CA2247370A1 - Use of hexahydrolupulones as antibacterial and anticancer agents - Google Patents
Use of hexahydrolupulones as antibacterial and anticancer agents Download PDFInfo
- Publication number
- CA2247370A1 CA2247370A1 CA002247370A CA2247370A CA2247370A1 CA 2247370 A1 CA2247370 A1 CA 2247370A1 CA 002247370 A CA002247370 A CA 002247370A CA 2247370 A CA2247370 A CA 2247370A CA 2247370 A1 CA2247370 A1 CA 2247370A1
- Authority
- CA
- Canada
- Prior art keywords
- cells
- hhc
- medicament
- cell
- growth
- 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.)
- Abandoned
Links
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
- A61K31/12—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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The present invention provides pharmaceutical compositions comprising an amount of hydrogenated lupulones or derivatives or analogs thereof, effective to inhibit cancer cell growth, and methods of use thereof.
Description
W O 97t31630 PCTrUS97103073 USE OF ~n~AHYlDROLU~JUD~S AS ANnlBACl~KlAL A~nD ANm CANCER AGENrS
s ~ r~round of the lnveDtion The ~ t and/or cure of cancer has been intensely inve,ctigslted clllminAtin~ in a wide range of therapies. Cancer has been typically treated with surgery, r~ tion and chernolh~ia~y, alone or in conjunction with various th~ ics10 employing drugs, biologic agents, stntibo~i~s, and radioactive immt~nocQnjugateS~
among others. The comml-n goal of cmcer lle~ has beerL and CC~ C5 to be, the elimination or stm~lion~ n of .,~ce~ tumors and cells with minim~l I - rlerc~nt or life~ t~ side effects, due to toxicity to normal tissues and cells.
However, despite efforts, these goals remain largely unrnet.
Even where effective non-invasive drug th~,la~;cs have been developed, patients having solid ms~ nt tu nors and hematological ms~li~s~nt disorders often develop multidrug re~i~ts~n~ Current therapies involve the ~1minictration of in~.-,aced ~los~ges of the snnti~s~nrer drug or drugs to which the cancer cells have become resis~ll andlor s~flmini c~tion of agents ~lesi~Pd to reverse drug 20 acctln llk tion defects in drug ~ l cells. However, these the.~;cs are limited by risk of toxicity to the patie,nt.
Alpha- and beta-a~:ids, derived ~om hops, possess the ability to inhibit the growth of microor~stni~m~;. Some of these acids have been used as antibiotic and~ntifimgs l agents. In addition, alpha acids (also known as hl~ lon~) have been 25 shown to inhibit the tumor promoting effect of 12-O-tetr~dec~noylphorbol-13-acetate in mice (Yasukawa et al., Oncology 52:156-158 (1995)). Colupulone has been reported to be active against Hela cells, CEM lellkPmi~ cells, and adriamycin-and vinblastin-resistant CEM cells (Mannering et al., Food, Nutrition and Chemical Toxicity, Parke et al., eds, Smith Gordon, G.B. (1993) at ch. 28), W O 97131630 PCT~US97/03073 Thus, a need exists for new and effective ~rug theraples for treating cancer which have minim~l side effects and are also effective against multidrug resistant cancer cells.
S Summary of the Ir.vention The present invention provides a method to inhibit cancer cell growth, and thereby to treat cancer comprising ~r~ministering lo a m~mm~1 afflicted with cancer an effective arnounl of the compound OH O
/ " '--' ~ R
o ~ OH
(I) wherein R is (C3 C~)alkyl, preferably -CH2CH(CH3)2, -CH(CH3)2~ or -CH(CH3)CH2CH3, or a l)h~ .eutically acceptable salt thereof. In a pl~r~lled embodiment of the invention, R is -CH(CH3)2. The present invention further provides a method of inhibiting bacterial cell growth by c~mt~ctin~; bacterial cells with an effective amount of a compound of formula (I~. The present invention also provides ph~ eutica] compositions, such as compositions adapted for topical ~t1mini~tration, cornprising an effective amount of a compound of formula (I) incombination with a pha~maceutically acceptable carrier. The compounds of the invention are particularly effective against gram positive bacteria, such as mycobacterium strains, including drug-resistant ~. tuberculosis strains or Mycobacterial avi~m complex (MHC). The compounds are also active against S.
aureus, including methocillin-resistant S. aureus.
W O 97J31630 PCTrUS97/03073 -As used herein, the term alkyl encompasses branched and straight-chain alkyl groups, as well as c ycloalkyl and (cycloalkyl)alk~ l.
Brief Description of t~e Drawings S Figure 1 shows the activity of HHC against Staphylococcus aureus.
DPt~ Descri~ion of the Invention The present invention provides a method of killing cancer cells andlor inhibiting their growth through the use of certain ~-acids. In a plefell~d embo-liment, the method utilizes lupulones or analogs thereof. Generally ~,ref~l~ed are hydrogenated derivatives of lupulones, e.g., compounds of the formula OH O
O~OH
(I) are used, wherein R is (C3-C~)alkyl, preferably -(~H2CH(CH3)2 (hexahydrolupulone), -CH(CH3)2 (hexahydrocolupulone or "HHC"), or -CH(CH3)CH2CH3 (hexahydroadlupulone), or a ph~ eutically acceptable salt thereof. In a prer.,lled embodiment of the invention, R is -CH(CH3)2. The compounds may also be ~ministered as a mixture comprising one or more compounds of formula (I). The tautomeric forms of the compounds of formula (I) are also within the scope of the present method.
Beta-acids (also known as lupulones), and tetrahydroisohumulone and hexahydrocolupulone in particular, can inhibit the growth of food pathogens, such as Listeria monomcyto~ enes (U.S. patent nos. 5,286,506; 5,455,038). In addition, hexahydrolupulone inhibits the growth of certain Lactobacilli (U.S. patent W O 97131630 PCT~US97103073 no. 5,082,975). HoweveI, the antineoplastic effects of hydrogenated lupulones have not been previously repoIted.
These compounds have been found to be highly ef~ective cytostatic and cytotoxic agents which are active against cells of a wide variety of cancers. For example, hexahydrocolupulone has been found to have cytotoxic and/or cytostatic effect against human breast adenocarcinoma cells human acute Iymphoblastic leukemia cells, vinblastin resistant cells, human Burl~itt Iymphoma cells, human oral epidermoid carcinoma cells, and human cervical epithelioid carcinoma cells.
Hexahydrocolupl;llone (HHC) has also demonstrated effectiveness against drug resistant human cancer cell lines, for example, vinblastin resistant human acute Iymphoblastic Ie~ emi~ cells. Multidrug resiet~nce against anticancer compounds is one of the most forrnidable problems in cancer chemotherapy. In addition to their broad spectrum anticancer activily, the compounds of the present invention have potential clinical application due to their high potency, as shown by their relatively low IC50 concentrations (IC50 represents the concentration of compound required to ki:ll 50% of cancer cells in an in vitro assay). Moreover, as a derivative of a generally-recognized safe hop extract, HHC can be considered negligibly toxic when inpested, an advantage over many existing chemotherapeuticagents.
Hydrogenated lupulones appear to be more active and stable than their non-hydrogenated parent corrlpounds. For ~ ple, hexahydrocolupulone is more active than colupulone (see Mannering et al., cited supra) while hexahydrolupulone has been found to be more stable than lupulone (Carson, J. Amer Chem. Soc. 73 :1850-1852 (1951)).
According to the invention, cancer cells are inhibited by ~lministration to a m~mm~l afflicted with cancer of an effective amount of the compounds of Formula (I). The "effec1ive amount" will ultimately depend upon whether inhibition of growth or killin~ of c;mcer cells is goal of the treatment. However, as described W O 97131630 PCTrUS97/03073 herein, a suitable dose will be in the range of about 0.5 to about 100 mg/kg of body weight per day.
The composltions described herein are believed to be effective in the treatment of solid m~mm~ n tumors or hematologic m~lign~ncies, and include those which can develop multidrug resistance. These solid tumors include cancersof the head and nec~;, lung, mesothelioma, mediastinum, esophagus, stomach, pancreas, hepatobiliary system, small intestine, colon, rectum, anus, kidney, ureter~
bladder, prostate, urethra, penis, testis, gynecological organs, ovarian, breast, endocrine system, skin central nervous system; sarcomas of the soft tissue and bone;
and melanoma of cutaneous and intraocular origin. Hematological m~ n~ieS
include childhood leukemia and lymphom~, Hodgkin's (1isç~e' Iymphomas of lymphocytic and cutaneous origin, acute and chronic lellk~mi:~ plasma cell neoplasm and cancers associated with AIDS. The pl~f.,.led m~mm~ n species for lled~ elll are h~ n~ and domesticated ~nim~
Without being bound by any particular theory regarding mel~h~ni~m of action, prelimin~ry resull:s using the fluorescent activated cell sorting (FACS)method suggest that HHC has an effect upon the (Jl-S phase transition of the cell cycle. Other studies suggest that the cytotoxic eff'ect of HHC on cancer cells is not due to major effects upon DNA, RNA or protein synthesis.
In another embodiiment of the invention, a compound of formula (I), or a salt or ph~rm~ce~ltic~l composition thereof, is used to inhibit bacterial cell growth. In a pfc;relled method7 the ba~,teria is a gram positive ~acteria. In a more pref~,edembodiment, the gram p~DSitiVe bacteria is S~aphylococcus Aureus. In another preferred embodiment, the bacteria is a mycobacterium. In a more pl~r~ d 2~ embodiment, the mycobacteria is tuherculosis or mycobacterial avian complex (MHC).
In a further embodiment of the invention, a compound of forrnula (1), or a salt or a pharmaceutical composition thereof, is useful as a pdl~scilicidal agent, for W O 97/31630 PCT~US97103073 example as shown in Example VII hereinbelow. 1 he compounds of the invention can be used against the hemoflagellates, such as against lei.~hmAniA
Hexahydrolupulones are hydrogenated derivatives of lupulones, a constituent of hops (up to 5-7%). He~ahydrocolupulone can be made via the chemical 5 hydrogenation of colupulone using a number of methods known in the art. For example, hydrogenation can be achieved with platinum (IV) oxide as a catalyst asdescribed by Riedl (Ber. 89:1863 (1956)) or by Carson (J. Am Chem. Soc. 73:1850 (1951)).
~n a ~lcl~ d method, beta-acids are purified as described in U.S. patent no.
