electron microscopy
  • B10.MLM cells were cultured and infected with C. trachomatis with or without Lycopene addition in six -well plates for 48 hours of post-infection period and then harvested from the plates with trypsin-versene solution.
  • Cell pellets obtained by centrifugation for 10 min at 1500 r.p.m. ( otanta 460R;Hettich) were fixed with Ito-Karnovsky fixative solution, followed by post-fixation with OsC>4 and treatment with aqueous uranyl acetate to provide contrast.
  • the specimens were subsequently dehydrated in an ascending series of alcohol concentrations (50, 70, 96 and 100 % ethanol), infiltrated in a 1 : 1 (v/v) mixture of LR White resin and 100 % ethanol for 1 h and in a pure resin for 12 h at 4 uC. Resin polymerization was performed at 56 uC for 24 h. Ultrathin sections were prepared, treated with a lead solution to provide contrast (Reynolds, 1963) and analyzed using a JEOL 100B transmission electron microscope with an accelerating voltage of 80 kV (Jeol, Japan). 2. Results
  • Lycopene inhibits chlamydial infection in dose-dependent manner
  • lycopene oil-formulated and microencapsulated leads to the dose- dependent inhibition of chlamydial infection and accumulation of inclusion bodies in the cultured cells (Fig. 1). Both formulations of lycopene inhibited chlamydial infection even in 24 hours of post-infection period. After 42 hours after pathogen inoculation the number of the infected cells was reduced by 95% after addition of oil -formulated lycopene. Remarkably, the size of the inclusion bodies was smaller than in infected cells untreated with lycopene.
  • Microencapsulated lycopene at concentration of 0.5 mg/ml reduced the number of infected cells by 92% and lead to the significant reduction of inclusion bodies at lower concentrations.
  • Addition of lycopene has been performed in all cases after finalized adhesion and internalization of C. trachomatis by cultured cells. Inhibition of chlamydial infection observed in the studies described herein therefore does not develop due to any direct effect of lycopene on the bacterial pathogen and the suppression of its infective abilities. Rather, lycopene has an inhibitory effect on the development of the intracellular infection process.
  • Electron microscopy studies conducted with infected alveolar macrophages showed that after inoculation of C. trachomatis there is an increase in cell size, which did not take place in the control group. Nevertheless the shape of the cells was unchanged and remains round-shaped with a small number of membrane extensions. There were multiple inclusion bodies containing chlamydial particles at different stages of the life cycle (Fig.3A).
  • lipid droplets were located in the cytoplasm and even in some chlamydial inclusion bodies. In the cytoplasm of some alveolar macrophages were seen some typical chlamydial particles. However the majority of chlamydial particles were atypical (Fig. 3B).
  • CHD Coronary Heart Disease
  • the first formulation comprised 7 mg from Nestle Inc. (Lacto-Lycopene, LL, Switzerland);
  • the second formulation comprised 15 mg lycopene from LycoRed (Lyc-O-Mato, Switzerland);
  • the third formulation comprised 7 mg lycopene with 1 mg phosphatidylcholine; • The fourth formulation comprised 7 mg lycopene with 10 mg phosphatidylcholine ("GA");
  • the fifth formulation comprised 7 mg lycopene with 40 mg phosphatidylcholine;
  • the sixth formulation comprised 7 mg of lycopene with 10 mg of phosphatidylcholine and 50 mg of ascorbic acid.
  • the first formulation comprised 12 mg lutein-meso-zeaxanthin*, LMZ, only
  • the second formulation comprised lOmg LMZ and 2 mg of zeaxanthin;
  • the third formulation comprised 7 mg of LMZ, 1.4 mg of zeaxanthin and 10 mg of phosphatidylcholine;
  • the fourth formulation comprised 7 mg of LMZ, 1.4 mg of zeaxanthin and 40 mg of phosphatidylcholine.
  • lutein used in these preparations contained up to 50% of naturally occurring its isomer meso-zeaxanthin.
  • the first formulation comprised 7 mg of astaxanthin only,
  • the second formulation comprised 7 mg of astaxanthin and 10 mg of phosphatidylcholine.
  • BMI Body mass index
  • SBP systolic and diastolic blood pressure
  • Flow -Mediated Dilation Endothelium-dependent flow mediated vasodilatation was measured in accordance with widely accepted guidelines [2]. Patients were screened under ambient conditions at the same time of the morning in a supine position. High resolution ultrasound was applied at the same anatomical landmark of a section of the brachial artery for a period of 30 seconds before and during the peak of reactive hyperemia. It was positioned prior to sphygmomanometer cuff occlusion and 1 min after its deflation. The level of inflation was 50 mm Hg above the patient' s systolic blood pressure and it continued for 5 minutes. Arterial diameter was imaged above the antecubital fossa in a longitudinal scan by duplex ultrasound with linear phase-array transducer. FMD was calculated as a change in post-stimulus diameter as a percentage of the baseline diameter [3].
  • ABSI Ankle-Brachial Index
  • Tissue Oxygenation Thenar eminence and forearm muscles of the patients were used as a tissue target for the assessment of oxygen saturation, St0 2 , or combined level of oxygenated haemoglobin and myoglobin. StC was assessed by continuous wavelength near-infrared spectroscopy, MRS, with wide-gap second-derivative (In Spectra, Hutchinson Technology, MN, USA). The measurements were taken at different time points. The recording was initiated after 15 min of rest in a supine position before occlusion of the brachial artery. It was then continued during stagnant ischemia induced by rapidly inflating the cuff to 50 mm Hg above systolic BP.
  • MRS continuous wavelength near-infrared spectroscopy
  • the ischemia lasted for 3 min, and the recording period lasted for another 5 min after that until StCh was stabilized [5, 6].
  • the area under the hyperaemic curve, AUC, of the recorded signal for the settling time in the post-occlusion period was then calculated as described earlier in % 02/minute [7, 8].
  • Blood Collection Blood was collected in the morning from the arm veins of patients following night fast. The serum was separated from the rest of the clotted mass by centrifugation, aliquots were then stored at -80°C prior to analysis. Biochemistry and Inflammatory Markers.
  • Glucose, total cholesterol, TC, triglycerides, TG, high density cholesterol, HDL, low density cholesterol, LDL, C- reactive protein, CRP and Chlamydophila pneumoniae IgG were determined using commercially available analytical kits according to the manufacturers' instructions (BioSystems, Medac, R&D Systems).
  • Lycopene Quantitative Analysis The lycopene concentration in all serum samples was measured in duplicate by high-performance liquid chromatography with modifications. Briefly, 400 ⁇ of serum was mixed with 400 ⁇ of ethanol and was extracted twice with 2 ml hexane. The combined hexane layers were evaporated to dryness in a vacuum (Scan Speed 32 centrifuge) and the residue reconstituted to a volume of 100 ⁇ in sample solution (absolute ethanol - methylene chloride, 5: 1, v/v). The specimens were centrifuged again (15 minutes at 10,000 g) and clear supernatant was transferred to HPLC vials. Five microliters of the extract was injected into an Acquity HSS T3 75x2.
  • IOD Inflammatory Oxidative Damage
  • Results presented in Table 1 demonstrate that lycopene and phosphatidylcholine at a ratio of at least 1 : 0.1 mg/mg allows the product to perform its anti -chlamydia properties, in terms of reduction of Chlamydophila pneumoniae IgG. This is also accompanied by significant inhibition of the marker oxidative damage.
  • the further increase of lycopene to phosphatidylcholine to the ratio of 1 : 1 mg/mg resulted in improved anti-chlamydial and improved anti-oxidative damage effects.
  • Further increase of the presence of PC did not significantly increase this improved lycopene efficacy. PC alone did not have any effect either on Chlamydophila pneumoniae IgG titers or on the markers of oxidative damage.
  • lycopene concentration in the blood itself is not sufficient for anti-chlamydial and/or anti-oxidant activity.
  • the intake of 15 mg of lycopene alone, for one month resulted in a substantial increase of this carotenoid in the blood, by 95 ng/ml. This was accompanied by reduction of the marker of oxidation by 19% with p ⁇ 0.05.
  • Lycosomes are the organelles of the cells which are responsible for proteolytic degradation of infective agents and own damaged cells and tissues. When lysosomes release their acidic content, the pH of the local microenvironment is reduced. While acidic pH helps proteolytic and hydrolytic enzymes perform their function, it may at the same time negatively affect integrity of such useful molecules as the carotenoids, which can accelerate their oxidation and degradation.
  • the phospholipid chaperone was also essential for the anti-chlamydia effect of astaxanthin.
  • the addition of PC provided a significant boost in astaxanthin
  • Coronary Heart Disease is understood to be associated with Chlamydia pneumoniae infection [10-14], However, clinical trials have not (or have failed to show
  • Chlamydia has a causative effect in the development of
  • Lycopene measurements did not show any significant differences between the two groups of the study in the pre-treatment period. Ingestion of lactolycopene did not affect serum lycopene level after 2 weeks of consumption (median value of 58.0 ng/mg; 5/95% CI: 69.2/48.4; versus pre-treatment value of 58.0 ng/mg; 5/95% CI:76.4/55.0, P>0.05). However, there was some increase in serum lycopene content by the end of the observational period (up to median value of 87.0 ng/mg; 5/95% CL93.9/72.2, PO.05).
  • the addition of the chaperone PC enhanced the bioavailability of lycopene, and resulted in a substantial anti-chlamydia effect.
  • Treatment with GA lycopene led to a 3- fold reduction of Chlamydia pneumoniae IgG and lowering to the same degree of the IOD marker. The reduction of oxidized LDL was 5 fold.
  • the observations described herein may be of significant importance for safe nutraceutical management of cardiovascular patients, since oxidative stress plays a pivotal role in the initiation, development and outcomes of cardiovascular disease.
  • the significant anti-chlamydia effect of GA lycopene, accompanied by strong antioxidant activity also translates into a positive effect on some cardiovascular parameters. In particular, such treatments resulted in a significant increase of tissue oxygenation and flow-mediated dilation by the end of the observational period.
  • Example 3 Mecrobiota as a new additional targets for carotenoids in the treatment of bacterial infections
  • Figures 8 and 9 panels A
  • Figures 8 and 9 panel B
  • Figures 8 and 9 panel B
  • the results demonstrate a significant increase in levels of probiotic bacterial species such as lactobacilli in the gut after administration of GA lycopene.
  • probiotic bacterial species such as lactobacilli in the gut after administration of GA lycopene.
  • Figures 8 and 9 are only a small representative of bacterial species in the samples for illustration.
  • microbiota is an aggregate of microrganisms that resides on or within any number of tissues and biofluids of an individual, including the skin and gut of individuals such as human individuals.
  • beneficial bacterial species i.e., nonpathogenic
  • other bacteria i.e., pathogenic bacteria
  • pathogenic bacteria obligate intracellular bacteria
  • carotenoids are an important part of their metabolism.
  • the invention relates to the unexpected finding that the administration of the formulations of the invention or carotenoids such as lycopene to an individual acts to boost (i.e., increases the activity and/or number) of carotenoid-metabolizing bacteria in the individual and thereby help to prevent, control and/or treat bacterial infections, particularly obligate intracellular bacterial infections caused by Chlamydia, Ehrlichia and/or Rickettsia
  • the formulations of the invention or carotenoids such as lycopene enhance the activity and/or number of carotenoid-metabolizing bacteria in the microbiota of an individual.
  • Carotenoid-metabolizing bacteria may act locally within one or more organs such as the gastrointestinal tract.
  • carotenoid-metabolizing bacteria may release antibacterial, anti-infective, antiinflammatory molecules or metabolites, which can be absorbed and be circulated around the whole organism, to prevent and treat bacterial infections or inflammatory conditions in other organs and tissues, or systemically, in the whole body.
  • increases the activity of carotenoid-metabolizing bacteria is understood to mean an increase in the rate or the amount of anti -bacterial, anti-infective, anti-inflammatory molecules or metabolites that are produced by the carotenoid- metabolizing bacteria upon administration of a formulation of the invention and/or carotenoid such as lycopene as compared to one or more control individual(s).
  • increases the number of carotenoid-metabolizing bacteria is understood to mean a statistically significant increase in the number (i.e., total number in a population) of carotenoid-metabolizing bacteria in the microbiota of the individual as compared to one or more control individual(s).
  • control individual may be same individual, prior to or at an earlier stage of treatment with the formulation of the invention and/or carotenoids such as lycopene.
  • control individual may be a different individual, i.e., an individual receiving a control composition that lacks the formulation of the invention and/or carotenoid such as lycopene.
  • the carotenoid-metabolizing bacteria may be any bacteria that is capable of metabolizing any one or more carotenoids as defined herein.
  • the carotenoid-metabolizing bacteria is a Actinobacteria or Proteohacteria species such as Micrococcus luteus .
  • the carotenoid-metabolizing bacteria is a probiotic.
  • the carotenoid-metabolizing bacteria is a lactobacilli such as lactobacillus acidophilus, lactobacillus brevis, lactobacillus bulgaricus, lactobacillus plantarum, and/or lactobacillus rhamnosus.
  • the carotenoid-metabolizing bacteria is bifidobacteria such as bifidobacerium bifidum, streptocuccus such as streptococcus thermophilius or enter ococcus such as enter ococcus faecium.
  • the formulations of the invention and/or carotenoids such as lycopene may lead to anti-bacterial effects (such as the enhanced anti-Chlamydial effect observed following treatment with the "GA” lycopene) by having a direct effect on bacterial propagation (i.e., directly reducing levels or numbers of pathogenic bacteria).
  • the formulations of the invention and/or carotenoids such as lycopene may lead to anti-bacterial effects (such as the enhanced mti-Chlamydial effect observed following treatment with the "GA” lycopene) by indirectly boosting carotenoid-metabolizing members of microbiota in an individual (e.g. increasing the numbers or levels of carotenoid-metabolizing microorganisms, including bacteria, in the individual).