10 4,918,240 and hydrogenated as described in U.S. patent no. 5,082,975 (both ofwhich are herein incorporated in their entireties). ~ccording to a preferred method of purifying beta acids, palladium or plAtimlm catalyst poisons are removed from an aqueous Alk~line beta acid solution by agitating the solution at a pH of at least about l 0, preferably between about l l and l 2.9, in the presence of polyvalent metals other 15 than palladium and platinum. The insoluble catalyst poison contAining materials are then separated.
Preferably, the polyvalent metals used are edible metal ions, preferably magnesium and calcium, and may also be zinc, alllminl-rn or iron. Preferably, the pH is bet~,veen about 11 and l 2.9. Although a solvent is not necessA. ~ for removal 20 of the catalyst poisons, water-immiscible food-grade solvents may be used.
Examples include hvdrocarbons such as hexane, esters, liquid fatty alcohols~ andterpenes such as limonen In pl~p~lion for the ensuing hydrogenation step, the purified beta acids may be recovered from the ~lkAline aqueous phase by the CO2 method of Cowles 25 (U.S. patent no. 4,590,296). Alternatively, the bela-acid solution is mixed with about an equal volume oi' an organic solvent such as hexane. The beta-acids are extracted into solvent by slow acidification with agitation, using an acid such as phosphoric acid, until a pH of about 9.5-9.8 is reached. Substantially all of the beta acids are removed from the aqueous phase by this procedure. The beta acids may W O 97~31630 PCTAJS97/03073 then be recovered from the solvent by partial evaporation and cooling, or by removal of the hexane under vacuum, by procedures known to the art.
Any known method of hydrogenation ma~ be used, although preferably one is used which results in a high purity of beta acids. Preferably, the purified beta-S acids are hydrogenated under non-acidic conditions. According to a preferred method, the beta-acids are dissolved in solvent and water. Any non-toxic, non-reactive solvent may be used such as lower alkanols, an organic water miscible or THF. A hydrogenation catalyst such as palladium or platinum is added. The vesselis evacuated, hydrogen ~as is introduced, and the vessel is agitated until hydrogen 10 uptake has ceased. The hydro~ton~ted product is filtered to remove the catalyst. A
solution of water and a hydrocarbon solvent such as hexane is then added. The mixture is ~git~ted and tl~ hydrogenated beta acid is extracted into the hexane phase. After partial evaporation of the hexane, the mixture is seeded and cooled, allowing the product to crystallize.
To allow conversion to pure hydrogenated beta acids, the minimum pH used is about 3, preferably above 4, with the most preferred range between about 7 to 9.
The rate of hyclrogenation may be accelerated through the use of additional amounts of catalyst or by raising the te~ c~ c.
An aqueous liquid ~lk~line solution of hydrolupulones may be prepared as described in U.S. patent 5,082,975. Alternatively, propylene glycol, glycerine, similar stable alcohols and polyols, or rnixtures thereof with or ~,vithout water, may be substituted for the water of the aqueous solution. In addition, the foregoingaqueous solution may be mixed with glycol or glycerine, etc., to form a standardized solution of product, which is readily-dispersible in water~ and stable as well.
Ph~ eutically acceptable salts of the biologically active compounds described herein may be used as well in practicing the claimed methods.
Pharmaceutically acceptable salts may be formed using organic or inorganic bases, such as NaOH, Na(CO3)2, NaHCO3, KOH, amines and the like.
W O 97/31630 PCTrUS97103073 Although the compounds described herein and/or their its salts may be ministered as the pure chemicals, it is preferable to present the active ingredient as a pharmaceutical composition. The invention thus further provides the use of a ph~rm~ce.~ltical composition compri~i~g one or more compounds and/or a 5 pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable carriers therefor and, optionally, other therapeuticand/or prophylactic ingredients. The carrier(s) must be 'acceptable' in the sense of being compatible wlth the other ingredients of the composition and not deleterious to the recipient thereof.
Pharmaceutical compositions include those suitable for oral or parenteral (including intramuscular, ~lbcul;~leous and intravenous) ~lrnini~tration The compositions may, where a~pro~ ate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound ~,vith liquid carriers, solid m~triceS, semi-solid carriers, finely divided solid carriers or combination thereof, and then, if necess~ry, shaping the product into the desired delivery system. Such methods also include encapsulation of a therapeutically effective amount of the active ingredient in liposomes, by methods known to the art.
Pharmaceutlcal cDmpositions suitable for oral ~lmini~t~ation may be presented as discrete unit dosage forms such as hard or soft gelatin capsules, cachets or tablets each cont~ining a predetçrmin~d amount of the active ingredient; as apowder, as liposomal ~ dlions, or as granules as a solution, a suspension or as an emulsion.
Compositions suitable for topical ~mini~tration in the mouth include unit dosage forms such as loi~enges comprising active ingredient in a flavored base, usually sucrose and acadlia or fr~r~nth; pastilles comprising the active ingredient in an inert base such as ~elatin and glycerin or sucrose and acacia; muco~(lherent gels, and mouthwashes comprising the active ingredient in a suitable liquid carrier.
W O 97/31630 PCTrUS97/03073 When desired, the abo~,-e-described compositions can be adapted to provide sustained release of'the ac:tive ingredient employe(l, e.g., by combination thereof vvith certain hydrophilic polymer matrices, e.g., comprising natural gels, synthetic polymer gels or mixtures thereof.
For topical ~lmin i~tratïon to the epidermis, the active ingredients may be formulated as ointments, creams or lotions, or patc hes. Ointments and creams may, for example, be formulated with an a~ueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also conta~in one or more emulsifying agents, stabilizing agents., dispe~ g agents, s~ en.l ;.~g agents, thicl~nin~ agents, or coloring agents. The active ingredient can also be delivered via iontophoresis from a suitable reservoir, e.g., as disclosed in U.S. Patent Nos. 4,140,122;
4,383,529; or4,051.842.
The active ingredient can be delivered via patches for transdermal ~ imini~tration. 'Suitable tr~n.c~l~rmal de~ivery systems are disclosed, for exarnple, in U.S. PatentNo. 4,788,603, U.S. PatentNo. 4,931,279, U.S. PatentNo. 4,668,506, U.S. PatentNo. 4,713,224 and U.S. PatentNo. 5,560,922. Patches fortr~ clc-rnn~l delivery can comprise a backing layer and a polymer matrix which has dispersed or dissolved therein an effec,tive amiount of the active ingredient, along with one or more skin permeation çnln~ncers. The backing layer can be made of any suitable material which is imperrmeable to the analogs or derivatives of the invention. The backing layer serves as a protective cover for the matrix layer and provides also a support function. The baicking can be formed so that it is ec.centi~lly the same size layer as the polymer matrix or it ca]n be of larger ~iimen.~jon so that it can extend beyond the side of the polymer matrix or overlay the side or sides of the polymer matrix and then can exte:nd outwardly in a manner that the surface of the extension of the backing layer can be the base for an adhesive means. Alternatively, the polymer matrix can contain, or be form~ te~l of, an adhesive polymer, such as polyacrylate or acrylate/vinyl acetate copolymer. For long-term applications it W O 97/31630 PCTrUS97/03073 I() might be desirable to use microporous andlor breathable backing l~min:~te~, so hydration or maceration of the skin can be minimi7çd.
Examples ol' materials suitable for makin~ the backing layer are films of high and low density polyethylene, polypropylene, polyurethane, polyvinylchloride, polyesters such as poly(ethylene phth~l~te), metal foils, metal foil l~min~tes of such suitable polymer films, and the like. Preferably, the materials' used for the backing layer are l~min~tes of such polymer films with a metal foil such as al--minl-m foil.
In such l~minAtec, a polymer film of the l~min~te will usually be in contact with the a&esive polymer matrix. The backing layer can be any ap~ ,p.iate thickness whichwill provide the desired pr~ Ic.;li~e and support functions. A suitable thickness will be from about 10 to about 200 microns.
Generally. those polymers used to form the biologically acceptable adhesive polymer layer are those c apable of forming shaped bodies, thin walls or coatings through which therapeutic agents can pass at a controlled rate. Suitable polymers are biologically and pharmaceutically compatible? nonallergenic and insoluble inand compatible with body fluids or tissues with which the device is contacted. The use of soluble polymers i s to be avoided since dissolution or erosion of the matrix by skin moisture would affect the release rate of the therapeutic agents as well as the capability of the dosage lmit to remain in place for convenience of removal.
Exemplary materials for fabricating the adhesive polymer layer include polyethylene, polypropy]ene, polyurethane, ethylene/propylene copolymers, ethylene/ethylacrylate copolymers, ethylene/vinyl acetate copolymers, silicone elastomers, especially the medical-grade polydimethylsiloxanes, neoprene rubber,polyisobutylene, polyacrylates, chlorinated polyethyleae, polyvinyl chloride, vinyl chloride-vinyl acetate copolymer, crosslinked polymethacrylate polymers (hydro-gel), polyvinylidene chloride, poly(ethylene terephth~l~te), butyl rubber, epichlorohydrin rubbers, ethylenvinyl alcohol copolymers, ethylene-vinyloxyethanol copolymers; silicone copolymers, for example, polysiloxane-polycarbonate copolymers, polysiloxanepolyethylene oxide copolymers, W O 97/31630 PCT~US97/03073 polysiloxane-polymethaclylate copolymers, polysiloxane-alkylene copolymers (e.g,polysiloxane-ethylene copolymers), polysiloxane-~lkylenesilane copolymers (e.g.,polysiloxane-ethylenesilane copolymers), and the like; cellulose polymers, for example methyl or ethyl c:ellulose, hydroxypropyl methyl cellulose, and cellulose 5 esters; polycarbonates; polytetrafluoroethylene; and the like.
Preferably, a biolagically acceptable adheslve polymer matrix should be selected from polymers with glass transition t~ cldLIlres below room temperature.
The polymer may, but need not neces~rily, have a degree of crystallinity at roomte.~ dlule. Cross-linkirlg monomeric units or sites can be incol~ul~led into such 10 polymers. For example, c ross-linking monomers can be incorporated into polyacrylate polymers, which provide sites for cross-linking the matrix after dispersing the th~ ulic agent into the polymer. Known cross-linking monomers for polyacrylate polymers include polym~th~.~rylic esters of polyols such as butylene diacrylate and dimethacrylate, trimethylol propane trimethacrylate and the l;ke.15 Other monomers which p:rovide such sites include allyl acrylate, allyl methacrylate, diallyl maleate and the lik:e.
Preferably, a plasticizer and/or humectant is dispersed within the adhesive polymer matrix. Water-soluble polyols are generally suitable for this purpose.