Abstract

The invention relates to new formulations of carotenoids and chaperone molecules, and relates uses and methods.

Description

COMPOSITIONS COMPRISING CAROTENOIDS AND
PHOSPHATIDYLCHOLINE
Field of the Invention
The invention relates to new formulations of carotenoids and chaperone molecules. The invention also relates to the use of these formulations for preventing or treating any bacterial infection, especially obligate intracellular bacterial infections such as those of the class Chlamydiae, as well as conditions associated with obligate intracellular bacterial infections. The invention also relates to methods of treating bacterial infections and associated conditions using these formulations.
Background of the Invention
Obligate intracellular bacteria such as those of the class Chlamydiae are responsible for a wide range of diseases in different hosts organs and tissues, including genital and eye inflammatory diseases, arthritis, inflammatory diseases of the respiratory and cardiovascular system. Such intracellular bacteria may also be associated with development of neurodegenerative and behavioral disorders. For example, there is evidence that Chlamydophila pneumoniae may promote differentiation of pre-adipocyte cells into mature fat cells, and be associated with development of metabolic syndrome, fatty liver or non-alcoholic steatohepatitis.
Even though obligate intracellular bacteria such as Chlamydiae display many resemblances with certain Gram-negative bacteria, they represent a unique phylogenetic and genetic entity distinct from other types of bacteria. The developmental cycle of all members belonging to Chlamydiae is remarkably similar. Intracellular infection becomes initiated with infectious but metabolically inert elementary bodies which differentiate within the membrane-associated vacuole of the cell into metabolically active but non-infective reticulate bodies. The infective cycle terminates within 48-72 hours by lysis of eukaryotic cells and release of infective progeny to neighboring cells, and subsequent repeat of cellular infections in adjacent epitheliocytes. The whole chlamydial infectious cycle is highly dependent on host cell energy homeostasis and metabolism, since chlamydial species lack crucial enzymes for ATP biosynthesis and are defective in biosynthesis of lipids and many other organic substances.
Due to the growing resistance of obligate intracellular bacteria such as
Chlamydiae to conventional antibiotics and anti -bacterial drugs, there remains a need for alternative therapies to treat or prevent such bacterial infections. Summary of the Invention
The invention relates to the finding that the high dependence of the obligate intracellular bacterial developmental cycle upon the host's cell metabolism creates an opportunity to control these species by modulating the host cell's metabolic pathway. In particular, the inventors have surprising shown that carotenoids target metabolic pathways essential for survival and propagation of obligatory intracellular bacteria. Targeting such metabolic pathways using carotenoids therefore unexpectedly provides an alternative means to prevent or treat such bacterial infections as compared to conventional antibiotics and anti-bacterial drugs.
The invention also relates to the finding that lipoprotein metabolism may be compromised when obligate intracellular bacteria such as Chlamydiae hijack the lipid metabolism of the host. Therefore, in order to make carotenoid treatment of such bacteria effective, it is essential to target these molecules to the sites of infection.
Through extensive in vivo studies, the inventors have developed novel formulations that more effectively assist the incorporation of carotenoids into lipoproteins. These formulations act to assist the delivery of carotenoids to target infection sites, and surprisingly improve anti-bacterial efficacy.
The inventors have also shown that the formulations of the invention not only target obligate intracellular bacterial infections themselves, but may also be used to treat or prevent their complications and associated conditions such as metabolic syndrome, fatty liver, steatohepatitis, atherosclerosis, cardiovascular pathologies, cerebrovascular, neurodegenerative conditions and/or functional carotenoid deficiency.
The invention also relates to the unexpected finding that the formulations of the invention act to boost carotenoid-metabolizing bacteria and thereby inhibit pathogenic bacteria.
As such, the invention provides:
A composition comprising a carotenoid and phosphatidylcholine (PC) at a weight ratio of at least about 1 : 0.1.
A method of treating or preventing an obligate intracellular bacterial infection and/or a condition associated with an obligate intracellular bacterial infection in an individual, the method comprising administering a therapeutically or prophylactically effective amount of a composition as defined herein to the individual, and thereby treating or preventing the infection and/or condition. A composition as defined herein for use in a method of treating or preventing an obligate intracellular bacterial infection and/or a condition associated with an intracellular bacterial infection in an individual.
Use of a composition as defined herein in the manufacture of a medicament for treating or preventing an obligate intracellular bacterial infection and/or a condition associated with an intracellular bacterial infection in an individual.
A method of reducing the titre of antibodies against an obligate intracellular bacterial infection in an individual, the method comprising administering a therapeutically or prophylactically effective amount of a composition as defined herein to the individual and thereby reducing the titre of antibodies against the bacterial infection.
A method of treating or preventing an obligate intracellular bacterial infection and/or a condition associated with an intracellular bacterial infection in an individual, the method comprising administering a therapeutically or prophylactically effective amount of a composition comprising lycopene to the individual and thereby treating or preventing the infection.
A composition comprising lycopene for use in a method of treating or preventing an obligate intracellular bacterial infection and/or a condition associated with an intracellular bacterial infection in an individual.
Use of a composition comprising lycopene in the manufacture of a medicament for the treatment or prevention of an obligate intracellular bacterial infection and/or a condition associated with an intracellular bacterial infection in an individual.
A method of producing a pharmaceutical formulation comprising formulating composition components as defined herein, together with a pharmaceutically acceptable excipient.
A method of producing a nutraceutical formulation comprising formulating composition components as defined herein, together with a nutraceutically acceptable excipient.
A method of treating or preventing a bacterial infection in an individual, the method comprising administering a therapeutically or prophylactically effective amount of a composition comprising a carotenoid to the individual, wherein the composition increases the activity and/or number of carotenoid-metabolizing bacteria in the individual and thereby treats or prevents the bacterial infection.
The invention additionally provides a composition comprising a carotenoid for use in a method of treating or preventing a bacterial infection in an individual, wherein the composition increases the activity and/or number of carotenoid-metabolizing bacteria in the individual. Any of the carotenoid-comprising compositions defined herein may be used in any such method of treatment or prevention. The carotenoid-metabolizing bacteria may be any carotenoid-metabolizing bacteria described herein, such as lactobacilli and/or bifidobacteriacea. The bacterial infection which may be treated or prevented with any of such compositions is any bacterial infection comprising any bacterium or bacteria which are susceptible to inhibition by carotenoid-metabolizing bacteria, e.g. as described in more detail herein. The bacterial infection may be caused by an obligate intracellular bacteria such as Chlamydia, Ehrlichia and/or Rickettsia. In any such method of treatment or prevention, any of the carotenoid-comprising compositions defined herein may be used to increase any of the activities described herein of the carotenoid-metabolizing bacteria.
Brief Description of the Figures
Figure 1. Dose-Dependent inhibition of C. trachomatis growth in B IO.MLM cells in presence of oil-formulated (A) and microencapsulated (B) lycopene. Panel A- C. trachomatis Bu-434 infection in BIO.MLM cells at 42 h.p.i. (1); growth in the presence of 0. 015% olive oil in DMSO (2); growth in the presence of 0.75
(4) and 3.0 μg/ml of oil-formulated lycopene (5). Panel B - C. trachomatis infection in B IO.MLM cells at 42 h.p.i. (1); growth in the presence of 1.0% cyclodextrin in H20(2); growth in the presence of 0.125 mg/ml (3), 0.25 mg/ml (4) and 0.5 mg/ml of microencapsulated lycopene (5). Scale bar ΙΟΟμηι.
Figure 2. Inhibition of C. pneumoniae growth in B 10.MLM cells in presence of oil-formulated and microencapsulated lycopene. C. pneumoniae K-6 infection in B I O.MLM cells at 72 h.p.i. (1); growth in the presence of 0.015% olive oil in DMSO (2) and 3.0 μg/ml of oil-formulated lycopene (3); growth in the presence of 1.0% cyclodextrin in H2O (4) and 0.5 mg/ml of microencapsulated lycopene (5). Scale bar ΙΟΟμιτι.
Figure 3. Panel A - C. trachomatis cells within a vacuole of the infected macrophages, 48h, χ 17,000. Panel B - Infected microphages + oil form lycopene, 48h, x6,300. Fragments of damaged Chlamydia reticulate body. Panel C - Infected microphages + microencapsulated lycopene, 48h, χ 17,000. Fragmented reticulate body and lysis destroyed Chlamydia bacteria.
Figure 4. Reversibility of the apparent decline in lycopene concentration in inflammatory environment, in ex vivo experimental, human blood serum. Figure 5. Box and whisker analysis of serum lycopene levels.
Figure 6. Box and whisker analysis of serum LDL.
Figure 7 Inflammatory oxidative damage (A) and oxidized LDL (B) in patients treated with lycopene.
Figure 8. Changes in the gut microbiota after daily ingestion of 7mg GA lycopene for 4 weeks. Panel A - analysis of the most powerful probiotics in the gut. Panel B - analysis of health negative, potentially pathogenic bacteria. The results demonstrate a significant increase in probiotic bacterial species such as lactobacilli in the gut after administration of the GA lycopene.
Figure 9. Changes in the gut microbiota after daily ingestion of 30mg GA lycopene for 4 weeks. Panel A - analysis of the most powerful probiotics in the gut. Panel B - analysis of health negative, potentially pathogenic bacteria. The results demonstrate a significant increase in levels of probiotic bacterial species such as lactobacilli in the gut after administration of the GA lycopene.
Detailed Description of the Invention
Herein, any reference to a term in the singular also encompasses its plural.
Where the term "comprising", "comprise" or "comprises" is used in a particular embodiment, also encompassed are the embodiments wherein said term is substituted for "consisting of, "consist of or "consists of respectively, as well as embodiments where the term "comprising", "comprise" or "comprises" is substituted for "consisting essentially of, "consist essentially of or "consists essentially of respectively. Any reference to a numerical range or single numerical value also includes values that are about that range or single value, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Anywhere herein where a compound is referred to, if a salt of such compound may also be employed, that is also encompassed in the invention, particularly the use of such physiological acceptable salts. Where a given entity is referred to herein for use in a particular method, the method itself is also provided as is use of the entity in the manufacture of a medicament for use in such a method.
Carotenoids The invention provides compositions comprising one or more carotenoids. The compositions of the invention may include any carotenoid. Carotenoid compounds are a class of tetraterpenoids which contain long polyene chains. Carotenoids include carotenes, such as beta-carotene, alpha-carotene, zeto-carotene, and lycopene and related molecules, including l -HO-3', 4'-didehydrolycopene, 3, l '-(HO)2 -gamma-carotene, 1, 1'- (HO)2-3, 4, 3', 4'-tetradehydrolycopene, 1 , l'-(HO)2-3, 4-didehydrolycopene.
Carotenoids also include xanthophylls such as lutein, meso-zeaxanthin, zeaxanthin and astaxanthin.