Incorporation of a hllm~c1~nt in the form~ tion allows the dosage unit to absorb20 moisture on the sur~Face o,F skin which in turn helps to reduce skin irritation and to prevent the adhesive polymer layer of the delivery system from failing.
The~a~ulic agents released from a transdermal delivery system must be capable of penetrating each layer of skin. In order to increase the rate of permeation of a therapeutic agent, a penetration agent, such as a fatty alcohol or a glycol such as 25 propylene glycol can be used to increase the permeability of the outermost layer of skin, the stratum corneum, to the th.,. a~;ulic agent.
The active agent or derivative may also be formulated so as to be suitable for ~1mini~tration by inhalation or in~llffl~tion or for nasal, intraocular or other topical (including buccal and sub-lingual) ~rlmini~tration. For example, for ~lmini~tration W O 97/31630 PCT~US97tO3073 to the upper (nasal) or lovver respiratory tract by inhalation, the active agent or derivative is conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient :means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such ~ dichlorodifluoromethane, 5 trichlorofluoromethane, dichlorotetrafluoroethane. carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.
Alternativel~, for ~tlmini~tration by inhalation or insuffl~tion, the active agent may take the form of a dry powder composition, for example, a powder mix of 10 the active agent and a suitable powder base such as lactose or starch. The powder composition may be l,lest;~ d in unit dosage form in, for example, capsules or cartridges or, e.g., gelatin or blister packs from which the powder may be z~r1mini~tered ~,vith the aid of an inh~l~tor, in~ tor or a metered-dose inhaler.
For intra-nasal ~f~mini.~tration, the active agent may also be ~mini~tered via 15 nose drops, a liquid spray, such as via a plastic bottle ~mi;~el or metered-dose inhaler. Typical of atomizers are the Mistometer(~' (Wintrop) and the Medihaler~~Riker).
The pharmac;eutical compositions according to the invention may also contain other adjuvants such as flavorings, coloring, antirnicrobial agents, or 20 preservatives.
It will be further appreciated that the amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of a~lmini~tration, the nature of the condition being treated and the age and condition of the patient and will be 25 ultimately at the discretion of the a1tenl1~nt physician or clinician.
In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mglkg, e.g., from about 10 to about 75 mglkg of body weight per day, such as 3 to about 51) mg per kilogram body weight of the recipient per day, W O 97131630 PCT~US97/03073 preferably in the range of 6 to 90 mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.
The compound is conveniently ~1mini~tered in unit dosage form; for example, co~ g 5 to 1000 mg, conveniently 1() to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage i'orm.
Ideally, the active ingredient should be ~11mini~tered to achieve peak plasma concentrations of the acti ve compound of from about 0.5 to about 75 IlM, preferably, about 1 to 50 ~lM, most preferably~ about 2 to about 30 ~M. This maybe achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally ~rlmini.et~ed as a bolus c~ about 1-100 mg of the active ingredient. Desirable blood levels may be m~int~in.ocl by continuous infilsion to provide about 0.01-5.0 mglkg/hr or by inte~ Lenl infusions cont~inin~ about 0.4-15 mgJkg of the active ingredient(s).
The desired dose may conveniently be presented in a single dose or as divided doses ~-innini~tt~.red at a~rop.;ate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced ~mini~trations, such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.
All publications, pates~ts and patent docurnents are incorporated by reference herein, as though individually incoll,ulaled by reference. The invention has been described with reference to various specific and p~ ef~ d embodiments and techniques. However, it should be understood that many variations and modifications may be made while rern~ining within the spirit and scope of the invention.
The following examples are intended to illustrate but not limit the invention.
EX~MPLES
EXAMPLE I - Cytostatic Acti~ib W O97/31630 PCT~US97/03073 HHC was tested for cytostatic activity using the trypan blue exclusion assay.
The purpose of the assay i s to determine the concentration of HHC at which 50~/O of Gell growth is inhibited (i.e., ID50) when cells are continuously exposed to the HHC.
I~ive ml of a mixture of 2 x 105 cells/ml were placed in T-25 flasks. HHC or control 5 ~no HHC or DMSO vehicle) was added. The cells were incubated at 37~C under 5% CO~ for 24 or 4~ hours. Monolayer cells which are attached need to be incubated for a minimum of 14 hours before HHC is added in order to allow the cells to attach. The cells are detached by pancre~tini7ing (~t~Zlchf~d cells) and or suspended well (lm~ hed cells). A 100 ~11 sample was removed from the cell 10~ suspension and placed in a borosilicate glass test tube. One hundred ~11 of .4%
trypan blue solution (trypan blue (Sigma) in phosphate buffered saline) was added to the cell suspension sample and mixed well. The cells were counted using a hemocytometer. ID~o was calculated by choosing a drug concentration that was below 50% inhibition as well as a drug con~Pntration that was above 50% inhibition 15 and then interpolating those values to 50% inhibition. The following formula was used:
% inhibition = 100 - % growth, where % growth = [(Ct - C0)/(CC, - C0)~ x ] 00, where C0 is the cell count at time "0";
C, is the cell count of the HHC treated sample at time "t"; and CCt is the c~ll count of the control sample at time "t".
Table I presents d.~ta on the activity of HHC against various cancer cell lines.25 MCF-7 cells are human breast adenocarcinoma cells which have some characteristics of differentiated m~mmZ~ry epithelium (i.e., dome formation and estradiol processing via cytoplasmic estrogen receptors). CEM cells are human acute Iymphoblastic leukemia cells which morphologically resemble Iymphoblastic cells. CEM vinblastin resistant cells contain the P-glycoprotein therefore enabling 30 them to exhibit multidrug resistance to many structurally unrelated compounds.
Raji cells are human Burlcitt Iymphoma cells which are Iymphoblast-like. KB cells CA 02247370 l998-08-26 W O 97/31630 PCTrUS97/03073 are human oral epidermoiid earcinoma cells. HeLa S3 cells are human cervical epithelioid careinoma cells.
Table I
5 Cell type no. of ~c.;l.lents ID50 (~lM) standard deviation MCF-7' 3 1.23 0.17 CEM' 4 1.97 0.34 CEM vin resistant' 4 2.61 0.33 Raji2 4 2.19 0.40 KB' 3 0.85 0.07 HeLa S32 3 1.21 0.03 'period of incubation = 4,Bh; 2period of ineubation = 24h.
As the results indieate, HHC is an effeetive and potent cytostatic agent with respect to a wide variety of eancer eell types.
EXAMPLE II - Cytotoxic Activity HHC was tested for cytotoxic activity using a colony formation assay, using 20 an 18 hour pulse exposure. The purpose of the clonogenic assay is to determine the eoneentration of HHC at whieh 50% of eell growth is inhibited (i.e., IC50) when eells are exposed to the HHC for a defined amount of time (i.e., a pulse exposure to the xenobiotic).
Five ml of a mixture of 2 x 10~ eells/ml (MCF-7 eells) were placed in eaeh 25 of 4 T-25 flasks in a media of RPMI (Gibeo). The cells were incubated at 37 "C
under 5 % CO2 for a minimnn~ of 14 hours to allow the cells to attaeh. The cellswere pulsed for 18 hours with HHC coneentrations of 0, 1.5, and 25 ~lM, or with DMSO.
The media was removed from the flasks (i.e., media with floating cells) and 30 placed in 15 ml polypropylene centrifuge tubes 1-4 (summarized in Table II). The W O 97131630 PCT~US97/03073 flasks were washed in 1 ml of PBS and the wash was placed in the ap~rol"iate tubes 1-4. Tubes 1-4 were centrifuged at l 50 rpm and the media was aspirated. Cells were detached from 1he flasks by pancre~tini7~tion Approximately 1 ml of the cell suspension from the flask, were added to the appropriate tubes (1-4) and the pellet 5 suspended. The cells in tubes 1-4 were pooled with the cells in the flask. The cells were then resuspended.
One ml of ce~l sus]pension from the flask was added to the applopliate l0x tube (tubes 5-8) along with 9 ml of media and suspended well. One ml of the suspension in each l0x tube was then placed in the a~plop.iate 50x tube (tubes 9-l0 12) along with 4 ml of media.
Table II
controlb DMSO 1.5 ,uM HHC 25 ~lMHHC
floating cells ] a 2 3 4 l0x dilution .5 6 7 8 50x dilution '~ 10 l l l2 a tube no. b[HHC]=0 The cells in the original cell sl.~p~n.~ion (i.e., in the flask) were counted usingtrypan blue exclusion assay (described in Example II). Based on this count, the number of cells in the 50x tubes were calculated. The volume n~cess~ to acquire approximately 5400 cells was then determine~l This volume was removed from the 25 50x suspension and placed into 50 ml polypropylene tubes (tubes 13- l 6). Media was added to attain a volume of l 8 ml. The cells were then suspended.
Five ml of suspension from each of tubes l 3-16 was transferred to 60 x 15 m~n tissue culture dishes l~in triplicate). The cells were incubated until colonies of at least 50 cells were present (approx. 6-8 days). The cells were then stained with30 Giemsa stain (a solution of 1% Giemsa stain (Sigma) in 100% methanol). The W O 97131630 PCT~US97/03073 culture dishes were remo~red from the incubator and the media aspirated off. Thedishes were washed 2x with phosphate buffered saline (3-4 ml). Three to four ml of' 100% ethanol was added l~o each dish, an arnount sufficient to cover the bottom of the dish, covered and allo-wed to stand for 30 minutes. The stain was washed out5 with cold H2O. The water was poured off, without loss of colonies. The plates were inverted and allowed to dry. The cells were counted in those colonies which consisted of at least ~0 cells. The ID50 was determined as described in Example II.
The data is shown in Tab]e III.
10 Table III
Cell type no. oi ~ IDso (~lM)Std. deviation MCF-7 2 1.62 0.28 . ..
As the data shows, HHC is able to effectively kill MCF-7 cancer cells at low 1 5 dosages.
Preliminary data suggests that the in vitro ID50 of HHC against C1300 cells ~mouse neuroblastoma) is approximately 1.94 ~M after 48 hours of continuous exposure.
20 EXAMPLE III - In Vivo Studies The maximal tole:rated dose (MTD) of HHC was deterrnined in BDF 1 mice by i.p. injection. Mlce w~re able to tolerate the equivalent of 200 ~Lmolar HHC with no apparent side et'fects.