Other suitable carotenoid compounds which may be used as described herein include hydrocarbons, such as lycopersene (7,8, 1 1, 12, 15,7', 8', 1 Γ, 12', 15'-decahydro-Y,y- carotene), phytofluene, hexahydrolycopene (15-cz's-7,8, l 1 , 12,7', 8'-hexahydro-y,Y- carotene), torulene (3',4'-didehydro-P,y-carotene) and a-zeacarotene (7',8'-dihydro-s,y- carotene); alcohols, such as alloxanthin, cynthiaxanthin, pectenoxanthin,
cryptomonaxanthin, ((3r,3'r)-7,8,7',8'-tetradehydro-P,P-carotene-3,3'-diol), crustaxanthin (P,-carotene-3,4,3',4'-tetrol), gazaniaxanthin ((3r)-5'-cis-P,y-caroten-3-ol), oh- chlorobactene (l ',2'-dihydro-f,Y-caroten-l '-ol), loroxanthin (P,s-carotene-3, 19,3'-triol), lycoxanthin (Y,y-caroten-16-ol), rhodopin (l,2-dihydro-y,Y-caroten-l-ol), rhodopinol (aka warmingol; 13-c/s-l,2-dihydro-y,y-carotene-l ,20-diol), saproxanthin (3',4'-didehydro- l',2'-dihydro-P,Y-carotene-3, l '-diol) and zeaxanthin; glycosides, such as oscillaxanthin (2,2'-bis(P-l-rhamnopyranosyloxy)-3, 4,3', 4'-tetradehydro- 1 ,2, 1 ',2'-tetrahy dro-γ,γ- carotene-l , l'-diol), and phleixanthophyll (l'-( -d-glucopyranosyloxy)-3',4'-didehydro- l',2'-dihydro-P,Y-caroten-2'-ol); ethers, such as rhodovibrin (l'-methoxy-3',4'- didehydro-l,2, l ',2'-tetrahydro-Y,Y-caroten- l -ol) and spheroidene (l -methoxy-3,4- didehydro-l,2,7',8'-tetrahydro-y,Y-carotene), epoxides, such as diadinoxanthin (5,6- epoxy-7',8'-didehydro-5,6-dihydro— carotene-3,3-diol), luteoxanthin (5,6: 5',8'-diepoxy- 5,6,5',8'-tetrahydro- , -carotene-3,3'-diol), mutatoxanthin, citroxanthin, zeaxanthin (furanoxide 5,8-epoxy-5,8-dihydro- , -carotene-3,3'-diol), neochrome (5',8'-epoxy-6,7- didehydro-5,6,5',8'-tetrahydro-P,P-carotene-3,5,3'-triol), foliachrome, trollichrome, and vaucheriaxanthin (5',6'-epoxy-6,7-didehydro-5,6,5',6'-tetrahydro- ,P-carotene-3,5, 19,3'- tetrol); aldehydes, such as rhodopinal, wamingone (13-cis- l -hydroxy-l,2-dihydro-Y,Y- caroten-20-al), torularhodinaldehyde (3',4'-didehydro-P,Y-caroten-16'-al); acids and acid esters, such as torularhodin (3',4'-didehydro-P,Y-caroten-16'-oic acid) and torularhodin methyl ester (methyl 3',4'-didehydro- ,Y-caroten-16'-oate); ketones, such as astaxanthin, canthaxanthin (aka aphanicin), chlorellaxanthin (P,P-carotene-4,4'-dione), capsanthin ((3r,3's,5'r)-3,3'-dihydroxy-P,K-caroten-6'-one), capsorubin ((3s,5r,3's,5'r)-3,3'- dihydroxy-K,K-carotene-6,6'-dione), cryptocapsin ((3'r,5'r)-3'-hydroxy-P,K-caroten-6'- one), 2,2'-diketospirilloxanthin (l,r-dimetrioxy-3,4,3',4'-tetradehydro-l,2,r,2'- tetrahydro-Y,y-carotene-2,2'-dione), flexixanthin (3, l'-dihydroxy-3',4'-didehydro-l',2'- dihydro- ,y-caroten-4-one), 3-oh-canthaxanthin (aka adonirubin; aka phoenicoxanthin; 3-hydroxy-P,P-carotene-4,4'-dione), hydroxyspheriodenone (l'-hydroxy- l-methoxy-3,4- didehydro-l,2, ,2',7',8'-hexahydro-y,Y-caroten-2-one), okenone (l'-methoxy- ,2'- dihydro-c,Y-caroten-4'-one), pectenolone (3,3'-dihydroxy-7',8'-didehydro-P,P-caroten-4- one), phoeniconone (aka dehydroadonirubin; 3-hydroxy-2,3-didehydn>P,P-carotene- 4,4'-dione), phoenicopterone (P,s-caroten-4-one), rubixanthone (3-hydroxy-P,y-caroten- 4'-one), siphonaxanthin (3, 19,3'-trihydroxy-7,8-dihydro-P,e-caroten-8-one); esters of alcohols, such as astacein (3,3'-bispalmitoyloxy-2,3,2',3'-tetradehydro-P,P-carotene-4,4'- dione or 3,3'-dihydroxy-2,3,2',3'-tetradehydro-P,P-carotene-4,4'-dione dipalmitate), fucoxanthin (3'-acetoxy-5,6-epoxy-3,5'-dihydroxy-6',7'-didehydro-5,6,7,8,5',6'- hexahydro-P,P-caroten-8-one), isofucoxanthin (3'-acetoxy-3,5,5'-trihydroxy-6',7'- didehydro-5,8,5',6'-tetrahydro-P,P-caroten-8-one), physalien, zeaxanthin dipalmitate ((3r,3'r)-3,3'-bispalmitoyloxy- , -carotene or (3r,3'r)- , -carotene-3,3'-diol dipalmitate) and siphonein (3,3'-dihydroxy-19-lauroyloxy-7,8-dihydro-P,e-caroten-8-one or 3,19,3'- trihydroxy-7,8-dihydro-P,s-caroten-8-one 19-laurate); apo carotenoids, such as P-apo-2'- carotenal (3',4'-didehydro-2'-apo-b-caroten-2'-al), apo-2-lycopenal, apo-6'-lycopenal (6'- apo-y-caroten-6'-al), azafrinaldehyde (5,6-dihydroxy-5,6-dihydro-10'-apo- -caroten-10'- al), bixin (6'-methyl hydrogen 9'-cis-6,6'-diapocarotene-6,6'-dioate), citranaxanthin (5',6'-dihydro-5'-apo-P-caroten-6'-one or 5',6'-dihydro-5'-apo-18'-nor-P-caroten-6'-one or 6'-methyl-6'-apo-P-caroten-6'-one), crocetin (8,8'-diapo-8,8'-carotenedioic acid), crocetinsemialdehyde (8'-oxo-8,8'-diapo-8-carotenoic acid), crocin (digentiobiosyl 8,8'- diapo-8,8'-carotenedioate), hopkinsiaxanthin (3-hydroxy-7,8-didehydro-7',8'-dihydro-7'- apo-b-carotene-4,8'-dione or 3-hydroxy-8'-methyl-7,8-didehydro-8'-apo-b-carotene-4,8'- dione), methyl apo-6'-lycopenoate (methyl 6'-apo-y-caroten-6'-oate), paracentrone (3,5- dihydroxy-6,7-didehydro-5,6,7',8'-tetrahydro-7'-apo-b-caroten-8'-one or 3,5-dihydroxy- 8'-methyl-6,7-didehydro-5,6-dihydro-8'-apo-b-caroten-8'-one) and sintaxanthin (7',8'- dihydro-7'-apo-b-caroten-8'-one or 8'-methyl-8'-apo-b-caroten-8'-one); nor and seco carotenoids, such as actinioerythrin (3,3'-bisacyloxy-2,2'-dinor-b,b-carotene-4,4'-dione), β-carotenone (5,6:5',6'-diseco-b,b-carotene-5,6,5',6'-tetrone), peridinin (3'-acetoxy-5,6- epoxy-3,5'-dihydroxy-6',7'-didehydro-5,6,5',6'-tetrahydro-12', 13',20'-trinor-b,b-caroten- 19,11 -olide), pyrrhoxanthininol (5,6-epoxy-3,3'-dihydroxy-7',8'-didehydro-5,6-dihydro- 12', 13',20'-trinor-b,b-caroten-19, l 1 -olide), semi-a-carotenone (5,6-seco-b,e-carotene- 5,6-dione), semi-P-carotenone (5,6-seco-b,b-carotene-5,6-dione or 5',6'-seco-b,b- carotene-5',6'-dione) and triphasiaxanthin (3-hydroxysemi-b-carotenone 3'-hydroxy-5,6- seco-b,b-carotene-5,6-dione or 3-hydroxy-5',6'-seco-b,b-carotene-5',6'-dione); retro carotenoids and retro apo carotenoids, such as eschscholtzxanthin (4',5'-didehydro-4,5'- retro-b,b-carotene-3,3'-diol), eschscholtzxanthone (3'-hydroxy-4',5'-didehydro-4,5'-retro- b,b-caroten-3-one), rhodoxanthin (4',5'-didehydro-4,5'-retro-b,b-carotene-3,3'-dione) and tangeraxanthin (3-hydroxy-5'-methyl-4,5'-retro-5'-apo-b-caroten-5'-one or 3-hydroxy- 4,5'-retro-5'-apo-b-caroten-5'-one); and higher carotenoids, such as nonaprenoxanthin (2-(4-hydroxy-3-methyl-2-butenyl)-7',8', 1 , 12'-tetrahydro-e,y-carotene),
decaprenoxanthin (2,2'-bis(4-hydroxy-3-methyl-2-butenyl)-e,e-carotene), c.p. 450 (2-[4- hydroxy-3-(hydroxymethyl)-2-butenyl]-2'-(3 -methyl -2-butenyl)-b,b-carotene), c.p. 473 (2l-(4-hydroxy-3-methyl-2-butenyl)-2-(3-methyl-2-butenyl)-3',4'-didehydro-r,2l- dihydro-b,y-caroten-l'-ol) and bacterioruberin (2,2'-bis(3-hydroxy-3-methylbutyl)- 3,4,3',4'-tetradehydro-l,2,r,2'-tetrahydro-y,y-carotene-l, -dio).
A composition as described herein may contain a single carotenoid compound or more than one carotenoid compound. For instance, a composition as described herein may comprise, one, two, three, four, five, six or more carotenoids, such as any of those numbers of the specific carotenoids specified here. A composition may in one instance comprise one, two or three carotenoids, for instance any of the specific carotenoids specified herein. In one preferred instance, a composition may comprise one carotenoid, for example where the carotenoids is any of those specified herein. In one instance, each carotenoid may be, for instance, present in a range of different isomeric forms.
In one particularly preferred embodiment the carotenoid compound is lycopene, hence in any of the embodiments described herein where a carotenoid is present, in a preferred instance the carotenoid is lycopene, or where more than one carotenoid is present lycopene may be one of the carotenoids present. Lycopene is an open-chain unsaturated C40 carotenoid of structure I (Chemical Abstracts Service Registry Number 502-65-8).
Structure I
Lycopene occurs naturally in plants such as tomatoes, guava, rosehip, watermelon and pink grapefruit. The lycopene of the invention may comprise one or more different isomers. For example, lycopene may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% , at least 80% , at least 90% , or at least 95% (Z)-isomers, (all-E)-isomers, or cz's-isomers, such as 5-cis- or 9-cis- or 13-c/s-isomers, which have improved bioavailability relative to trans isomers. Trans isomers may isomerise into cis forms in vivo, or during storage and processing.
The carotenoid compounds of the invention may be natural i .e., obtained from a natural source, for example, extracted from a plant, such as a tomato or melon. A range of methods for extracting, concentrating and/or purifying carotenoids from plants are known in the art. For example, solvent extraction using ethanol, DMSO, ethyl acetate, hexane, acetone, soya or other vegetable oil, or non-vegetable oils may be employed. A carotenoid compound may be isolated i.e. free or substantially free of other molecules found in its natural source or environment.
Carotenoid compounds for use as described herein may be synthetic i.e.
produced by artificial means, for example, by chemical synthesis or fermentation. A range of methods for chemical synthesis of lycopene and other carotenoids are known in the art. For example, a three-stage chemical synthesis based on the standard Wittig olefination reaction scheme for carotenoid synthesis may be employed, in which an organic solution of C15 phosphonium methanesulfonate in dichloromethane (DCM) and an organic solution of Cio dialdehyde in toluene are produced, and the two organic solutions are gradually combined with sodium methoxide solution and undergo a condensation reaction to form crude lycopene. The crude lycopene may then be purified using routine techniques, for example by adding glacial acetic acid and deionized water to the mixture, stirring vigorously, allowing the aqueous and organic phases to separate, and extracting the organic phase containing DCM and crude lycopene with water.
Methanol is added to the organic phase and the DCM removed via distillation under reduced pressure. The crude methanolic lycopene solution is then be heated and cooled to crystalline slurry that is filtered and washed with methanol. The lycopene crystals may then be recrystallized and dried under heated nitrogen. Synthetic carotenoids, such as lycopene, are also available from commercial suppliers (e.g. BASF Corp, NJ USA, DSM Nutritional Products, Basel, CH).
Synthetic carotenoids may comprise an increased proportion of cis isomers relative to natural carotenoids. For example, synthetic forms of carotenoids such as lycopene may be up to 25% 5-cis, 1% 9-cis, 1% 13-cis, and 3% other cis isomers, whilst natural forms of carotenoids, for example lycopene produced by tomatoes, may be 3-5% 5-cis, 0-1% 9-cis, 1% 13-cis, and <1% other cis isomers. Since cis-carotenoids, such as cis-lycopene, have increased bioavailability relative to trans-carotenoids, such as trans- lycopene, synthetic carotenoids may be preferred in some embodiments.
Derivatives of carotenoids as described above may be produced by chemical synthesis analogous to the synthesis described above; by chemical modification of natural carotenoids extracted from plant material or by microbial, yeast, algal, or fungal fermentation. For example, lycopene may be produced by fermentation of the fungus Blakeslea trispora (e.g. Lyconat™, Vitatene SA).
The composition may comprise 0.05 to 90% by weight of the carotenoid compound, preferably 0.1% to 10% by weight. For example, the population may be 0.01% or more, 0.05% or more, 0.1% or more, 0.2% or more, 0.5% or more, 1% or more, 10% or more, or 20% or more by weight of carotenoid compound. The population may be up to 90%, up to 80%, up to 70%, up to 60% up to 50%, up to 40%, up to 30%, up to 20% or up to 10% by weight of carotenoid compound.
The composition may contain the same or similar amounts of carotenoid compound or the amount of carotenoid compound may vary between particles in the population. Each carotenoid particle in the population may comprise 0.05 to 90% by weight of carotenoid compound. For example, each carotenoid particle in the population may be 0.05% or more, 0.1% or more, 1% or more, 10% or more, or 20% or more by weight of carotenoid molecules. Each carotenoid particle may be up to 90%, up to 80%, up to 70%, up to 60% up to 50%, up to 40% or up to 30%, up to 90% or more by weight of carotenoid compound.
In one particularly preferred embodiment of the invention, the carotenoid employed is one or more selected from lycopene, meso-zeaxanthin, zeaxanthin, astaxanthin, canthaxanthin and/or lutein. In a particularly preferred embodiment, the carotenoid lycopene is employed. Phosphatidylcholine (PC)
The compositions of the invention further comprise phosphatidylcholine (PC). Any phosphatidylcholine may be used in the invention. Phosphatidylcholine is a component of cell membrane bilayers and the main phospholipid circulating in the plasma. Phosphatidylcholine is highly absorbable and supplies choline which is needed to facilitate movement of fats and oils across the cell membrane, and to maintain the cell membranes. Phosphatidylcholine acts as a chaperone molecule for the delivery of carotenoids in the practice of the present invention.
The phosphatidylcholine of the invention may be obtained from various sources such as egg yolk or soybeans. The term "phosphatidylcholine" is understood herein to include lecithin, l,2-Diacyl-sn-glycero-3-phosphocholine, choline phosphatide, lecithol, posphatidyl-N-trimethylethanolamine, phospholutein. In certain embodiments, the phosphatidylcholine is l,2-diacyl-sw-glycero-3-phosphocholine, 10- (perfluorobutyl)decyl phosphatidylcholine, dioleoyl phosphatidylcholine. Typically, the phosphatidylcholine of the invention is a commercially available purified form.
Phosphatidylcholine is a glycerophosphocholine compound of structure II having O-acyl substituents at both the 1- and 2-positions of the glycerol.