25 EXAIVIP~E IV - Determination of Mechanism of Action Fluorescence activated cell sorting (FACS) was used to determine what percentage of cells could be found in the designated cell cycle phase after 48 hours of continuous HHC exposure. There are four distinct phases in the cell cycle which lead to duplication of the cell and its genetic material. In the G0/GI phase the cell is W O 97/31630 PCTrUS97/03073 at rest or is performing miscellaneous biosynthetic fimctions. L)uring the S phase, DNA synthesis occurs which leads to replication o~'the chromosomes. G2 is the pre-mitotic interval urhile the M phase is the time during which the cell divides.
Cells were exposed to concentrations of 1.5 ~lM and 25 IlM of HHC, and S DMSO. DMSO had no significant effect on the cell cycle, with a profile almost identical to that of the "no drug" control. The results indicate that low [HHC~'s (i.e., 1.5 ~M) increase the number of cells in G0/GI and decrease the number of cells in S
phase. At high [HHC~]'s (i.e., 25 ~lM) there is a decrease in the number of cells in GOIG1 and an increase in lhe nurnber of cells in S phase. This indicates that HHC
10 has an al)pd~el1t effect upon the Gl-S phase transition of the cell cycle.
EXAMPLE V - Antih~terial Activity (S'~ ' J)lococcus aureus) A single colony of Staphylococcus aureus was grown in 2 ml of Luria-Bertani (LB) broth overnight at 37~C in a bacterial shaker with vigorous ~h~king.
15 0.5 ml of the culture was transferred to 250 ml LB and allowed to grow at 37 ~C
until the OD600 (optical density) reading was about 0.2 (a reading of I OD600 indicates a cell concentrati on of approximately 8 x l o8 cells/ml). Cultures were aliquoted into 50 ml tubes co..1Slini~g 13 ml of bacterial culture per tube. 0.5, 1.0, 2.5, 5.0, 10 and 25 ~M of HHC was added to the tubes. No-drug and DMSO
solvent were used as controls. Aliquots of the bacterial culture were taken every 2 hours to obtain the OD60() reading. As the results ïndicate (Figure 1) HHC is aneffective inhibitor of bacterial growth.
EXAMPLE VI - Antibacterial Activity (M. tu~erculosis) Drug susceptibility was tested using the BACTEC method (Siddigi, BACTEC TB System Product and Procedure Manual, Becton Dickinson Corp ( 1989); Inderlied, Antimycobac terial Agents in Antibiotics in l,aboratory Medicine, ed. Lorian, 3rd edition, Williams and Wilkins). The growth curve of mycobacteriain liquid media with and without HHC is plotted for 4 to 12 days. Resistance is W O97t31630 PCT~US97/03073 determined by modification of the 1% proportional method. The control vial is inoculated with a l: l O0 d:ilution of org~ni.~ms, and a growth rate greater than vials with HHC is interpreted as evidence of susceptibility. Resistance occurs where less than 99% of the org~ni.cm.c are inhibited by the HHC.
Materials:
1 ) BACTEC I 2B vials 2) HHC
s ~ r~round of the lnveDtion The ~ t and/or cure of cancer has been intensely inve,ctigslted clllminAtin~ in a wide range of therapies. Cancer has been typically treated with surgery, r~ tion and chernolh~ia~y, alone or in conjunction with various th~ ics10 employing drugs, biologic agents, stntibo~i~s, and radioactive immt~nocQnjugateS~
among others. The comml-n goal of cmcer lle~ has beerL and CC~ C5 to be, the elimination or stm~lion~ n of .,~ce~ tumors and cells with minim~l I - rlerc~nt or life~ t~ side effects, due to toxicity to normal tissues and cells.
However, despite efforts, these goals remain largely unrnet.
Even where effective non-invasive drug th~,la~;cs have been developed, patients having solid ms~ nt tu nors and hematological ms~li~s~nt disorders often develop multidrug re~i~ts~n~ Current therapies involve the ~1minictration of in~.-,aced ~los~ges of the snnti~s~nrer drug or drugs to which the cancer cells have become resis~ll andlor s~flmini c~tion of agents ~lesi~Pd to reverse drug 20 acctln llk tion defects in drug ~ l cells. However, these the.~;cs are limited by risk of toxicity to the patie,nt.
Alpha- and beta-a~:ids, derived ~om hops, possess the ability to inhibit the growth of microor~stni~m~;. Some of these acids have been used as antibiotic and~ntifimgs l agents. In addition, alpha acids (also known as hl~ lon~) have been 25 shown to inhibit the tumor promoting effect of 12-O-tetr~dec~noylphorbol-13-acetate in mice (Yasukawa et al., Oncology 52:156-158 (1995)). Colupulone has been reported to be active against Hela cells, CEM lellkPmi~ cells, and adriamycin-and vinblastin-resistant CEM cells (Mannering et al., Food, Nutrition and Chemical Toxicity, Parke et al., eds, Smith Gordon, G.B. (1993) at ch. 28), W O 97131630 PCT~US97/03073 Thus, a need exists for new and effective ~rug theraples for treating cancer which have minim~l side effects and are also effective against multidrug resistant cancer cells.
S Summary of the Ir.vention The present invention provides a method to inhibit cancer cell growth, and thereby to treat cancer comprising ~r~ministering lo a m~mm~1 afflicted with cancer an effective arnounl of the compound OH O
/ " '--' ~ R
o ~ OH
(I) wherein R is (C3 C~)alkyl, preferably -CH2CH(CH3)2, -CH(CH3)2~ or -CH(CH3)CH2CH3, or a l)h~ .eutically acceptable salt thereof. In a pl~r~lled embodiment of the invention, R is -CH(CH3)2. The present invention further provides a method of inhibiting bacterial cell growth by c~mt~ctin~; bacterial cells with an effective amount of a compound of formula (I~. The present invention also provides ph~ eutica] compositions, such as compositions adapted for topical ~t1mini~tration, cornprising an effective amount of a compound of formula (I) incombination with a pha~maceutically acceptable carrier. The compounds of the invention are particularly effective against gram positive bacteria, such as mycobacterium strains, including drug-resistant ~. tuberculosis strains or Mycobacterial avi~m complex (MHC). The compounds are also active against S.
aureus, including methocillin-resistant S. aureus.
W O 97J31630 PCTrUS97/03073 -As used herein, the term alkyl encompasses branched and straight-chain alkyl groups, as well as c ycloalkyl and (cycloalkyl)alk~ l.
Brief Description of t~e Drawings S Figure 1 shows the activity of HHC against Staphylococcus aureus.
DPt~ Descri~ion of the Invention The present invention provides a method of killing cancer cells andlor inhibiting their growth through the use of certain ~-acids. In a plefell~d embo-liment, the method utilizes lupulones or analogs thereof. Generally ~,ref~l~ed are hydrogenated derivatives of lupulones, e.g., compounds of the formula OH O
O~OH
(I) are used, wherein R is (C3-C~)alkyl, preferably -(~H2CH(CH3)2 (hexahydrolupulone), -CH(CH3)2 (hexahydrocolupulone or "HHC"), or -CH(CH3)CH2CH3 (hexahydroadlupulone), or a ph~ eutically acceptable salt thereof. In a prer.,lled embodiment of the invention, R is -CH(CH3)2. The compounds may also be ~ministered as a mixture comprising one or more compounds of formula (I). The tautomeric forms of the compounds of formula (I) are also within the scope of the present method.
Beta-acids (also known as lupulones), and tetrahydroisohumulone and hexahydrocolupulone in particular, can inhibit the growth of food pathogens, such as Listeria monomcyto~ enes (U.S. patent nos. 5,286,506; 5,455,038). In addition, hexahydrolupulone inhibits the growth of certain Lactobacilli (U.S. patent W O 97131630 PCT~US97103073 no. 5,082,975). HoweveI, the antineoplastic effects of hydrogenated lupulones have not been previously repoIted.
These compounds have been found to be highly ef~ective cytostatic and cytotoxic agents which are active against cells of a wide variety of cancers. For example, hexahydrocolupulone has been found to have cytotoxic and/or cytostatic effect against human breast adenocarcinoma cells human acute Iymphoblastic leukemia cells, vinblastin resistant cells, human Burl~itt Iymphoma cells, human oral epidermoid carcinoma cells, and human cervical epithelioid carcinoma cells.
Hexahydrocolupl;llone (HHC) has also demonstrated effectiveness against drug resistant human cancer cell lines, for example, vinblastin resistant human acute Iymphoblastic Ie~ emi~ cells. Multidrug resiet~nce against anticancer compounds is one of the most forrnidable problems in cancer chemotherapy. In addition to their broad spectrum anticancer activily, the compounds of the present invention have potential clinical application due to their high potency, as shown by their relatively low IC50 concentrations (IC50 represents the concentration of compound required to ki:ll 50% of cancer cells in an in vitro assay). Moreover, as a derivative of a generally-recognized safe hop extract, HHC can be considered negligibly toxic when inpested, an advantage over many existing chemotherapeuticagents.
Hydrogenated lupulones appear to be more active and stable than their non-hydrogenated parent corrlpounds. For ~ ple, hexahydrocolupulone is more active than colupulone (see Mannering et al., cited supra) while hexahydrolupulone has been found to be more stable than lupulone (Carson, J. Amer Chem. Soc. 73 :1850-1852 (1951)).
According to the invention, cancer cells are inhibited by ~lministration to a m~mm~l afflicted with cancer of an effective amount of the compounds of Formula (I). The "effec1ive amount" will ultimately depend upon whether inhibition of growth or killin~ of c;mcer cells is goal of the treatment. However, as described W O 97131630 PCTrUS97/03073 herein, a suitable dose will be in the range of about 0.5 to about 100 mg/kg of body weight per day.
The composltions described herein are believed to be effective in the treatment of solid m~mm~ n tumors or hematologic m~lign~ncies, and include those which can develop multidrug resistance. These solid tumors include cancersof the head and nec~;, lung, mesothelioma, mediastinum, esophagus, stomach, pancreas, hepatobiliary system, small intestine, colon, rectum, anus, kidney, ureter~
bladder, prostate, urethra, penis, testis, gynecological organs, ovarian, breast, endocrine system, skin central nervous system; sarcomas of the soft tissue and bone;
and melanoma of cutaneous and intraocular origin. Hematological m~ n~ieS
include childhood leukemia and lymphom~, Hodgkin's (1isç~e' Iymphomas of lymphocytic and cutaneous origin, acute and chronic lellk~mi:~ plasma cell neoplasm and cancers associated with AIDS. The pl~f.,.led m~mm~ n species for lled~ elll are h~ n~ and domesticated ~nim~
Without being bound by any particular theory regarding mel~h~ni~m of action, prelimin~ry resull:s using the fluorescent activated cell sorting (FACS)method suggest that HHC has an effect upon the (Jl-S phase transition of the cell cycle. Other studies suggest that the cytotoxic eff'ect of HHC on cancer cells is not due to major effects upon DNA, RNA or protein synthesis.
In another embodiiment of the invention, a compound of formula (I), or a salt or ph~rm~ce~ltic~l composition thereof, is used to inhibit bacterial cell growth. In a pfc;relled method7 the ba~,teria is a gram positive ~acteria. In a more pref~,edembodiment, the gram p~DSitiVe bacteria is S~aphylococcus Aureus. In another preferred embodiment, the bacteria is a mycobacterium. In a more pl~r~ d 2~ embodiment, the mycobacteria is tuherculosis or mycobacterial avian complex (MHC).
In a further embodiment of the invention, a compound of forrnula (1), or a salt or a pharmaceutical composition thereof, is useful as a pdl~scilicidal agent, for W O 97/31630 PCT~US97103073 example as shown in Example VII hereinbelow. 1 he compounds of the invention can be used against the hemoflagellates, such as against lei.~hmAniA
Hexahydrolupulones are hydrogenated derivatives of lupulones, a constituent of hops (up to 5-7%). He~ahydrocolupulone can be made via the chemical 5 hydrogenation of colupulone using a number of methods known in the art. For example, hydrogenation can be achieved with platinum (IV) oxide as a catalyst asdescribed by Riedl (Ber. 89:1863 (1956)) or by Carson (J. Am Chem. Soc. 73:1850 (1951)).
~n a ~lcl~ d method, beta-acids are purified as described in U.S. patent no.
10 4,918,240 and hydrogenated as described in U.S. patent no. 5,082,975 (both ofwhich are herein incorporated in their entireties). ~ccording to a preferred method of purifying beta acids, palladium or plAtimlm catalyst poisons are removed from an aqueous Alk~line beta acid solution by agitating the solution at a pH of at least about l 0, preferably between about l l and l 2.9, in the presence of polyvalent metals other 15 than palladium and platinum. The insoluble catalyst poison contAining materials are then separated.
Preferably, the polyvalent metals used are edible metal ions, preferably magnesium and calcium, and may also be zinc, alllminl-rn or iron. Preferably, the pH is bet~,veen about 11 and l 2.9. Although a solvent is not necessA. ~ for removal 20 of the catalyst poisons, water-immiscible food-grade solvents may be used.
Examples include hvdrocarbons such as hexane, esters, liquid fatty alcohols~ andterpenes such as limonen In pl~p~lion for the ensuing hydrogenation step, the purified beta acids may be recovered from the ~lkAline aqueous phase by the CO2 method of Cowles 25 (U.S. patent no. 4,590,296). Alternatively, the bela-acid solution is mixed with about an equal volume oi' an organic solvent such as hexane. The beta-acids are extracted into solvent by slow acidification with agitation, using an acid such as phosphoric acid, until a pH of about 9.5-9.8 is reached. Substantially all of the beta acids are removed from the aqueous phase by this procedure. The beta acids may W O 97~31630 PCTAJS97/03073 then be recovered from the solvent by partial evaporation and cooling, or by removal of the hexane under vacuum, by procedures known to the art.
Any known method of hydrogenation ma~ be used, although preferably one is used which results in a high purity of beta acids. Preferably, the purified beta-S acids are hydrogenated under non-acidic conditions. According to a preferred method, the beta-acids are dissolved in solvent and water. Any non-toxic, non-reactive solvent may be used such as lower alkanols, an organic water miscible or THF. A hydrogenation catalyst such as palladium or platinum is added. The vesselis evacuated, hydrogen ~as is introduced, and the vessel is agitated until hydrogen 10 uptake has ceased. The hydro~ton~ted product is filtered to remove the catalyst. A
solution of water and a hydrocarbon solvent such as hexane is then added. The mixture is ~git~ted and tl~ hydrogenated beta acid is extracted into the hexane phase. After partial evaporation of the hexane, the mixture is seeded and cooled, allowing the product to crystallize.
To allow conversion to pure hydrogenated beta acids, the minimum pH used is about 3, preferably above 4, with the most preferred range between about 7 to 9.
The rate of hyclrogenation may be accelerated through the use of additional amounts of catalyst or by raising the te~ c~ c.
An aqueous liquid ~lk~line solution of hydrolupulones may be prepared as described in U.S. patent 5,082,975. Alternatively, propylene glycol, glycerine, similar stable alcohols and polyols, or rnixtures thereof with or ~,vithout water, may be substituted for the water of the aqueous solution. In addition, the foregoingaqueous solution may be mixed with glycol or glycerine, etc., to form a standardized solution of product, which is readily-dispersible in water~ and stable as well.
Ph~ eutically acceptable salts of the biologically active compounds described herein may be used as well in practicing the claimed methods.
Pharmaceutically acceptable salts may be formed using organic or inorganic bases, such as NaOH, Na(CO3)2, NaHCO3, KOH, amines and the like.
W O 97/31630 PCTrUS97103073 Although the compounds described herein and/or their its salts may be ministered as the pure chemicals, it is preferable to present the active ingredient as a pharmaceutical composition. The invention thus further provides the use of a ph~rm~ce.~ltical composition compri~i~g one or more compounds and/or a 5 pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable carriers therefor and, optionally, other therapeuticand/or prophylactic ingredients. The carrier(s) must be 'acceptable' in the sense of being compatible wlth the other ingredients of the composition and not deleterious to the recipient thereof.
Pharmaceutical compositions include those suitable for oral or parenteral (including intramuscular, ~lbcul;~leous and intravenous) ~lrnini~tration The compositions may, where a~pro~ ate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound ~,vith liquid carriers, solid m~triceS, semi-solid carriers, finely divided solid carriers or combination thereof, and then, if necess~ry, shaping the product into the desired delivery system. Such methods also include encapsulation of a therapeutically effective amount of the active ingredient in liposomes, by methods known to the art.
Pharmaceutlcal cDmpositions suitable for oral ~lmini~t~ation may be presented as discrete unit dosage forms such as hard or soft gelatin capsules, cachets or tablets each cont~ining a predetçrmin~d amount of the active ingredient; as apowder, as liposomal ~ dlions, or as granules as a solution, a suspension or as an emulsion.
Compositions suitable for topical ~mini~tration in the mouth include unit dosage forms such as loi~enges comprising active ingredient in a flavored base, usually sucrose and acadlia or fr~r~nth; pastilles comprising the active ingredient in an inert base such as ~elatin and glycerin or sucrose and acacia; muco~(lherent gels, and mouthwashes comprising the active ingredient in a suitable liquid carrier.
W O 97/31630 PCTrUS97/03073 When desired, the abo~,-e-described compositions can be adapted to provide sustained release of'the ac:tive ingredient employe(l, e.g., by combination thereof vvith certain hydrophilic polymer matrices, e.g., comprising natural gels, synthetic polymer gels or mixtures thereof.
For topical ~lmin i~tratïon to the epidermis, the active ingredients may be formulated as ointments, creams or lotions, or patc hes. Ointments and creams may, for example, be formulated with an a~ueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also conta~in one or more emulsifying agents, stabilizing agents., dispe~ g agents, s~ en.l ;.~g agents, thicl~nin~ agents, or coloring agents. The active ingredient can also be delivered via iontophoresis from a suitable reservoir, e.g., as disclosed in U.S. Patent Nos. 4,140,122;
4,383,529; or4,051.842.
The active ingredient can be delivered via patches for transdermal ~ imini~tration. 'Suitable tr~n.c~l~rmal de~ivery systems are disclosed, for exarnple, in U.S. PatentNo. 4,788,603, U.S. PatentNo. 4,931,279, U.S. PatentNo. 4,668,506, U.S. PatentNo. 4,713,224 and U.S. PatentNo. 5,560,922. Patches fortr~ clc-rnn~l delivery can comprise a backing layer and a polymer matrix which has dispersed or dissolved therein an effec,tive amiount of the active ingredient, along with one or more skin permeation çnln~ncers. The backing layer can be made of any suitable material which is imperrmeable to the analogs or derivatives of the invention. The backing layer serves as a protective cover for the matrix layer and provides also a support function. The baicking can be formed so that it is ec.centi~lly the same size layer as the polymer matrix or it ca]n be of larger ~iimen.~jon so that it can extend beyond the side of the polymer matrix or overlay the side or sides of the polymer matrix and then can exte:nd outwardly in a manner that the surface of the extension of the backing layer can be the base for an adhesive means. Alternatively, the polymer matrix can contain, or be form~ te~l of, an adhesive polymer, such as polyacrylate or acrylate/vinyl acetate copolymer. For long-term applications it W O 97/31630 PCTrUS97/03073 I() might be desirable to use microporous andlor breathable backing l~min:~te~, so hydration or maceration of the skin can be minimi7çd.
Examples ol' materials suitable for makin~ the backing layer are films of high and low density polyethylene, polypropylene, polyurethane, polyvinylchloride, polyesters such as poly(ethylene phth~l~te), metal foils, metal foil l~min~tes of such suitable polymer films, and the like. Preferably, the materials' used for the backing layer are l~min~tes of such polymer films with a metal foil such as al--minl-m foil.
In such l~minAtec, a polymer film of the l~min~te will usually be in contact with the a&esive polymer matrix. The backing layer can be any ap~ ,p.iate thickness whichwill provide the desired pr~ Ic.;li~e and support functions. A suitable thickness will be from about 10 to about 200 microns.
Generally. those polymers used to form the biologically acceptable adhesive polymer layer are those c apable of forming shaped bodies, thin walls or coatings through which therapeutic agents can pass at a controlled rate. Suitable polymers are biologically and pharmaceutically compatible? nonallergenic and insoluble inand compatible with body fluids or tissues with which the device is contacted. The use of soluble polymers i s to be avoided since dissolution or erosion of the matrix by skin moisture would affect the release rate of the therapeutic agents as well as the capability of the dosage lmit to remain in place for convenience of removal.