The composition of the invention comprises a carotenoid and
phosphatidylcholine at weight ratio of at least about 1 : about 0.1. After extensive studies, the inventors have shown that inclusion of carotenoid to phosphatidylcholine at least this weight ratio is essential for anti -bacterial and/or anti-oxidative effects. In certain embodiments, the composition of the invention comprises a carotenoid and phosphatidylcholine at weight ratio of at least about 1.00 : 0.14.
Typically, the composition comprises a carotenoid and phosphatidylcholine at a weight ratio of at least about 1 : 0.2, 1 : 0.4, 1 : 0.6, 1 : 0.8, 1 : 1, 1 : 1.2, 1 : 1.4, 1 : 1.6, 1 : 1.8, 1 : 2, 1 : 3, 1 : 4 , 1 : 5 , 1 : 6 or 1 : 7 or more. In a preferred embodiment, the weight ratio of carotenoid to phosphatidylcholine is at least about 1 : about 1. In even more preferred embodiments, the composition of the invention comprises a carotenoid and phosphatidylcholine at a weight ratio of at least about 1.0 : 1.4. The inventors have shown that, unexpectedly, inclusion of a carotenoid with phosphatidylcholine at a weight ratio of at least about 1 : 1 leads to a significant improvement in anti -bacterial and/or anti-oxidative effects.
In any of the compositions described herein the weight ratio may be expressed as mg/mg.
Typically, the composition of the invention includes at least about lmg, 2mg,
3mg, 4mg, 5mg, 6mg, 7mg or more of carotenoid, and so at least about O. lmg, 0.2mg, 0.3mg, 0.4mg, 0.5mg, 0.6mg, 0.7mg or more of phosphatidylcholine respectively. In certain embodiments, the composition of the invention includes at least about 4mg of carotenoid and at least about lmg, 2mg, 3mg, 4mg, 5mg, 6mg or more of
phosphatidylcholine. In certain embodiments, the composition of the invention includes at least about 7mg of carotenoid and at least about 7mg of phosphatidylcholine.
Preferably, the composition of the invention comprises at least about 7mg of carotenoid and at least about lOmg of phosphatidylcholine.
In preferred embodiments, the carotenoid is lycopene. In preferred
embodiments, the ratio of lycopene to phosphatidylcholine in the composition of the invention includes any of the ratios discussed above. For example, in preferred embodiments the composition of the invention comprises lycopene and
phosphatidylcholine at a weight ratio of at least about 1 to about 0.1, typically at least about 1 : 0.2, 1 : 0.4, 1 : 0.6, 1 : 0.8, 1 : 1, 1 : 1.2, 1 : 1.4, 1 : 1.6, 1 : 1.8, 1 : 2, 1 : 3, 1 : 4 , 1 : 5, 1 : 6 or 1 : 7 or more. In a preferred embodiment, the ratio of lycopene to phosphatidylcholine is at least about 1 : 1 (i.e., at a weight ratio of at least about 1.0 : 1.4). The inventors have shown that, unexpectedly, inclusion of lycopene with phosphatidylcholine at a weight ratio of at least about 1 : 1 leads to a significant improvement in anti-bacterial and/or anti-oxidative effects. In any of these
compositions the weight ratio may be expressed as mg/mg. Preferably, the composition of the invention comprises at least about 7mg of lycopene and at least about lOmg of phosphatidylcholine.
Ascorbic acid In preferred embodiments, the composition of the invention further comprises ascorbic acid (vitamin C). Ascorbic acid is a naturally occurring organic compound with antioxidant properties The inventors have shown that, unexpectedly, the addition of ascorbic acid to the composition of the invention acts to protect and extend the active life of carotenoid. The inventors have also shown that, unexpectedly, the addition of ascorbic acid to the composition of the invention leads to synergistic anti -bacterial and/or anti-oxidative effects. Although the inventors tested a number of different antioxidants including tocpherol, glutathione, propyl gallate, catalase and superoxide dismutase, only the addition of ascorbic acid led to such effects.
In certain embodiments, the composition of the invention comprises a carotenoid and ascorbic acid at a weight ratio of at least about 1 : about 5. For example, in preferred embodiments the composition of the invention comprises a carotenoid (such as lycopene) and ascorbic acid at a weight ratio of at least about 1 to about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more.
In preferred embodiments, the composition of the invention comprises a carotenoid (such as lycopene), phosphatidylcholine and ascorbic acid at a weight ratio of at least about 1 : about 0.1 : about 5. In preferred embodiments, the composition of the invention comprises a carotenoid (such as lycopene), phosphatidylcholine and ascorbic acid at a weight ratio of at least about 1 : 0.1 : 6, 1 : 0.1 : 7, 1 : 0.1 : 8, 1 : 0.1 : 9, 1 : 0.1 : 10, 1 : 0.1 : 11, 1 : 0.1 : 12, 1 : 0.1 : 13, 1 : 0.1 : 14, 1 : 0.1 : 15 or more. In even more preferred embodiments, the ratio of carotenoid to phosphatidylcholine to ascorbic acid in the composition of the invention is at least about 1 : 1 : 5, 1 : 1 : 6, 1 : 1 : 7, 1 : 1 : 8, 1 : 1 : 9, 1 : 1 : 10, 1 : 1 : 11, 1 : 1 : 12, 1 : 1 : 13, 1 : 1 : 14, 1 : 1 : 15 or more. In preferred embodiments, the carotenoid is lycopene, lutein-meso-zeaxanthin, zeaxanthin, astaxanthin, or any combination thereof. Typically, the carotenoid is lycopene.
Typically, the composition of the invention includes at least about 4mg, 5mg, 6mg, 7mg or more of carotenoid, and so at least about 0.4mg, 0.5mg, 0.6mg, 0.7mg or more of phosphatidylcholine respectively and at least about 20 mg, 25mg, 30mg, 35mg or more ascorbic acid respectively. In certain embodiments, the composition of the invention includes at least about 7mg of carotenoid, at least about lOmg of
phosphatidylcholine and at least about 20mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, 70mg or more ascorbic acid.
In preferred embodiments, the carotenoid is lycopene. In preferred
embodiments, the ratio of lycopene to phosphatidylcholine to ascorbic acid in the composition of the invention includes any of the ratios discussed above. For example, in preferred embodiments the composition of the invention comprises lycopene, phosphatidylcholine and ascorbic acid at weight ratio of at least about 1 : 1 : 7. For example, the composition of the invention may comprise at least 7mg or more lycopene, at least lOmg or more phosphatidylcholine and at least 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg or more ascorbic acid.
In any of the compositions described herein further comprising ascorbic acid the weight ratio may be expressed as mg/mg, or mg/mg/mg.
The invention provides compositions as described herein, including the following.
A composition comprising a carotenoid and phosphatidylcholine (PC) at a weight ratio of at least about 1 : 0.14, preferably wherein the weight ratio is mg/mg.
A composition comprising lycopene and phosphatidylcholine (PC) at a weight ratio of at least about 1 : 0.14, preferably wherein the weight ratio is mg/mg.
A composition comprising about 7mg lycopene and about lmg
phosphatidylcholine (PC).
A composition comprising a carotenoid, phosphatidylcholine (PC) and ascorbic acid at a weight ratio of at least about 1 : 0.14 : 7.1, preferably wherein the weight ratio is mg/mg.
A composition comprising lycopene, phosphatidylcholine (PC) and ascorbic acid at a weight ratio of at least about 1 : 0.14 : 7.1, preferably wherein the weight ratio is mg/mg.
A composition comprising about 7mg lycopene, about lmg phosphatidylcholine (PC) and about 50mg ascorbic acid.
A composition comprising a carotenoid and phosphatidylcholine (PC) at a weight ratio of at least about 1 : 1.4, preferably wherein the weight ratio is mg/mg.
A composition comprising lycopene and phosphatidylcholine (PC) at a weight ratio of at least about 1 : 1.4, preferably wherein the weight ratio is mg/mg.
A composition comprising about 7mg lycopene and about lOmg
phosphatidylcholine (PC).
A composition comprising a carotenoid, phosphatidylcholine (PC) and ascorbic acid at a weight ratio of at least about 1 : 1.4 : 7.1, preferably wherein the weight ratio is mg/mg. A composition comprising lycopene, phosphatidylcholine (PC) and ascorbic acid at a weight ratio of at least about 1 : 1.4 : 7.1, preferably wherein the weight ratio is mg/mg.
A composition comprising about 7mg lycopene, about lOmg
phosphatidylcholine (PC) and about 50mg ascorbic acid.
Formulations
Compositions of the invention may typically comprise, for instance, one or more pharmaceutically or nutraceutically acceptable carriers, excipients, buffers, adjuvants, stabilizers, or other materials, as described herein.
The term "pharmaceutically acceptable" as used herein typically pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.
The term "nutraceutically acceptable" as used herein typically pertains to compounds, materials, compositions, and/or dosage forms which are in common or widespread usage in food and dietary products and are generally considered non-toxic, for example, compounds may have the US FDA designation "GRAS" (Generally Recognized as Safe), or equivalent food additive status in other jurisdictions.
The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy, food science or nutrition. Such methods include the step of incorporating a carrier which may constitute one or more accessory ingredients.
Formulations may be in the form of food products, beverages, liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, lozenges, granules, powders, capsules, cachets, pills, ampoules, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols. A composition of the invention may be preferably in a form which is suitable for administration orally for delivery via the gastro-intestinal tract. Formulations suitable for oral administration (e.g., by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste. In one particularly preferred instance, a formulation of the invention may be provided in a capsule, hence the present invention provides a capsule comprising a composition of the invention. Formulations of the invention will, in particular, be suitable for oral administration. Oral administration is the most preferred route of administration for the invention.
A tablet may be made by conventional means, e.g. , compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g., povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica); disintegrants (e.g., sodium starch glycolate, cross- linked povidone, cross-linked sodium carboxymethyl cellulose); surface-active or dispersing or wetting agents (e.g., sodium lauryl sulfate); and preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid). Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Compositions for oral administration may further comprise sweeteners, texture modifiers, colorings and flavorings.
As used herein, the phrase "pharmaceutical composition" encompasses
"nutritional compositions" or "nutritional supplements." However, any of the compositions described herein may be provided as a nutritional composition or supplement. A composition of the invention may be a "nutraceutical" and that term may include: food products, foodstuffs, dietary supplements, nutritional supplements or a supplement composition for a food product or a foodstuff.
The composition of the invention may be provided in an enteric soft capsule shell. The shell of a capsule may be, for instance, made of naturally occurring ingredients. The composition of the invention may be taken by an individual after a meal. A composition of the invention may be, for instance, given on a daily basis, for examples after meals, or for instance at any appropriate intervals such as at weekly, fortnightly or monthly intervals.
In one preferred instance, a composition of the invention may be one that does not need to be prescribed by a doctor to be administered. For instance, in a preferred embodiment of the invention a composition of the invention is a supplement. It may be that the composition is one sold as an over the counter medicine. It may be that the composition is a nutraceutical. In one preferred instance, a composition of the invention is not one that requires regulatory approval prior to marketing. However, the invention may be also applied to pharmaceutical products, such as those that have to be prescribed. A composition of the invention may be one with an active agent such as that the composition requires regulatory approval.
As used herein, the term "effective amount" refers to a quantity sufficient to achieve a desired effect and in particular a desired therapeutic and/or prophylactic effect. A "therapeutically effective amount" may be, for instance, the amount needed to reduce or eliminate the presence, frequency, or severity of one or more signs, or symptoms of the conditions mentioned herein. In some embodiments, the amount of a formulation administered to the subject will depend on the type, degree, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled person will be able to determine appropriate dosages depending on these and other factors. Also provided herein is an oral pharmaceutical dosage form comprising any of the compositions described herein, particular a capsule comprising one of the compositions described herein, particularly a capsule provided a daily dose of the composition as described herein.
Any suitable amount of a composition of the invention may be administered. For instance, as discussed above the carotenoid may be administered at an amount from about lmg to about 50 mg, preferably from about 1 to about 25 mg, such as from about 1 to about 15 mg, such as from about 1 to about 10 mg. In a preferred embodiment, a composition provides at least about 4 mg of carotenoid. A composition of the invention may be in unit dose form, and so provide the recommended daily amount of the carotenoid.
In certain embodiments, the invention provides a method of producing a pharmaceutical formulation comprising formulating composition components as defined herein, together with a pharmaceutically acceptable excipient. In certain embodiments, the invention provides a method of producing a nutraceutical formulation comprising formulating composition components as defined herein, together with a nutraceutically acceptable excipient.
Individuals and Conditions to be treated
The invention provides a method of treating or preventing an obligate intracellular bacterial infection in an individual, comprising administering a
therapeutically or prophylactically effective amount of a composition of the invention, and thereby treating or preventing the infection. In other words, the invention provides a method of treating or preventing any bacterial infection wherein the bacteria cannot reproduce outside it' s host cell, meaning that it's reproduction is entirely reliant on intracellular resources. In certain embodiments, the bacterial infection is caused by Chlamydiae, Rickettsia, Ehrlichia and/or Coxiella.
In the methods described herein, the composition may act to reduce or inhibit the formation of infective progeny of the obligate intracellular bacteria in the individual. In other words, the composition may act to reduce or inhibit lysis of eukaryotic cells and the release of infective progeny to neighboring cells. The composition may therefore act to prevent the subsequent repeat of cellular infections in adjacent epitheliocytes. In certain embodiments, the composition acts to reduce or inhibit the release of infective progeny into the bloodstream of the individual.
The invention therefore provides a method of inhibiting the formation of infective progeny of obligate intracellular bacteria in an individual, the method comprising administering a therapeutically or prophylactically effective amount of a composition of the invention, and thereby reducing or inhibiting the formation of infective progeny of obligate intracellular bacteria in the individual. T
he invention also provides a method of inhibiting the release of infective progeny of obligate intracellular bacteria into the bloodstream of an individual, the method comprising administering a therapeutically or prophylactically effective amount of a composition of the invention, and thereby reducing or inhibiting the release of infective progeny into the bloodstream of the individual.