Exemplary materials for fabricating the adhesive polymer layer include polyethylene, polypropy]ene, polyurethane, ethylene/propylene copolymers, ethylene/ethylacrylate copolymers, ethylene/vinyl acetate copolymers, silicone elastomers, especially the medical-grade polydimethylsiloxanes, neoprene rubber,polyisobutylene, polyacrylates, chlorinated polyethyleae, polyvinyl chloride, vinyl chloride-vinyl acetate copolymer, crosslinked polymethacrylate polymers (hydro-gel), polyvinylidene chloride, poly(ethylene terephth~l~te), butyl rubber, epichlorohydrin rubbers, ethylenvinyl alcohol copolymers, ethylene-vinyloxyethanol copolymers; silicone copolymers, for example, polysiloxane-polycarbonate copolymers, polysiloxanepolyethylene oxide copolymers, W O 97/31630 PCT~US97/03073 polysiloxane-polymethaclylate copolymers, polysiloxane-alkylene copolymers (e.g,polysiloxane-ethylene copolymers), polysiloxane-~lkylenesilane copolymers (e.g.,polysiloxane-ethylenesilane copolymers), and the like; cellulose polymers, for example methyl or ethyl c:ellulose, hydroxypropyl methyl cellulose, and cellulose 5 esters; polycarbonates; polytetrafluoroethylene; and the like.
Preferably, a biolagically acceptable adheslve polymer matrix should be selected from polymers with glass transition t~ cldLIlres below room temperature.
The polymer may, but need not neces~rily, have a degree of crystallinity at roomte.~ dlule. Cross-linkirlg monomeric units or sites can be incol~ul~led into such 10 polymers. For example, c ross-linking monomers can be incorporated into polyacrylate polymers, which provide sites for cross-linking the matrix after dispersing the th~ ulic agent into the polymer. Known cross-linking monomers for polyacrylate polymers include polym~th~.~rylic esters of polyols such as butylene diacrylate and dimethacrylate, trimethylol propane trimethacrylate and the l;ke.15 Other monomers which p:rovide such sites include allyl acrylate, allyl methacrylate, diallyl maleate and the lik:e.
Preferably, a plasticizer and/or humectant is dispersed within the adhesive polymer matrix. Water-soluble polyols are generally suitable for this purpose.
Incorporation of a hllm~c1~nt in the form~ tion allows the dosage unit to absorb20 moisture on the sur~Face o,F skin which in turn helps to reduce skin irritation and to prevent the adhesive polymer layer of the delivery system from failing.
The~a~ulic agents released from a transdermal delivery system must be capable of penetrating each layer of skin. In order to increase the rate of permeation of a therapeutic agent, a penetration agent, such as a fatty alcohol or a glycol such as 25 propylene glycol can be used to increase the permeability of the outermost layer of skin, the stratum corneum, to the th.,. a~;ulic agent.
The active agent or derivative may also be formulated so as to be suitable for ~1mini~tration by inhalation or in~llffl~tion or for nasal, intraocular or other topical (including buccal and sub-lingual) ~rlmini~tration. For example, for ~lmini~tration W O 97/31630 PCT~US97tO3073 to the upper (nasal) or lovver respiratory tract by inhalation, the active agent or derivative is conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient :means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such ~ dichlorodifluoromethane, 5 trichlorofluoromethane, dichlorotetrafluoroethane. carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.
Alternativel~, for ~tlmini~tration by inhalation or insuffl~tion, the active agent may take the form of a dry powder composition, for example, a powder mix of 10 the active agent and a suitable powder base such as lactose or starch. The powder composition may be l,lest;~ d in unit dosage form in, for example, capsules or cartridges or, e.g., gelatin or blister packs from which the powder may be z~r1mini~tered ~,vith the aid of an inh~l~tor, in~ tor or a metered-dose inhaler.
For intra-nasal ~f~mini.~tration, the active agent may also be ~mini~tered via 15 nose drops, a liquid spray, such as via a plastic bottle ~mi;~el or metered-dose inhaler. Typical of atomizers are the Mistometer(~' (Wintrop) and the Medihaler~~Riker).
The pharmac;eutical compositions according to the invention may also contain other adjuvants such as flavorings, coloring, antirnicrobial agents, or 20 preservatives.
It will be further appreciated that the amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of a~lmini~tration, the nature of the condition being treated and the age and condition of the patient and will be 25 ultimately at the discretion of the a1tenl1~nt physician or clinician.
In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mglkg, e.g., from about 10 to about 75 mglkg of body weight per day, such as 3 to about 51) mg per kilogram body weight of the recipient per day, W O 97131630 PCT~US97/03073 preferably in the range of 6 to 90 mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.
The compound is conveniently ~1mini~tered in unit dosage form; for example, co~ g 5 to 1000 mg, conveniently 1() to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage i'orm.
Ideally, the active ingredient should be ~11mini~tered to achieve peak plasma concentrations of the acti ve compound of from about 0.5 to about 75 IlM, preferably, about 1 to 50 ~lM, most preferably~ about 2 to about 30 ~M. This maybe achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally ~rlmini.et~ed as a bolus c~ about 1-100 mg of the active ingredient. Desirable blood levels may be m~int~in.ocl by continuous infilsion to provide about 0.01-5.0 mglkg/hr or by inte~ Lenl infusions cont~inin~ about 0.4-15 mgJkg of the active ingredient(s).
The desired dose may conveniently be presented in a single dose or as divided doses ~-innini~tt~.red at a~rop.;ate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced ~mini~trations, such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.
All publications, pates~ts and patent docurnents are incorporated by reference herein, as though individually incoll,ulaled by reference. The invention has been described with reference to various specific and p~ ef~ d embodiments and techniques. However, it should be understood that many variations and modifications may be made while rern~ining within the spirit and scope of the invention.
The following examples are intended to illustrate but not limit the invention.
EX~MPLES
EXAMPLE I - Cytostatic Acti~ib W O97/31630 PCT~US97/03073 HHC was tested for cytostatic activity using the trypan blue exclusion assay.
The purpose of the assay i s to determine the concentration of HHC at which 50~/O of Gell growth is inhibited (i.e., ID50) when cells are continuously exposed to the HHC.
I~ive ml of a mixture of 2 x 105 cells/ml were placed in T-25 flasks. HHC or control 5 ~no HHC or DMSO vehicle) was added. The cells were incubated at 37~C under 5% CO~ for 24 or 4~ hours. Monolayer cells which are attached need to be incubated for a minimum of 14 hours before HHC is added in order to allow the cells to attach. The cells are detached by pancre~tini7ing (~t~Zlchf~d cells) and or suspended well (lm~ hed cells). A 100 ~11 sample was removed from the cell 10~ suspension and placed in a borosilicate glass test tube. One hundred ~11 of .4%
trypan blue solution (trypan blue (Sigma) in phosphate buffered saline) was added to the cell suspension sample and mixed well. The cells were counted using a hemocytometer. ID~o was calculated by choosing a drug concentration that was below 50% inhibition as well as a drug con~Pntration that was above 50% inhibition 15 and then interpolating those values to 50% inhibition. The following formula was used:
% inhibition = 100 - % growth, where % growth = [(Ct - C0)/(CC, - C0)~ x ] 00, where C0 is the cell count at time "0";
C, is the cell count of the HHC treated sample at time "t"; and CCt is the c~ll count of the control sample at time "t".
Table I presents d.~ta on the activity of HHC against various cancer cell lines.25 MCF-7 cells are human breast adenocarcinoma cells which have some characteristics of differentiated m~mmZ~ry epithelium (i.e., dome formation and estradiol processing via cytoplasmic estrogen receptors). CEM cells are human acute Iymphoblastic leukemia cells which morphologically resemble Iymphoblastic cells. CEM vinblastin resistant cells contain the P-glycoprotein therefore enabling 30 them to exhibit multidrug resistance to many structurally unrelated compounds.
Raji cells are human Burlcitt Iymphoma cells which are Iymphoblast-like. KB cells CA 02247370 l998-08-26 W O 97/31630 PCTrUS97/03073 are human oral epidermoiid earcinoma cells. HeLa S3 cells are human cervical epithelioid careinoma cells.
Table I
5 Cell type no. of ~c.;l.lents ID50 (~lM) standard deviation MCF-7' 3 1.23 0.17 CEM' 4 1.97 0.34 CEM vin resistant' 4 2.61 0.33 Raji2 4 2.19 0.40 KB' 3 0.85 0.07 HeLa S32 3 1.21 0.03 'period of incubation = 4,Bh; 2period of ineubation = 24h.
As the results indieate, HHC is an effeetive and potent cytostatic agent with respect to a wide variety of eancer eell types.
EXAMPLE II - Cytotoxic Activity HHC was tested for cytotoxic activity using a colony formation assay, using 20 an 18 hour pulse exposure. The purpose of the clonogenic assay is to determine the eoneentration of HHC at whieh 50% of eell growth is inhibited (i.e., IC50) when eells are exposed to the HHC for a defined amount of time (i.e., a pulse exposure to the xenobiotic).
Five ml of a mixture of 2 x 10~ eells/ml (MCF-7 eells) were placed in eaeh 25 of 4 T-25 flasks in a media of RPMI (Gibeo). The cells were incubated at 37 "C
under 5 % CO2 for a minimnn~ of 14 hours to allow the cells to attaeh. The cellswere pulsed for 18 hours with HHC coneentrations of 0, 1.5, and 25 ~lM, or with DMSO.
The media was removed from the flasks (i.e., media with floating cells) and 30 placed in 15 ml polypropylene centrifuge tubes 1-4 (summarized in Table II). The W O 97131630 PCT~US97/03073 flasks were washed in 1 ml of PBS and the wash was placed in the ap~rol"iate tubes 1-4. Tubes 1-4 were centrifuged at l 50 rpm and the media was aspirated. Cells were detached from 1he flasks by pancre~tini7~tion Approximately 1 ml of the cell suspension from the flask, were added to the appropriate tubes (1-4) and the pellet 5 suspended. The cells in tubes 1-4 were pooled with the cells in the flask. The cells were then resuspended.
One ml of ce~l sus]pension from the flask was added to the applopliate l0x tube (tubes 5-8) along with 9 ml of media and suspended well. One ml of the suspension in each l0x tube was then placed in the a~plop.iate 50x tube (tubes 9-l0 12) along with 4 ml of media.
Table II
controlb DMSO 1.5 ,uM HHC 25 ~lMHHC
floating cells ] a 2 3 4 l0x dilution .5 6 7 8 50x dilution '~ 10 l l l2 a tube no. b[HHC]=0 The cells in the original cell sl.~p~n.~ion (i.e., in the flask) were counted usingtrypan blue exclusion assay (described in Example II). Based on this count, the number of cells in the 50x tubes were calculated. The volume n~cess~ to acquire approximately 5400 cells was then determine~l This volume was removed from the 25 50x suspension and placed into 50 ml polypropylene tubes (tubes 13- l 6). Media was added to attain a volume of l 8 ml. The cells were then suspended.