Herein, "reducing or inhibiting the formation of infective progeny" means reducing or inhibiting the formation or release of infective progeny in the individual as compared to one or more control individual(s). In other words, "reducing or inhibiting" the formation or release of infective progeny is understood to mean a statistically significant reduction or inhibition in the formation or release of infective progeny in the individual as compared to one or more control individual(s).
In certain embodiments, the number of infective progeny being formed and/or released in the individual may be reduced by at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more as compared to one or more control individual(s). The control individual may be same individual, prior to or at an earlier stage of treatment with the composition of the invention. Alternatively, the control individual may be a different individual, i.e., receiving a control composition that includes a carotenoid but does not include PC and/or ascorbic acid.
Any standard method may be used to determine the number of infective progeny being produced or released in the individual, such as those described in the Examples. For instance, in clinical cases where the obligate intracellular bacteria are not present in the circulating blood and collection of tissue biopsy of the infected tissues is limited or not practical, the reduction or inhibition of the formation or release of infective progeny can be determined by the standard diagnostic practice of measuring titers of specific antibodies against the obligate intracellular bacteria. As discussed further below, the invention therefore further provides a method of measuring the titers of antibodies against the obligate intracellular bacteria in a sample taken from the individual, as compared to one or more control samples.
The control sample may be from the same individual, prior to or at an earlier stage of treatment with the composition of the invention. Alternatively, the control sample may be from a different individual receiving a control composition that includes a carotenoid but does not include PC and/or ascorbic acid. The sample may be from any tissue or bodily fluid. The sample typically comprises a body fluid and/or cells of the individual and may, for example, be obtained using a needle. The sample may be, or be derived from, plasma, serum or whole blood from the individual. The sample is typically processed prior to being assayed, for example by centrifugation or by passage through a membrane that filters out unwanted molecules or cells, such as red blood cells. The sample may be measured immediately upon being taken. The sample may also be stored prior to assay, preferably below -70°C.
The titer of antibodies in the one or more sample(s) may be measured by any standard technique. For example, ELISA assays may be used to measure the antibodies against the obligate intracellular bacteria. Typically, the antibodies are against a surface protein of the obligate intracellular bacteria. Typically, the antibodies are against Chlamydophilia. More typically, the antibodies are anti -Chlamydia IgG or IgA.
Obligate intracellular bacterial infections such as Chlamydia and/or conditions associated with obligate intracellular bacterial infections are associated with depressed tissue oxygenation and/or impaired flow-mediated dilation in the infected individual. The administration of a therapeutically or prophylactically effective amount of a composition of the invention may increase depressed tissue oxygenation and/or impaired flow-mediated dilation in the individual, thereby restoring such parameters to their physiological norm.
Herein, "increasing" depressed tissue oxygenation and/or impaired flow- mediated dilation means increasing depressed tissue oxygenation and/or increasing impaired flow-mediated dilation in the individual as compared to one or more control individual(s). In other words, "increasing" tissue oxygenation and/or flow-mediated dilation is understood to mean a statistically significant increase in tissue oxygenation and/or flow-mediated dilation in the individual as compared to one or more control individual(s). For example, tissue oxygenation and/or flow-mediated dilation may be increased by at least about 5%, 10%, 20%, 30%, 40% , 50%, 60% or more as compared to one or more control individuals. The control individual may be same individual, prior to or at an earlier stage of treatment with the composition as defined herein.
Alternatively, the control individual may be a different individual, i.e., receiving a control composition that includes a carotenoid such as lycopene but does not include PC and/or ascorbic acid.
Obligate intracellular bacterial infections such as Chlamydia and/or conditions associated with obligate intracellular bacterial infections are also associated with tissue hypoxia, impaired microcirculation, pre-tension and/or hypertension in the infected individual. The administration of a therapeutically or prophylactically effective amount of a composition of the invention may reduce or inhibit tissue hypoxia, impaired microcirculation, pre-tension and/or hypertension, thereby restoring such parameters to their physiological norm.
Any standard method may be used to determine tissue oxygenation, flow-mediated dilation, tissue hypoxia, microcirculation, pre-tension and/or hypertension in an individual, such as those described in the Examples. For instance, Near-Red
Spectroscopy, Laser Doppler Flowmetry, transcutaneous oxygen monitoring, magnetic resonance imaging and/or angiography may be used to measure such parameters. Typically, measurements are made from a sample taken from the individual, as compared to one or more control samples. Any sample may be used for such measurements, such as those described herein.
In preferred embodiments, the bacterial infection is caused by Chlamydiae. In more preferred embodiments, the bacterial infection is caused by Chlamydophilia, typically Chlamydophila psittaci, Chlamydophila trachomatis or Chlamydophila pneumoniae. In preferred embodiments, the composition used to treat or prevent such Chlamydial bacterial infections comprises lycopene and phosphatidylcholine (PC) at a weight ratio of at least 1 : 1 , preferably mg/mg. In even more preferred embodiments, the composition used to treat or prevent such Chlamydial infections further comprises ascorbic acid, typically at a weight ratio of at least 1 : 1 : 7, preferably mg/mg/mg.
The invention also provides a method of treating or preventing any condition associated with an obligate intracellular bacterial infection in an individual, comprising administering a therapeutically or prophylactically effective amount of a composition of the invention, and thereby treating or preventing the condition. In certain embodiments, the composition is selected from a metabolic syndrome, fatty liver, steatohepatitis, coronary heart disease (CHD), coronary vascular disease (CVD), atherosclerosis, cardiovascular pathology, cerebrovascular pathology and/or a neurodegenerative condition.
In certain embodiments, the individual that is treated is not infected with an obligate intracellular bacteria and/or the individual does not display any symptoms of the bacterial infection.
In preferred embodiments, the individual that is treated is infected with an obligate intracellular bacteria and/or the individual displays symptoms of the bacterial infection. Typically, an individual who is infected with an obligate intracellular bacteria and/or who displays symptoms of the bacterial infection has increased serum titer of antibodies against the bacterial infection, as compared to one or more control serum samples.
In preferred embodiments, the composition used to treat the condition associated with an obligate intracellular bacterial infection comprises lycopene and
phosphatidylcholine (PC), typically at a weight ratio of at least about 1 : 1, preferably mg/mg. In even more preferred embodiments, the composition further comprises ascorbic acid, typically at a weight ratio of at least about 1 : 1 : 7, preferably mg/mg/mg.
The invention also provides a method of treating or preventing a functional carotenoid deficiency in an individual, comprising administering a therapeutically or prophylactically effective amount of a composition of the invention, and thereby treating or preventing the functional carotenoid deficiency.
Any individual may have a functional carotenoid deficiency. For example, lipoprotein metabolism may be compromised by ageing, and individuals older than approximately 50 years may lose the ability to assemble new lipoproteins. This may lead to functional carotenoid deficiency, leading to age-associated subclinical inflammation and oxidation, especially in cells such as enterocytes and hepatocytes and associated tissues where these processes occur.
The invention therefore provides a method of increasing the bioavailability of carotenoids by administering a therapeutically or prophylactic effective amount of a composition of the invention.
Typically, the individual having a functional carotenoid deficiency is at least approximately 50, at least approximately 55, at least approximately 60, at least approximately 65, at least approximately 70 years in age or more.
In certain embodiments, the individual having a functional carotenoid deficiency is not infected with an obligate intracellular bacteria and/or the individual does not display any symptoms of the bacterial infection.
In preferred embodiments, the individual having a functional carotenoid deficiency is infected with an obligate intracellular bacteria and/or the individual displays symptoms of the bacterial infection. Typically, an individual who is infected with an obligate intracellular bacteria and/or who displays symptoms of the bacterial infection has increased serum titer of antibodies against the bacterial infection, as compared to one or more control serum samples.
In preferred embodiments, the composition used to treat functional carotenoid deficiency comprises lycopene and phosphatidylcholine (PC), typically at a weight ratio of at least about 1 : 1, preferably mg/mg. In even more preferred embodiments, the composition further comprises ascorbic acid, typically at a weight ratio of at least about 1 : 1 : 7, preferably mg/mg/mg.
Any composition of the invention as described above may be administered in the methods described herein. In certain embodiments, the compositions of the invention are employed as supplements, such as nutritional supplements or nutraceuticals. Hence, in one embodiment, the individual may be a healthy individual. In certain
embodiments, compositions of the invention may be used prophylactically, to help prevent or reduce the risk of developing an intracellular bacterial infection and/or a condition associated with an intracellular bacterial infection, such as any of those described above. Any of the methods discussed herein may be used to prevent, or delay the onset of, a condition, such as the conditions specified or to treat the condition once it has arisen in an individual. In some instances, treatment may include, for instance, elimination of a condition or reducing the severity of the condition. It may, for instance, involve elimination or reduction of a symptom or symptoms of the condition. Treatment may include bringing about regression of a disorder.
In one instance, the effect of administering a carotenoid (such as lycopene) and ascorbic acid is a synergistic effect than if the same amount of a composition was administered by adding a carotenoid (such as lycopene) or ascorbic acid individually with PC. For example, administration of lycopene in conjunction with ascorbic acid agent leads to an improved effect in preventing or treating the obligate intracellular bacterial infection and/or associated conditions, and so is greater than when lycopene is administered individually with PC.
The invention may be applied to any suitable individual. The individual may be an individual organism, a vertebrate, a mammal, or a human. In a particularly preferred instance, the invention is applied to a human. However, the invention may, for instance, also be applied to non-human animals, such a pets or commercial animals. Such animals include, for instance, dogs, cats, cattle, pigs and sheep. In some instances, the individual may be elderly, for instance over 50, 55, 60, 65, 70, 75 or 80 years of age. The subject may be male or female. In some instances, the subject is pregnant.
Methods of reducing serum titer of antibodies
The invention further provides a method of reducing the titer of antibodies against a bacterial infection in an individual, comprising administering a therapeutically or prophylactically effective amount of a composition of the invention.
The term "reducing" is understood herein to mean, for example, a statistically significant reduction in the titer of antibodies against a bacterial infection in a sample from an individual as compared to a control sample. In certain embodiments, the invention provides a method of reducing the titer of antibodies by at least about 45%, 50%, 55%, 60% or more as compared to one or more control samples. The control sample may be from the same individual, prior to or at an earlier stage of treatment with the composition of the invention. Alternatively, the control sample may be from a different individual receiving a control composition that includes a carotenoid but does not include PC and/or ascorbic acid. The sample may be from any tissue or bodily fluid. The sample typically comprises a body fluid and/or cells of the individual and may, for example, be obtained using a needle. The sample may be, or be derived from, plasma, serum or whole blood from the individual. The sample is typically processed prior to being assayed, for example by centrifugation or by passage through a membrane that filters out unwanted molecules or cells, such as red blood cells. The sample may be measured immediately upon being taken. The sample may also be typically stored prior to assay, preferably below -70°C.
Any method may be used to determine whether or not the titer of antibodies is reduced in the individual. For example, standard ELISA assays may be used to measure the titer of the antibodies against the bacterial infection. Typically, the antibodies are against a surface protein of the obligate intracellular bacteria. Typically, the bacterial infection is caused by Chlamydophilia. More typically, the antibodies are anti-Chlamydia IgG or IgA.
Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described. All documents mentioned in this specification are incorporated herein by reference in their entirety.
EXAMPLES
Example 1 - Cell Culture Infection 1. Materials and methods
Reagents. Stock oil solutions of lycopene (15%) was purchased from LycoRed (Switzerland) and kept at - 20°C. For studies in cultured cells the stock solution was dissolved in DMSO at concentrations 0.75, 1.5 and 3.0 mg/ml. Water dispersible microencapsulated lycopene was purchased from BASF. Its 10% suspension was mixed with DMEM at a final concentration of 5 mg/ml. Cell Lines. Cells were grown in 5% CO2 in DMEM supplemented with 2 mM glutamine and 10% FCS.
In Vitro Studies. C. trachomatis was initially propagated in McCoy cells and purified by Renografin gradient centrifugation as previously described [1]. Chlamydial titers were determined by infecting McCoy cells with 10-fold dilutions of thawed stock suspension. Purified elementary bodies (EB) of known titer were suspended in sucrose-phosphate- glutamic acid buffer (SPG) and used as inoculums for B IO.MLM cells. Cells were grown in 24 well plates until a confluence rate of 80% was reached. BIO.MLM plates were infected with C. trachomatis at multiplicity of infection (MOI) of 30 in DMEM with 5% FBS without cycloheximide and centrifuged for 0.5 hour at 1500 g. After 1 hour of incubation the cell monolayers were washed with DMEM and lycopene additions were made. Oil suspension of lycopene diluted with DMSO was tested at the final concentration of lycopene of 0.75, 1.5 and 3.0 μg/mL in medium. Lycopene microencapsulated in dextran was added in medium up to the final concentration of lycopene of 0.125, 0.25 and 0.5 mg/ml of DMEM. Control cells received additions of solvents or microencapsulating substances (DMSO, olive oil or cyclodextrin) as singular ingredients.
Immunofluorescence Staining. Infected BIO.MLM monolayers grown on coverslips in 24-well plates for 24, 30 and 42 hours were fixed with methanol. Permeabilised cells were stained by direct immunofluorescence (IF) using FITC— conjugated species-specific monoclonal antibody against the major outer-membrane protein of C. trachomatis (Bio-Rad). Inclusion-containing cells were visualized using a Nikon Eclipse 50i fluorescence microscope at x200 and x 1000 magnification.
Assessment of Infective Progeny. For the assessment of infective progeny accumulation, B IO.MLM cells were harvested for reinfection after 42 h of cultivation, as described before [22]. Serial dilutions of lysates were inoculated onto the McCoy cells. Infected McCoy cells were grown for 42 h on coverslips in 24-well plates, fixed with methanol and visualized with C. trachomatis species-specific monoclonal antibody (Bio- Rad). The semi -quantitative analysis was based on the counting of infected cells in 20 random visual fields at magnification of x200 and calculating the mean number of inclusion-forming units (IFU) per ml of the specimen. Every experiment was repeated three times. Lycopene toxicity verification. Lycopene toxicity was controlled in MTT test in 24 hours after lycopene addition using 96 well dishes.
Neutral lipid staining. For neutral lipid staining B10.MLM cells grown on coverslips were incubated with Lycopene for 24, 30 and 42 hours. Then cells were washed with PBS twice, fixed with 3% formaldehyde/ 0.025% glutaraldehyde at room temperature for 20 min and stained with BODIPY 493/503 (Molecular Probes, Invitrogen Life Technologies, Carlsbad, California, USA) according to manufacturer' s instructions. Cells were visualized using a Nikon Eclipse 50i fluorescence microscope at x lOOO magnification.
Transmission electron microscopy (TEM). B10.MLM cells were cultured and infected with C. trachomatis with or without Lycopene addition in six -well plates for 48 hours of post-infection period and then harvested from the plates with trypsin-versene solution. Cell pellets obtained by centrifugation for 10 min at 1500 r.p.m. ( otanta 460R;Hettich) were fixed with Ito-Karnovsky fixative solution, followed by post-fixation with OsC>4 and treatment with aqueous uranyl acetate to provide contrast. The specimens were subsequently dehydrated in an ascending series of alcohol concentrations (50, 70, 96 and 100 % ethanol), infiltrated in a 1 : 1 (v/v) mixture of LR White resin and 100 % ethanol for 1 h and in a pure resin for 12 h at 4 uC. Resin polymerization was performed at 56 uC for 24 h. Ultrathin sections were prepared, treated with a lead solution to provide contrast (Reynolds, 1963) and analyzed using a JEOL 100B transmission electron microscope with an accelerating voltage of 80 kV (Jeol, Japan). 2. Results
Lycopene inhibits chlamydial infection in dose-dependent manner
Inoculation of lycopene (oil-formulated and microencapsulated) leads to the dose- dependent inhibition of chlamydial infection and accumulation of inclusion bodies in the cultured cells (Fig. 1). Both formulations of lycopene inhibited chlamydial infection even in 24 hours of post-infection period. After 42 hours after pathogen inoculation the number of the infected cells was reduced by 95% after addition of oil -formulated lycopene. Remarkably, the size of the inclusion bodies was smaller than in infected cells untreated with lycopene.
Microencapsulated lycopene at concentration of 0.5 mg/ml reduced the number of infected cells by 92% and lead to the significant reduction of inclusion bodies at lower concentrations. Addition of lycopene has been performed in all cases after finalized adhesion and internalization of C. trachomatis by cultured cells. Inhibition of chlamydial infection observed in the studies described herein therefore does not develop due to any direct effect of lycopene on the bacterial pathogen and the suppression of its infective abilities. Rather, lycopene has an inhibitory effect on the development of the intracellular infection process.
There was a significant inhibition of infective progeny formation in the cells infected with C. trachomatis and treated with lycopene. Infected cell monolayers produced on average 8xl05 infective particles per ml, whereas the cells treated with oil- formulated and microencapsulated lycopene produced less infective progeny (2xl02 and lxlO3 particles/ml respectively). Inhibition of chlamydial growth was not caused by lycopene toxicity in cell B 10.MLM monolayers. It was shown in MTT assay that oil- formulated lycopene does not cause toxicity at concentration of 3 mg/ml. In this case 89% of cells were viable. Microencapsulated lycopene was also not toxic to the cells at concentration of 0.5 mg/ml. 98% of cells were viable and metabolically active in this case after 24 hour incubation.
Similar inhibitory effect of lycopene was observed on Chlamydophila pneumoniae infection (Fig. 2)
Electron microscopy studies conducted with infected alveolar macrophages showed that after inoculation of C. trachomatis there is an increase in cell size, which did not take place in the control group. Nevertheless the shape of the cells was unchanged and remains round-shaped with a small number of membrane extensions. There were multiple inclusion bodies containing chlamydial particles at different stages of the life cycle (Fig.3A).
When oil-formulated lycopene was introduced, lipid droplets were located in the cytoplasm and even in some chlamydial inclusion bodies. In the cytoplasm of some alveolar macrophages were seen some typical chlamydial particles. However the majority of chlamydial particles were atypical (Fig. 3B).
On electron micro-photos with microencapsulated lycopene there were enlarged vacuoles with singular chlamydial particles and membrane inclusions whereas lipid vesicles were absent (Fig. 3C). Therefore, incubation of infected alveolar macrophages with oil-formulated and microencapsulated lycopene leads to the reduction of chlamydial particle numbers in the inclusion bodies and causes destruction of chlamydial pathogen. Example 2 -Clinical Evidence 1. Materials and methods
Study Design. The study was conducted confidentially at the Institute of Cardiology, the Ministry of Health of the Russian Federation (Saratov, Russian Federation) by Lycotec Ltd (Cambridge, United Kingdom). The protocol was approved by the local ethics committee. All patients were informed of the purpose and goals of the study and had signed a consent form before enrolment and participation in the study.
Participants. There were two groups of participants for two different clinical trials. They were all positive for Chlamydia pneumoniae IgG. The first group comprised of clinically healthy volunteers, who participated in pharmacokinetic and pharmacodynamic studies to identify the most efficient formulations. The second group included patients with diagnosed Coronary Heart Disease, CHD, which was defined and diagnosed according to the American College of Cardiology and American Heart Association Guidelines. CHD patients eligible for the study were screened for levels of blood markers for oxidative stress and inflammation. 155 CHD patients were positive for both types of markers and were randomized into two groups for the study. 13 patients were unable to complete the study for reasons not related to the intake of the test products. Eligibility for the study was determined by the following inclusion/exclusion criteria. Inclusion Criteria were:
• Caucasian male or female subjects 45-73 years old,
• ability to sign an informed consent,
• light-to-moderate smokers (<10 cigarettes daily),
• Chlamydophila pneumoniae IgG with titers ELISA 103 > 100 [350 - 250 /200 "grey zone" / background reading],
• elevated serum markers for oxidative stress IOD > 40 μΜ/mL, • no participation in other dietary trials during the last 3 months before enrolment and duration of study,
• willingness and ability to comply with the study protocol for the duration of the study.
Exclusion criteria were:
• Unwillingness to sign informed consent,
• unable to comply with the protocol for the duration of the study,
• history of myocardial infarction in the 3 months preceding the study,
• ejection fraction (EF) < 45%,
• significant medical condition that would impact safety considerations (e.g., significantly elevated LFT, hepatitis, severe dermatitis, uncontrolled diabetes, cancer, severe GI disease, fibromyalgia, renal failure, recent CVA (cerebrovascular accident), pancreatitis, respiratory diseases, epilepsy, etc.),
• compulsive alcohol abuse (>10 drinks weekly), or regular exposure to other substances of abuse,
• participation in other nutritional or pharmaceutical studies,
• resting heart rate of >100 beats per minute or <50 beats per minute,
• positive test for tuberculosis, HIV, or hepatitis B,
• unable to tolerate phlebotomy,
• special diets in the 4 weeks prior to the study (e.g., liquid, protein, raw food diet), tomato intolerance.
Products. A number of carotenoid products were studied for the ability to inhibit Chlamydophila pneumoniae infection, in terms of inhibition its IgG level in blood of the patients.
Of six lycopene formulations tested, in patients:
• The first formulation comprised 7 mg from Nestle Inc. (Lacto-Lycopene, LL, Switzerland);
• The second formulation comprised 15 mg lycopene from LycoRed (Lyc-O-Mato, Switzerland);
• The third formulation comprised 7 mg lycopene with 1 mg phosphatidylcholine; • The fourth formulation comprised 7 mg lycopene with 10 mg phosphatidylcholine ("GA");
• The fifth formulation comprised 7 mg lycopene with 40 mg phosphatidylcholine;
• The sixth formulation comprised 7 mg of lycopene with 10 mg of phosphatidylcholine and 50 mg of ascorbic acid.
Four lutein-meso-zeaxanthin and zeaxanthin formulations were also tested, of which:
• The first formulation comprised 12 mg lutein-meso-zeaxanthin*, LMZ, only
• The second formulation comprised lOmg LMZ and 2 mg of zeaxanthin;
• The third formulation comprised 7 mg of LMZ, 1.4 mg of zeaxanthin and 10 mg of phosphatidylcholine;
• The fourth formulation comprised 7 mg of LMZ, 1.4 mg of zeaxanthin and 40 mg of phosphatidylcholine.
*It should be noted that lutein used in these preparations contained up to 50% of naturally occurring its isomer meso-zeaxanthin.
Two astaxanthin formulations were also tested, of which:
• The first formulation comprised 7 mg of astaxanthin only,
• The second formulation comprised 7 mg of astaxanthin and 10 mg of phosphatidylcholine.
For the trial on patients with CHD two products were used, 7 mg lycopene without phosphatidylcholine (Lacto-Lycopene, LL, Switzerland) and the same dose of lycopene but with 10 mg of this chaperone molecule ("GA"). The daily intake for all participants was 1 capsule of the product, which was to be taken with the main evening meal. The period of administration was 1 month.
BMI, Pulse Rate, and BP. Measurements of body mass index, BMI, body mass of the patients and their height were carried out in the morning and BMI was calculated in kg/m2. Pulse rate, systolic and diastolic blood pressure, SBP and DBP, were recorded three times on the left arm of the seated patient after 15 min of rest. The time between measurements was greater than 2 minutes. The mean result for each parameter was calculated. All body and vascular parameters were recorded in the morning between 8 and 10 am.
Flow -Mediated Dilation (FMD). Endothelium-dependent flow mediated vasodilatation was measured in accordance with widely accepted guidelines [2]. Patients were screened under ambient conditions at the same time of the morning in a supine position. High resolution ultrasound was applied at the same anatomical landmark of a section of the brachial artery for a period of 30 seconds before and during the peak of reactive hyperemia. It was positioned prior to sphygmomanometer cuff occlusion and 1 min after its deflation. The level of inflation was 50 mm Hg above the patient' s systolic blood pressure and it continued for 5 minutes. Arterial diameter was imaged above the antecubital fossa in a longitudinal scan by duplex ultrasound with linear phase-array transducer. FMD was calculated as a change in post-stimulus diameter as a percentage of the baseline diameter [3].
Ankle-Brachial Index (ABI). ABI was measured between left and right brachial arteries, the one with the highest SBP was chosen, and between left and right tibial arteries, the one with the highest SBP was also chosen for the assessment of ABI. For this purpose a continuous-wave Doppler probe was used after patients had been in a supine position for at least 15 minutes of rest [4].
Tissue Oxygenation. Thenar eminence and forearm muscles of the patients were used as a tissue target for the assessment of oxygen saturation, St02, or combined level of oxygenated haemoglobin and myoglobin. StC was assessed by continuous wavelength near-infrared spectroscopy, MRS, with wide-gap second-derivative (In Spectra, Hutchinson Technology, MN, USA). The measurements were taken at different time points. The recording was initiated after 15 min of rest in a supine position before occlusion of the brachial artery. It was then continued during stagnant ischemia induced by rapidly inflating the cuff to 50 mm Hg above systolic BP. The ischemia lasted for 3 min, and the recording period lasted for another 5 min after that until StCh was stabilized [5, 6]. The area under the hyperaemic curve, AUC, of the recorded signal for the settling time in the post-occlusion period was then calculated as described earlier in % 02/minute [7, 8]. Blood Collection. Blood was collected in the morning from the arm veins of patients following night fast. The serum was separated from the rest of the clotted mass by centrifugation, aliquots were then stored at -80°C prior to analysis. Biochemistry and Inflammatory Markers. Glucose, total cholesterol, TC, triglycerides, TG, high density cholesterol, HDL, low density cholesterol, LDL, C- reactive protein, CRP and Chlamydophila pneumoniae IgG (Chl.pn-lgG) were determined using commercially available analytical kits according to the manufacturers' instructions (BioSystems, Medac, R&D Systems).
Lycopene Quantitative Analysis. The lycopene concentration in all serum samples was measured in duplicate by high-performance liquid chromatography with modifications. Briefly, 400 μΐ of serum was mixed with 400 μΐ of ethanol and was extracted twice with 2 ml hexane. The combined hexane layers were evaporated to dryness in a vacuum (Scan Speed 32 centrifuge) and the residue reconstituted to a volume of 100 μΐ in sample solution (absolute ethanol - methylene chloride, 5: 1, v/v). The specimens were centrifuged again (15 minutes at 10,000 g) and clear supernatant was transferred to HPLC vials. Five microliters of the extract was injected into an Acquity HSS T3 75x2. lmm 1.8 μπι column (Waters, USA) preceded by a Acquity HSS T3 1.8 μιη VanGuard precolumn (Waters, USA) and eluted isocratically at 45°C with the mobile phase (acetonitrile - 0.08 % phosphoric acid solution - tert-Butyl methyl ether, 70:5:25, v/v/v) at a flow rate of 0.5 ml/min. The lycopene peak was detected by a Photodiode Array Detector (Waters, USA) at 474 nm. The peak area was measured using Empower 3 software (Waters, MA). The lycopene concentration in serum samples was calculated by reference analytical standard (lycopene from tomato, L9879, Sigma, USA).
Inflammatory Oxidative Damage (IOD). Serum samples were incubated overnight in 0.05 M PBS acetate buffer (pH 5.6) to imitate the type of oxidative damage which occurs during the release of lysosomes following neutrophil degranulation. The following morning the reaction was stopped using trichloroacetic acid. The concentration of the end products such as malonic dialdehyde (MDA), and other possible thiobarbituric acid reactive substances (TBARS), was then measured by colorimetry [9] using reagents and kits from Cayman Chemical (MC, USA). LDL-Px. Activity of serum LDL peroxidase proteins, which include IgG with superoxide dismutase activity, was measured as described previously [ 10].