Five ml of suspension from each of tubes l 3-16 was transferred to 60 x 15 m~n tissue culture dishes l~in triplicate). The cells were incubated until colonies of at least 50 cells were present (approx. 6-8 days). The cells were then stained with30 Giemsa stain (a solution of 1% Giemsa stain (Sigma) in 100% methanol). The W O 97131630 PCT~US97/03073 culture dishes were remo~red from the incubator and the media aspirated off. Thedishes were washed 2x with phosphate buffered saline (3-4 ml). Three to four ml of' 100% ethanol was added l~o each dish, an arnount sufficient to cover the bottom of the dish, covered and allo-wed to stand for 30 minutes. The stain was washed out5 with cold H2O. The water was poured off, without loss of colonies. The plates were inverted and allowed to dry. The cells were counted in those colonies which consisted of at least ~0 cells. The ID50 was determined as described in Example II.
The data is shown in Tab]e III.
10 Table III
Cell type no. oi ~ IDso (~lM)Std. deviation MCF-7 2 1.62 0.28 . ..
As the data shows, HHC is able to effectively kill MCF-7 cancer cells at low 1 5 dosages.
Preliminary data suggests that the in vitro ID50 of HHC against C1300 cells ~mouse neuroblastoma) is approximately 1.94 ~M after 48 hours of continuous exposure.
20 EXAMPLE III - In Vivo Studies The maximal tole:rated dose (MTD) of HHC was deterrnined in BDF 1 mice by i.p. injection. Mlce w~re able to tolerate the equivalent of 200 ~Lmolar HHC with no apparent side et'fects.
25 EXAIVIP~E IV - Determination of Mechanism of Action Fluorescence activated cell sorting (FACS) was used to determine what percentage of cells could be found in the designated cell cycle phase after 48 hours of continuous HHC exposure. There are four distinct phases in the cell cycle which lead to duplication of the cell and its genetic material. In the G0/GI phase the cell is W O 97/31630 PCTrUS97/03073 at rest or is performing miscellaneous biosynthetic fimctions. L)uring the S phase, DNA synthesis occurs which leads to replication o~'the chromosomes. G2 is the pre-mitotic interval urhile the M phase is the time during which the cell divides.
Cells were exposed to concentrations of 1.5 ~lM and 25 IlM of HHC, and S DMSO. DMSO had no significant effect on the cell cycle, with a profile almost identical to that of the "no drug" control. The results indicate that low [HHC~'s (i.e., 1.5 ~M) increase the number of cells in G0/GI and decrease the number of cells in S
phase. At high [HHC~]'s (i.e., 25 ~lM) there is a decrease in the number of cells in GOIG1 and an increase in lhe nurnber of cells in S phase. This indicates that HHC
10 has an al)pd~el1t effect upon the Gl-S phase transition of the cell cycle.
EXAMPLE V - Antih~terial Activity (S'~ ' J)lococcus aureus) A single colony of Staphylococcus aureus was grown in 2 ml of Luria-Bertani (LB) broth overnight at 37~C in a bacterial shaker with vigorous ~h~king.
15 0.5 ml of the culture was transferred to 250 ml LB and allowed to grow at 37 ~C
until the OD600 (optical density) reading was about 0.2 (a reading of I OD600 indicates a cell concentrati on of approximately 8 x l o8 cells/ml). Cultures were aliquoted into 50 ml tubes co..1Slini~g 13 ml of bacterial culture per tube. 0.5, 1.0, 2.5, 5.0, 10 and 25 ~M of HHC was added to the tubes. No-drug and DMSO
solvent were used as controls. Aliquots of the bacterial culture were taken every 2 hours to obtain the OD60() reading. As the results ïndicate (Figure 1) HHC is aneffective inhibitor of bacterial growth.
EXAMPLE VI - Antibacterial Activity (M. tu~erculosis) Drug susceptibility was tested using the BACTEC method (Siddigi, BACTEC TB System Product and Procedure Manual, Becton Dickinson Corp ( 1989); Inderlied, Antimycobac terial Agents in Antibiotics in l,aboratory Medicine, ed. Lorian, 3rd edition, Williams and Wilkins). The growth curve of mycobacteriain liquid media with and without HHC is plotted for 4 to 12 days. Resistance is W O97t31630 PCT~US97/03073 determined by modification of the 1% proportional method. The control vial is inoculated with a l: l O0 d:ilution of org~ni.~ms, and a growth rate greater than vials with HHC is interpreted as evidence of susceptibility. Resistance occurs where less than 99% of the org~ni.cm.c are inhibited by the HHC.
Materials:
1 ) BACTEC I 2B vials 2) HHC
3) Special Diluting Fluid (DF): 0.2% fatty acid free bovine serum albumin and 0.02% polysorbate 8() in distilled water, adjusted to pH 6.8 + 0.2 ~ ~n.ced and sterilized in the follo~,ving alloquots: a) 3.0 ml with 8-10 glass beads (1-2 mm/ea); b) 1.5 ml; c) 9.9 ml; and d) 9.0 ml 4) 0.5 McFarland standard in the same tube as (3) above 5) disposable sterile culture loops 6) 1.0 ml tuberculin syringes with fixed 22-26 gauge needle 7) individually packaged alcohol wipes 8) Vortex mixer 9) ATCC 27294 (S.I.R.E. susceptible M. tuberculosis) A quantity of growth of ATCC 27294 is removed from the solid media with a sterile applicator stick and placed in a tube with glass beads and DF. Vortex is used to emulsify bacteria~ clumps. 0.1 ml of this s..cp~n.~ion is used to inoculate new, pre-gassed BACTEIC vials. The vials are incubated at 37~C and monitored da~ily. The culture is readly for inoculation into the HHC and control via~s on the day when the growth index (GI) reaches 900-999.
Vials that have been at 999 for more than one day should not be used, as the bacterial load may be toa high. Control vials, inoculated with grow~ below an index of 900, do not relia~bly reach the test cutoff value of 30 in the required 12 day e~ ental period. Selial BACTEC-to-BACTEC transfers of the ATCC
org~ni.~m~ are acceptable within the confines of the experimental period.
W O97/31630 PCTrUS97103073 BACTEC vials ar- run on the BACTEC instrument to establish proper CO2 levels. The HHC is inoculated aseptically (0.1 ml HHC solution/vial) into labeled vials. Each HHC vial, and one DF vial Cont~inin~ 9.9 ml DF, is inoculated with 0. l ml of the ATCC BAC'TEC culture. The DF vial, now containing a 1:100 S dilution of the bacterial suspension, is inverted 1 OX to mix and 0.1 ml withdrawn to inoculate the controi 12B vial. A 1:10 control is made in the sarne manner as above, using a vial with 9.C ml r)F and 1.0 ml of the AT('C BACTEC culture. All test vials are incubatçd at 37~C ~ 1 ~C for 4 to 12 days. with daily reading on the BACTEC instrument. The vials must be run at approximately the sarne time each 1 0 day.
The test is fini~hPd when the GI of the control vial reaches 30 and at least S days have llc~llspiled. 1 he change in GI from one reading to another (delta change) is indicative of the growth rate. If the change is greater in the HHC vial compared to the control, the isolate is resistant to the HHC; if' about the same, the 15 isolate is borderline in susceptibility; and if less than the control, the isolate is susceptible to HHC If the GI in the HHC vials is initially very high ()300) and the GI in the control is ]ow, the inoculurn probably included clumps, and the test needs to be repeated. If the GI in a particular HHC vial has reached 500 and then declines when the control reaches 30, the isolate is considered resistant to HHC. If the GI in 20 a HHC vial reaches 900 cmd then declines before the control reaches 30, the isolate is considered resistant. If the GI of a vial reaches 999 before the control reaches 30, the inoculum was too heavy, and the test needs to be repeated. If the GI reaches 999 on the same day as the control reaches 30, and the delta GI is greater than the control, resistance can be reported. However susceptibility carmot be deterrnined 25 with a reading of 999, and the test would need to be repeated. If the delta GI for the HHC vial is close ( 10%) to the delta GI of the control this indicates partial resistance, and additional readings (1-3 days) should be taken. The 1:10 control is not used as part of a standard 1% proportional method, but is included here to measure smaller dmg effects that may not be ~I)pdlel1t with the standard 1% method.
wo 97/31630 PCT/US97/03073 EXAMPLE VII - Activity of HHC Against Leishmania Direct susceptibility of lei~hm~ni~ was examined using gross inhibition.
After 6 and 24 hours exposure of lei~hm~ni:~ to HHC, lei~hm~niA were fixed using5 formaldehyde and cells were counted on a hemacylometer. The results showed that after 24 hours exposure to 2.5 IlM HHC, 90% of lei~hm~nia growth was inhibited.
All publications? ~atents and patent documents are incorporated by reference herein, as though individu.ally incorporated by reference The invention has been10 described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while rem~inin~ within the spirit and scope of the invention.
Vials that have been at 999 for more than one day should not be used, as the bacterial load may be toa high. Control vials, inoculated with grow~ below an index of 900, do not relia~bly reach the test cutoff value of 30 in the required 12 day e~ ental period. Selial BACTEC-to-BACTEC transfers of the ATCC
org~ni.~m~ are acceptable within the confines of the experimental period.
W O97/31630 PCTrUS97103073 BACTEC vials ar- run on the BACTEC instrument to establish proper CO2 levels. The HHC is inoculated aseptically (0.1 ml HHC solution/vial) into labeled vials. Each HHC vial, and one DF vial Cont~inin~ 9.9 ml DF, is inoculated with 0. l ml of the ATCC BAC'TEC culture. The DF vial, now containing a 1:100 S dilution of the bacterial suspension, is inverted 1 OX to mix and 0.1 ml withdrawn to inoculate the controi 12B vial. A 1:10 control is made in the sarne manner as above, using a vial with 9.C ml r)F and 1.0 ml of the AT('C BACTEC culture. All test vials are incubatçd at 37~C ~ 1 ~C for 4 to 12 days. with daily reading on the BACTEC instrument. The vials must be run at approximately the sarne time each 1 0 day.