Statistics. For the assessment of normally distributed parameters the Shapiro-Wilk method was used. Student's t-test was then applied for both paired and unpaired samples. In cases where parameters were not normally distributed the Mann-Whitney test and Kruskal-Wallis test were used. ANOVA and ANCOVA were used with post hoc analysis (Statistica 9 suite, StatSoft; Inc.). Statistical significance between two-tailed parameters was considered to be P<0.05.
2. Results - Pharmacokinetic and pharmacodynamic studies
Phosphatidylcholine chaperone
Results presented in Table 1 demonstrate that lycopene and phosphatidylcholine at a ratio of at least 1 : 0.1 mg/mg allows the product to perform its anti -chlamydia properties, in terms of reduction of Chlamydophila pneumoniae IgG. This is also accompanied by significant inhibition of the marker oxidative damage. The further increase of lycopene to phosphatidylcholine to the ratio of 1 : 1 mg/mg resulted in improved anti-chlamydial and improved anti-oxidative damage effects. Further increase of the presence of PC did not significantly increase this improved lycopene efficacy. PC alone did not have any effect either on Chlamydophila pneumoniae IgG titers or on the markers of oxidative damage.
An important observation is that to a degree, lycopene concentration in the blood itself is not sufficient for anti-chlamydial and/or anti-oxidant activity. For example, the intake of 15 mg of lycopene alone, for one month, resulted in a substantial increase of this carotenoid in the blood, by 95 ng/ml. This was accompanied by reduction of the marker of oxidation by 19% with p < 0.05. However, there was no significant anti-chlamydial effect. In contrast, ingesting less than a half of the lycopene dose but with the presence of PC, either in ratio 1 : 0.1 mg/mg or 1 : 6 mg/mg, for the same period of one month resulted in a lower increase in blood lycopene. However, a significant inhibition of Chlamydia infection was observed. The titers of anti-chlamydia IgG were reduced by 67% and 75%, respectively. This also accompanied by more profound reduction of the oxidative damage, by 55% and 63%. These results clearly demonstrate a beneficial role of a phospholipid chaperone in boosting lycopene incorporation into its lipoprotein carriers in vivo. This has the effect of increasing lycopene serum concentration, in people of this age group, and in particular aggravated by the presence of dormant or even sub-clinical chlamydia infection.
Ascorbic acid
Inflammation is one of the key reactions of the body when it has an active infection. Lycosomes are the organelles of the cells which are responsible for proteolytic degradation of infective agents and own damaged cells and tissues. When lysosomes release their acidic content, the pH of the local microenvironment is reduced. While acidic pH helps proteolytic and hydrolytic enzymes perform their function, it may at the same time negatively affect integrity of such useful molecules as the carotenoids, which can accelerate their oxidation and degradation.
In order to protect carotenoids from this inflammatory damage we tested in an ex vivo experiment a number of different antioxidants, which included tocopherol, glutathione, propyl gallate, catalase, superoxide dismutase and some others. Of the range of different anti-oxidants tested, we found that the addition of ascorbic acid not only prevented but also reversed to a certain degree the decline in lycopene concentration in our model of inflammation (Fig. 4). Therefore, ascorbic acid helps to protect and extend the active life of the carotenoid's anti -bacteria efficacy, especially when active inflammation is taking place. The demonstration of this positive role of ascorbic acid is presented in Table 1. The addition of ascorbic acid into the lycopene-PC formulation did not change bioavailability of lycopene, but surprisingly further increased its anti- chlamydia efficacy. The higher level of antioxidant performance of this new formulations could be explained by synergetic effect of two powerful antioxidants - lycopene and ascorbic acid.
Lutein-Meso-Zeaxanthin and Zeaxanthin formulations
As for the above clinical studies on lycopene, the presence of phosphatidylcholine was important for LMZ and Zeaxanthin formulations to have anti -chlamydia activity in humans. Although the administration of the increased dose of these carotenoids, 12mg in combination, resulted in a statistically significant anti-oxidant effect, the lower combined dose of 8.4mg but with the presence of PC was twice as strong (Table 2). It is interesting to note that despite the lower intake of these carotenoid preparations their serum concentration was significantly higher than when they were taken without PC. These results indicate an important role of this phospholipid chaperone to facilitate inclusion of LMZ and zeaxanthin in their lipoprotein carriers.
Astaxanthin formulations
The phospholipid chaperone was also essential for the anti-chlamydia effect of astaxanthin. The addition of PC provided a significant boost in astaxanthin
performance. Without PC the inhibition of anti-chlamydia IgG level was 38%, whilst with PC the inhibition of anti-chlamydia IgG level was 77% (Table 3). Antioxidant activity and serum concentration of astaxanthin, after 4 weeks of supplementation, were on comparable level whether it was taken with or without phosphatidylcholine.
Table 1 - Effect of phosphatidylcholine and ascorbic acid on pharmacokinetic and pharmacodynamic performance of lycopene formulations
4 weeks Clinical trial on Inflammatory & Oxidative Damage - GA Astaxanthin comparison
difference with the baseline
Table 2 - Effect of phosphatidylcholine on pharmacokinetic and pharmacodynamic performance of Lutein-Meso-Zeaxanthin and Zeaxanthin formulations
* Lycopene formulation, "lactolycopene", by Nestle (Switzerland)
In all other formulation lycopene was supplied by LycoRed (Switzerland)
Table 3 - Effect of phosphatidylcholine on pharmacokinetic and pharmacodynamic performance of Astaxanthin formulations
4 weeks Clinical trial on Chlamydia pneumoniae IgG and Oxidative Damage - GA Lutein-Zeaxanthin comparison
Coronary Heart Disease
Coronary Heart Disease is understood to be associated with Chlamydia pneumoniae infection [10-14], However, clinical trials have not (or have failed to show
conclusively) that Chlamydia has a causative effect in the development of
atherosclerosis and its clinical complications [15-17], The failure of these studies may be explained by the use of antibiotic regimes. Such antibiotics are usually applied for acute forms of Chlamydia infection. However, Chlamydia may have persistent, asymptomatic or dormant types of infection during which these bacteria turn into antibiotic insensitive form and/or require a longer eradication treatment process. The duration of this period may exceed permitted safe application for the majority of antibiotics. Therefore, alternative treatment of the persistent Chlamydia infection with anti -bacterial preparations made of safe food ingredients would be a highly desirable for cardio-vascular medicine. For this purposes we used the formulation of 7mg lycopene with lOmg of phosphatidylcholine ("GA") as discussed above.
Clinical Groups and Randomization
As can be seen from Table 4, there was no significant difference in age, gender, body mass index, fasting glucose and lipids (total cholesterol, triglycerides, LDL, HDL) between the two clinical groups of the study. The patients belonging to the groups also had similar parameters of cardiovascular health suggesting an altogether successful and valid randomization. No significant differences were found in parameters of lipid peroxidation between the groups (results not shown). All patients remained normoglycemic from enrolment to the end point of the study.
Table 4
Pharmacokinetics Parameters
Lycopene measurements did not show any significant differences between the two groups of the study in the pre-treatment period. Ingestion of lactolycopene did not affect serum lycopene level after 2 weeks of consumption (median value of 58.0 ng/mg; 5/95% CI: 69.2/48.4; versus pre-treatment value of 58.0 ng/mg; 5/95% CI:76.4/55.0, P>0.05). However, there was some increase in serum lycopene content by the end of the observational period (up to median value of 87.0 ng/mg; 5/95% CL93.9/72.2, PO.05). In contrast, the GA lycopene markedly increased serum lycopene concentration with an increase in median over control level of 105.0 ng/mg and 182.0 ng/mg (P=0.0001) after 2 and 4 weeks of treatment respectively (Fig. 5). Lipid Profile
Supplementation with lactolycopene and GA lycopene did not significantly affect parameters of lipid profile in patients belonging to either group of the study (Table 5).
Table 5
(*) - p>0.05; (**)- p<0.05.
However, detailed analysis of variant distribution showed (Fig. 6) that ingestion of GA lycopene caused a tendency towards serum LDL reduction at the end point of the study with borderline level of statistical significance (P=0.028). Such an effect was not seen in the patients treated with lactolycopene.
Cardiovascular parameters
Neither lycopene formulation changed pulse rate or parameters of systemic blood pressure in patients by the end point of the study (Table 6). Similarly, no changes in Ankle- Brachial Index Test were observed in either group of the study. However, there were statistically significant increases in values for FMD (increase in median by 1.1 points or increase over baseline by 10.7%) and St02 (increase in median by 2.7 points, or increase over baseline by 24.3 %) by the end of the observational period in patients treated with the GA lycopene. Interestingly, such an increase did not take place when the lactolycopene formulation was ingested. Table 6
(*) - p>0.05; (**)- p<0.05.
Parameters of Inflammation and Oxidative Stress
Lycopene treatment had no impact on serum CRP level (Table 4). However, there was a differential pattern of regulation of other inflammatory parameters between the two formulations of lycopene (Table 7 and Fig. 7). Lactoycopene did not affect inflammatory markers by the end of the interventional period, whereas GA lycopene significantly reduced anti-chlamydial IgG levels (3 fold reduction, P=0.0001), concentration of oxidized LDL (5 fold decrease, P=0.0001) and IOD value (3 fold reduction, P=0.0001) as compared to pre-treatment values.
Table 7
(*) - p>0.05; (**)- p<0.05.
Discussion
Firstly, the studies show that ingestion of GA lycopene leads to significant increase in serum lycopene level, exceeding the baseline values by 2.9 and 4.3 fold after 2 and 4 weeks respectively. In contrast, supplementation with lactolycopene was less effective, there was no changes in the serum lycopene concentration after the first 2 weeks, and only half-fold increase after 4 week.
The addition of the chaperone PC enhanced the bioavailability of lycopene, and resulted in a substantial anti-chlamydia effect. Treatment with GA lycopene led to a 3- fold reduction of Chlamydia pneumoniae IgG and lowering to the same degree of the IOD marker. The reduction of oxidized LDL was 5 fold. The observations described herein may be of significant importance for safe nutraceutical management of cardiovascular patients, since oxidative stress plays a pivotal role in the initiation, development and outcomes of cardiovascular disease. The significant anti-chlamydia effect of GA lycopene, accompanied by strong antioxidant activity, also translates into a positive effect on some cardiovascular parameters. In particular, such treatments resulted in a significant increase of tissue oxygenation and flow-mediated dilation by the end of the observational period.
Example 3 -Microbiota as a new additional targets for carotenoids in the treatment of bacterial infections
Changes in the gut microbiota after daily ingestion of 7mg GA lycopene (Figure 8) or 30mg GA lycopene for 4 weeks (Figure 9) were also observed. The data shown in Figures 8 and 9 are average in two groups of 6 persons each, 45-65 years old, 50:50 male / female.
In particular, Figures 8 and 9 (panels A) shows results of an analysis of the most powerful probiotics in the gut. In contrast, Figures 8 and 9 (panel B) shows results of health negative, potentially pathogenic bacteria. The results demonstrate a significant increase in levels of probiotic bacterial species such as lactobacilli in the gut after administration of GA lycopene. Several hundred to thousand bacterial species were analyzed, and Figures 8 and 9 are only a small representative of bacterial species in the samples for illustration.
Discussion
The microbiota is an aggregate of microrganisms that resides on or within any number of tissues and biofluids of an individual, including the skin and gut of individuals such as human individuals.
In such microbiota, there are a number of beneficial bacterial species (i.e., nonpathogenic) which can produce metabolites and thereby inhibit other bacteria (i.e., pathogenic bacteria) including obligate intracellular bacteria such as Chlamydia, Ehrlichia and/or Rickettsia. Moreover, among these beneficial bacterial species which produce metabolites and inhibit other bacteria, there are some species where carotenoids are an important part of their metabolism.
The invention relates to the unexpected finding that the administration of the formulations of the invention or carotenoids such as lycopene to an individual acts to boost (i.e., increases the activity and/or number) of carotenoid-metabolizing bacteria in the individual and thereby help to prevent, control and/or treat bacterial infections, particularly obligate intracellular bacterial infections caused by Chlamydia, Ehrlichia and/or Rickettsia
For example, the formulations of the invention or carotenoids such as lycopene enhance the activity and/or number of carotenoid-metabolizing bacteria in the microbiota of an individual. Carotenoid-metabolizing bacteria may act locally within one or more organs such as the gastrointestinal tract. In addition or alternatively, carotenoid-metabolizing bacteria may release antibacterial, anti-infective, antiinflammatory molecules or metabolites, which can be absorbed and be circulated around the whole organism, to prevent and treat bacterial infections or inflammatory conditions in other organs and tissues, or systemically, in the whole body.
Herein, "increases the activity of carotenoid-metabolizing bacteria" is understood to mean an increase in the rate or the amount of anti -bacterial, anti-infective, anti-inflammatory molecules or metabolites that are produced by the carotenoid- metabolizing bacteria upon administration of a formulation of the invention and/or carotenoid such as lycopene as compared to one or more control individual(s).
In other words, "increases the activity" is understood to mean a statistically significant increase in the activity of carotenoid-metabolizing bacteria against other bacteria (i.e., pathogenic bacteria) as compared to one or more control individual(s).
Herein, "increases the number of carotenoid-metabolizing bacteria" is understood to mean a statistically significant increase in the number (i.e., total number in a population) of carotenoid-metabolizing bacteria in the microbiota of the individual as compared to one or more control individual(s).
The control individual may be same individual, prior to or at an earlier stage of treatment with the formulation of the invention and/or carotenoids such as lycopene. Alternatively, the control individual may be a different individual, i.e., an individual receiving a control composition that lacks the formulation of the invention and/or carotenoid such as lycopene.
The carotenoid-metabolizing bacteria may be any bacteria that is capable of metabolizing any one or more carotenoids as defined herein. In certain embodiments, the carotenoid-metabolizing bacteria is a Actinobacteria or Proteohacteria species such as Micrococcus luteus . In certain embodiments, the carotenoid-metabolizing bacteria is a probiotic. In certain embodiments, the carotenoid-metabolizing bacteria is a lactobacilli such as lactobacillus acidophilus, lactobacillus brevis, lactobacillus bulgaricus, lactobacillus plantarum, and/or lactobacillus rhamnosus. In certain embodiments, the carotenoid-metabolizing bacteria is bifidobacteria such as bifidobacerium bifidum, streptocuccus such as streptococcus thermophilius or enter ococcus such as enter ococcus faecium.
In summary, the formulations of the invention and/or carotenoids such as lycopene may lead to anti-bacterial effects (such as the enhanced anti-Chlamydial effect observed following treatment with the "GA" lycopene) by having a direct effect on bacterial propagation (i.e., directly reducing levels or numbers of pathogenic bacteria). In addition or alternatively, the formulations of the invention and/or carotenoids such as lycopene may lead to anti-bacterial effects (such as the enhanced mti-Chlamydial effect observed following treatment with the "GA" lycopene) by indirectly boosting carotenoid-metabolizing members of microbiota in an individual (e.g. increasing the numbers or levels of carotenoid-metabolizing microorganisms, including bacteria, in the individual).
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Claims

1. A composition comprising a carotenoid and phosphatidylcholine (PC) at a weight ratio of at least about 1 : 0.1.
2. The composition according to claim 1, wherein the composition comprises at least about 4 mg of the carotenoid.
3. The composition according to claim 1 or 2, wherein the carotenoid is a carotene.
4. The composition according to claim 3, wherein the carotene is lycopene.
5. The composition according to claim 1 or 2, wherein the carotenoid is xanthophyll.
6. The composition according to claim 5, wherein the xanthophyll is meso-zeaxanthin, zeaxanthin, astaxanthin, canthaxanthin, lutein or any combination thereof.
7. The composition according to any one of the preceding claims, wherein the
carotenoid and PC are at a weight ratio of about 1 : 1.
8. The composition according to any one of the preceding claims, wherein the
composition further comprises ascorbic acid.
9. The composition according to claim 8, wherein the carotenoid and ascorbic acid are at a weight ratio of about 1 : 7.
10. The composition according to claim 9, wherein the carotenoid, PC and ascorbic acid are at a weight ratio of about 1 : 1 : 7.
11. The composition according to any one of the preceding claims, wherein the
composition is provided as a capsule.
12. A method of treating or preventing an obligate intracellular bacterial infection
and/or a condition associated with an obligate intracellular bacterial infection in an individual, the method comprising administering a therapeutically or
prophylactically effective amount of a composition as defined in any one of claims 1 to 11 to the individual, and thereby treating or preventing the infection and/or condition.
13. The method of claim 12, wherein the composition reduces or inhibits (i) the
formation of infective progeny of the obligate intracellular bacteria in the individual and/or (ii) the release of infective progeny into the bloodstream of the individual.
14. The method of claim 12 or 13, wherein the composition:
(i) increases depressed tissue oxygenation and/or impaired flow-mediated
dilation associated with the bacterial infection and/or the condition in the individual; and/or
(ii) increases the activity and/or number of carotenoid-metabolizing bacteria in the individual.
15. The method of any one of claims 12 to 14, wherein the composition reduces or inhibits tissue hypoxia, impaired microcirculation, pre-tension and/or hypertension associated with the bacterial infection and/or the condition in the individual.
16. The method according to any one of claims 12 to 15, wherein the composition is administered once a day, typically after a meal.
17. The method according to any one of claims 12 to 16, wherein the intracellular bacterial infection is caused by Chlamydia, Ehrlichia and/or Rickettsia.
18. The method according to any one of claims 12 to 17, wherein the condition is
selected from:
(i) a metabolic syndrome, fatty liver, steatohepatitis, coronary heart disease (CHD), coronary vascular disease (CVD), atherosclerosis, cardiovascular pathology, cerebrovascular pathology and/or neurodegenerative condition; and/or
(ii) a functional carotenoid deficiency, typically a functional lycopene deficiency.
19. A composition as defined in any one of claims 1 to 1 1 for use in a method of
treating or preventing an obligate intracellular bacterial infection and/or a condition associated with an intracellular bacterial infection in an individual.
20. The composition for use in a method according to claim 19, wherein the composition reduces or inhibits (i) the formation of infective progeny of the obligate intracellular bacteria in the individual and/or (ii) the release of infective progeny into the bloodstream of the individual..
21. The composition for use in a method according to claim 19 or 20, wherein the
composition:
(i) increases depressed tissue oxygenation and/or impaired flow-mediated
dilation associated with the bacterial infection and/or the condition in the individual; and/or
(ii) increases the activity and/or number of carotenoid-metabolizing bacteria in the individual.
22. The composition for use in a method according to any one of claims 19 to 21,
wherein the composition reduces or inhibits tissue hypoxia, impaired
microcirculation, pre-tension and/or hypertension associated with the bacterial infection and/or the condition in the individual.
23. The composition for use in a method according to any one of claims 19 to 22,
wherein the composition is administered once a day, typically after a meal.
24. The composition for use in a method according to any one of claims 19 to 23,
wherein the intracellular bacterial infection is caused by Chlamydia, Ehrlichia and/or Rickettsia.
25. The composition for use in a method according to any one of claims 19 to 23,
wherein the condition is selected from:
(i) a metabolic syndrome, fatty liver, steatohepatitis, coronary heart disease (CHD), coronary vascular disease (CVD), atherosclerosis, cardiovascular pathology, cerebrovascular pathology and/or neurodegenerative condition; and/or
(ii) a functional carotenoid deficiency, typically a functional lycopene deficiency.
26. Use of the composition as defined in any one of claims 1 to 1 1 in the manufacture of a medicament for treating or preventing an obligate intracellular bacterial infection and/or a condition associated with an intracellular bacterial infection in an individual.
27. Use of the composition according to claim 26, wherein the composition reduces or inhibits (i) the formation of infective progeny of the obligate intracellular bacteria in the individual and/or (ii) the release of infective progeny into the bloodstream of the individual.
28. Use of the composition according to claim 26 or 27, wherein the composition:
(i) increases depressed tissue oxygenation and/or impaired flow-mediated
dilation associated with the bacterial infection and/or the condition in the individual; and/or
(ii) increases the activity and/or number of carotenoid-metabolizing bacteria in the individual.
29. Use of the composition according to claim 27 to 28, wherein the composition
reduces or inhibits tissue hypoxia, impaired microcirculation, pre-tension and/or hypertension associated with the bacterial infection and/or the condition in the individual.
30. Use of the composition according to any one of claims 26 to 29, wherein the
composition is administered once a day, typically after a meal.
31. Use of the composition according to any one of claims 26 to 30, wherein the
intracellular bacterial infection is caused by Chlamydia, Ehrlichia and/or Rickettsia.
32. Use of the composition according to any one of claims 26 to 31, wherein the
condition is selected from:
(i) a metabolic syndrome, fatty liver, steatohepatitis, coronary heart disease (CHD), coronary vascular disease (CVD), atherosclerosis, cardiovascular pathology, cerebrovascular pathology and/or neurodegenerative condition; and/or
(ii) a functional carotenoid deficiency, typically a functional lycopene
deficiency.
33. A method of reducing the titre of antibodies against an obligate intracellular bacterial infection in an individual, the method comprising administering a therapeutically or prophylactically effective amount of a composition according to any one of claims 1 to 1 1 to the individual and thereby reducing the titre of antibodies against the bacterial infection.
34. The method of claim 33, wherein the intracellular bacterial infection is caused by Chlamydia.
35. The method of claim 34, wherein the antibodies are anti-Chlamydia IgG or IgA.
36. A method of treating or preventing an obligate intracellular bacterial infection and/or a condition associated with an intracellular bacterial infection in an individual, the method comprising administering a therapeutically or prophylactically effective amount of a composition comprising lycopene to the individual and thereby treating or preventing the infection.
37. The method of claim 36, wherein the composition reduces or inhibits (i) the
formation of infective progeny of the obligate intracellular bacteria in the individual and/or release of infective progeny into the bloodstream of the individual.
38. The method of claim 36 or 37, wherein the composition:
(i) increases depressed tissue oxygenation and/or impaired flow-mediated
dilation associated with the bacterial infection and/or the condition in the individual; and/or
(ii) increases the activity and/or number of carotenoid-metabolizing bacteria in the individual.
39. The method of any one of claims 36 to 38, wherein the composition reduces or
inhibits tissue hypoxia, impaired microcirculation, pre-tension and/or hypertension associated with the bacterial infection and/or the condition in the individual.
40. The method according to any one of claims 36 to 39, wherein the composition
administered once a day, typically after a meal.
The method according to any one of claims 36 to 39, wherein the intracellular bacterial infection is caused by Chlamydia, Ehrlichia and/ 'or Rickettsia.
The method according to any one of claims 36 to 41, wherein the condition is selected from:
(i) a metabolic syndrome, fatty liver, steatohepatitis, coronary heart disease (CHD), coronary vascular disease (CVD), atherosclerosis, cardiovascular pathology, cerebrovascular pathology and/or neurodegenerative condition; and/or
(ii) a functional carotenoid deficiency, typically a functional lycopene
deficiency.
43. A composition comprising lycopene for use in a method of treating or preventing obligate intracellular bacterial infection and/or a condition associated with an intracellular bacterial infection in an individual.
44. The composition for use according to claim 43, wherein the composition reduces inhibits (i) the formation of infective progeny of the intracellular bacteria in the individual and/or the release of infective progeny into the bloodstream of the individual.
45. The composition of claim 43 or 44, wherein the composition:
(i) increases depressed tissue oxygenation and/or impaired flow-mediated
dilation associated with the bacterial infection and/or the condition in the individual; and/or
(ii) increases the activity and/or number of carotenoid-metabolizing bacteria in the individual.
46. The composition of any one of claims 43 to 45, wherein the composition reduces or inhibits tissue hypoxia, impaired microcirculation, pre-tension and/or hypertension associated with the bacterial infection and/or the condition in the individual.
47. The composition according to any one of claims 43 to 46, wherein the composition is administered once a day, typically after a meal.
48. The composition according to any one of claims 43 to 47, wherein the intracellular bacterial infection is caused by Chlamydia, Ehrlichia and/ 'or Rickettsia.
49. The composition according to any one of claims 43 to 48, wherein the condition is selected from:
(i) a metabolic syndrome, fatty liver, steatohepatitis, coronary heart disease (CHD), coronary vascular disease (CVD), atherosclerosis, cardiovascular pathology, cerebrovascular pathology and/or neurodegenerative condition; and/or
(ii) a functional carotenoid deficiency, typically a functional lycopene
deficiency.
50. Use of a composition comprising lycopene in the manufacture of a medicament for the treatment or prevention of an obligate intracellular bacterial infection and/or a condition associated with an intracellular bacterial infection in an individual.
51. The use according to claim 50, wherein the composition reduces or inhibits (i) the formation of infective progeny of the obligate intracellular bacteria in the individual and/or (ii) the release of infective progeny into the bloodstream of the individual.
52. The use according to claim 51, wherein the composition:
(i) increases depressed tissue oxygenation and/or impaired flow-mediated
dilation associated with the bacterial infection and/or the condition in the individual; and/or
(ii) increases the activity and/or number of carotenoid-metabolizing bacteria in the individual.
The use according to claim 51 or 52, wherein the composition reduces or inhibits tissue hypoxia, impaired microcirculation, pre-tension and/or hypertension associated with the bacterial infection and/or the condition in the individual.
The use according to any one of claims 51 to 53, wherein the composition is administered once a day, typically after a meal.
55. The use according to any one of claims 51 to 54, wherein the intracellular bacterial infection is caused by Chlamydia, Ehrlichia and/or Rickettsia.
56. The use according to any one of claims 51 to 55, wherein the condition is selected from:
(i) a metabolic syndrome, fatty liver, steatohepatitis, coronary heart disease (CHD), coronary vascular disease (CVD), atherosclerosis, cardiovascular pathology, cerebrovascular pathology and/or neurodegenerative condition; and/or
(ii) a functional carotenoid deficiency, typically a functional lycopene
deficiency.
57. A method of producing a pharmaceutical formulation comprising formulating
composition components as defined in any one of claims 1 to 1 1, together with pharmaceutically acceptable excipient.
58. A method of producing a nutraceutical formulation comprising formulating
composition components as defined in any one of claims 1 to 1 1, together with a nutraceutically acceptable excipient.
59. A method of treating or preventing a bacterial infection in an individual, the method comprising administering a therapeutically or prophylactically effective amount of a composition comprising a carotenoid to the individual, wherein the composition increases the activity and/or number of carotenoid-metabolizing bacteria in the individual and thereby treats or prevents the bacterial infection.
60. A composition comprising a carotenoid for use in a method of treating or preventing a bacterial infection in an individual, wherein the composition increases the activity and/or number of carotenoid-metabolizing bacteria in the individual.
61. The method of claim 59 or the composition for use according to claim 60, wherein the composition comprising a carotenoid is as defined in any one of claims 1 to 1 1.
62. The method or composition for use according to any one of claims 59 to 61, wherein the carotenoid-metabolizing bacteria are lactobacilli and/or bifidobacteriacea.
63. The method or composition for use according to any one of claims 59 to 62, wherein the bacterial infection is caused by an obligate intracellular bacteria such as Chlamydia, Ehrlichia and/or Rickettsia.
EP17792173.1A 2016-10-31 2017-10-31 Compositions comprising carotenoids and phosphatidylcholine Withdrawn EP3532027A1 (en)

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