The test is fini~hPd when the GI of the control vial reaches 30 and at least S days have llc~llspiled. 1 he change in GI from one reading to another (delta change) is indicative of the growth rate. If the change is greater in the HHC vial compared to the control, the isolate is resistant to the HHC; if' about the same, the 15 isolate is borderline in susceptibility; and if less than the control, the isolate is susceptible to HHC If the GI in the HHC vials is initially very high ()300) and the GI in the control is ]ow, the inoculurn probably included clumps, and the test needs to be repeated. If the GI in a particular HHC vial has reached 500 and then declines when the control reaches 30, the isolate is considered resistant to HHC. If the GI in 20 a HHC vial reaches 900 cmd then declines before the control reaches 30, the isolate is considered resistant. If the GI of a vial reaches 999 before the control reaches 30, the inoculum was too heavy, and the test needs to be repeated. If the GI reaches 999 on the same day as the control reaches 30, and the delta GI is greater than the control, resistance can be reported. However susceptibility carmot be deterrnined 25 with a reading of 999, and the test would need to be repeated. If the delta GI for the HHC vial is close ( 10%) to the delta GI of the control this indicates partial resistance, and additional readings (1-3 days) should be taken. The 1:10 control is not used as part of a standard 1% proportional method, but is included here to measure smaller dmg effects that may not be ~I)pdlel1t with the standard 1% method.
wo 97/31630 PCT/US97/03073 EXAMPLE VII - Activity of HHC Against Leishmania Direct susceptibility of lei~hm~ni~ was examined using gross inhibition.
After 6 and 24 hours exposure of lei~hm~ni:~ to HHC, lei~hm~niA were fixed using5 formaldehyde and cells were counted on a hemacylometer. The results showed that after 24 hours exposure to 2.5 IlM HHC, 90% of lei~hm~nia growth was inhibited.
All publications? ~atents and patent documents are incorporated by reference herein, as though individu.ally incorporated by reference The invention has been10 described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while rem~inin~ within the spirit and scope of the invention.
Claims (12)
1. The use of a compound of formula (I):
wherein R is (C3-C8)alkyl, or a pharmaceutically acceptable salt thereof to prepare a medicament effective to inhibit growth of mycobacterium avium complex.
wherein R is (C3-C8)alkyl, or a pharmaceutically acceptable salt thereof to prepare a medicament effective to inhibit growth of mycobacterium avium complex.
2. Claim 1 wherein R is -CH(CH3)2.
3. Claim 1 wherein R is -CH2CH(CH3)2, or -CH(CH3)(CH2CH3.
4. Claim 1 wherein the medicament comprises a mixture comprising two or more compounds of formula (I).
5. Claim 1 wherein the compound or its salt is combined with a pharmaceutically acceptable carrier.
6. Claim 5 wherein the carrier is a liquid vehicle.
7. Claim 6 wherein the medicament is adapted for parenteral administration.
8. Claim 5 wherein the medicament is adapted for topical administration.
9. Claim 5 wherein the medicament is adapted for administration by means of a transdermal patch.
10. Claim 8 wherein the medicament is adapted for administration by means of iontophoresis.
11. Claim 5 wherein the medicament is adapted for oral administration.
12. Claim 5 wherein the carrier is a liposome.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US60747396A | 1996-02-27 | 1996-02-27 | |
| US08/607,473 | 1996-02-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2247370A1 true CA2247370A1 (en) | 1997-09-04 |
Family
ID=24432432
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002247370A Abandoned CA2247370A1 (en) | 1996-02-27 | 1997-02-27 | Use of hexahydrolupulones as antibacterial and anticancer agents |
Country Status (5)
| Country | Link |
|---|---|
| US (2) | US5827895A (en) |
| EP (1) | EP0885003A1 (en) |
| JP (1) | JP2000505474A (en) |
| CA (1) | CA2247370A1 (en) |
| WO (1) | WO1997031630A1 (en) |
Families Citing this family (47)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5827895A (en) * | 1996-02-27 | 1998-10-27 | Regents Of The University Of Minnesota | Hexahydrolupulones useful as anticancer agents |
| US6165447A (en) * | 1999-06-15 | 2000-12-26 | Colgate-Palmolive Company | Synergistic antibacterial combination |
| US7005453B1 (en) | 1999-10-12 | 2006-02-28 | Miller Brewing Company | Use of hop acids to inhibit growth of Staphylococcus aureus and prevent toxic shock syndrome |
| US6547971B2 (en) | 2000-03-08 | 2003-04-15 | Hercules Incorporated | Methods of using hop acids to control organisms |
| US8293258B2 (en) * | 2000-05-18 | 2012-10-23 | John I. Hass, Inc. | Pesticide treatments made from hop extracts |
| US8153146B2 (en) * | 2000-05-18 | 2012-04-10 | John I. Haas | Pesticide and fungicide treatments made from hop extracts |
| US7105177B1 (en) | 2000-10-20 | 2006-09-12 | Miller Brewing Company | Antimicrobial diapers and wet wipes |
| US6352726B1 (en) * | 2001-01-09 | 2002-03-05 | S. S. Steiner, Inc. | Method of killing protozoa |
| CA2441074A1 (en) * | 2001-03-28 | 2002-10-10 | Hercules Incorporated | Method of using hop acids to control organisms |
| US7205151B2 (en) * | 2001-06-20 | 2007-04-17 | Metaproteomics, Llc | Complex mixtures exhibiting selective inhibition of cyclooxygenase-2 |
| US8168234B2 (en) * | 2001-06-20 | 2012-05-01 | Metaproteomics, Llc | Compositions that treat or inhibit pathological conditions associated with inflammatory response |
| US7901714B2 (en) * | 2001-06-20 | 2011-03-08 | Metaproteomics, Llp | Treatment modalities for autoimmune diseases |
| US7815944B2 (en) * | 2001-06-20 | 2010-10-19 | Metaproteomics, Llc | Anti-inflammatory pharmaceutical compositions for reducing inflammation and the treatment of prevention of gastric toxicity |
| US20040219240A1 (en) * | 2001-06-20 | 2004-11-04 | Babish John G. | Anti-inflammatory pharmaceutical compositions for reducing inflammation and the treatment or prevention of gastric toxicity |
| US7901713B2 (en) * | 2001-06-20 | 2011-03-08 | Metaproteomics, Llc | Inhibition of COX-2 and/or 5-LOX activity by fractions isolated or derived from hops |
| US7270835B2 (en) * | 2001-06-20 | 2007-09-18 | Metaproteomics, Llc | Compositions that treat or inhibit pathological conditions associated with inflammatory response |
| US8142819B2 (en) | 2002-10-21 | 2012-03-27 | Metaproteomics, Llc | Synergistic compositions that treat or inhibit pathological conditions associated with inflammatory response |
| US8206753B2 (en) * | 2001-06-20 | 2012-06-26 | Metaproteomics, Llc | Anti-inflammatory botanical products for the treatment of metabolic syndrome and diabetes |
| US7718198B2 (en) * | 2001-06-20 | 2010-05-18 | Metaproteomics, Llc | Treatment modalities for autoimmune diseases |
| US20040115290A1 (en) | 2001-06-20 | 2004-06-17 | Tripp Matthew L. | Modulation of inflammation by hops fractions and derivatives |
| US7279185B2 (en) | 2001-10-26 | 2007-10-09 | Metaproteonics, Llc | Curcuminoid compositions exhibiting synergistic inhibition of the expression and/or activity of cyclooxygenase-2 |
| US20090263522A1 (en) * | 2001-10-26 | 2009-10-22 | Babish John G | Curcuminoid Compositions Exhibiting Synergistic Inhibition Of The Expression And/Or Activity Of Cyclooxygenase-2 |
| US8158160B2 (en) | 2001-11-13 | 2012-04-17 | Eric Hauser Kuhrts | Anti-inflammatory cyclooxygenase inhibitors |
| US20030228814A1 (en) * | 2002-06-07 | 2003-12-11 | Barney Michael C. | Antibacterial packaging material including hop acids |
| NZ566511A (en) * | 2002-09-23 | 2009-10-30 | Haas Inc John I | Hop acids as a replacement for antibiotics in animal feed |
| JP2006508182A (en) * | 2002-10-21 | 2006-03-09 | メタプロテオミクス， エルエルシー | A composition for treating or inhibiting a pathological condition associated with an inflammatory response. |
| ATE421357T1 (en) * | 2003-05-22 | 2009-02-15 | Metaproteomics Llc | ANTI-INFLAMMATORY MEDICINAL PREPARATIONS FOR REDUCING INFLAMMATION AND THE THERAPY OR PROPHYLAXIS OF GASTRIC TOXICITY |
| US20050192356A1 (en) * | 2004-02-27 | 2005-09-01 | Babish John G. | Synergistic anti-inflammatory pharmaceutical compositions and methods of use |
| US7914831B2 (en) | 2004-02-27 | 2011-03-29 | Metaproteomics, Llc | Synergistic anti-inflammatory pharmaceutical compositions and related methods using curcuminoids or methylxanthines |
| NZ555219A (en) * | 2004-11-13 | 2011-03-31 | Metaproteomics Llc | Compositions exhibiting inhibition of cyclooxygenase-2 |
| US7324419B2 (en) * | 2004-12-11 | 2008-01-29 | Hewlett-Packard Development Company, L.P. | Focus control via AC input signal |
| MX2008001903A (en) * | 2005-08-09 | 2008-04-16 | Metaproteomics Llc | Protein kinase modulation by hops and acacia products. |
| US20070154576A1 (en) * | 2005-12-09 | 2007-07-05 | Tripp Matthew L | Protein kinase modulation by hops and Acacia products |
| CN101045678B (en) * | 2006-03-28 | 2010-05-26 | 新疆大学 | Method for producing hexahydrolupulicone |
| US7767234B2 (en) | 2006-03-31 | 2010-08-03 | John I. Haas, Inc. | Compositions and methods for controlling a honey bee parasitic mite |
| WO2007149504A2 (en) * | 2006-06-20 | 2007-12-27 | Metaproteomics, Llc | Isoalpha acid based protein kinase modulation cancer treatment |
| KR20090060360A (en) * | 2006-09-25 | 2009-06-11 | 하스, 존 아이. | Methods for treating or preventing infestation |
| EP2086312A2 (en) * | 2006-11-15 | 2009-08-12 | HAAS, John I. | Compositions and methods for inhibiting a honey bee pathogen infection on controlling a hive infestation |
| FR2910325B1 (en) * | 2006-12-22 | 2010-03-19 | Kronenbourg Brasseries | USE OF LUPULONES FOR THE PREVENTION AND THERAPY OF COLORECTAL CANCER. |
| WO2008115783A1 (en) * | 2007-03-19 | 2008-09-25 | Metaproteomics, Llc | Methods and compositions for promoting bone and joint health |
| AU2008251756B2 (en) | 2007-05-11 | 2013-06-06 | Metaproteomics, Llc | Methods and compositions for heavy metal detoxification |
| MX2010006425A (en) * | 2007-12-10 | 2010-08-31 | Metaproteomics Llc | Substituted 1,3-cyclopentadione multi-target protein kinase modulators of cancer, angiogenesis and the inflammatory pathways associated therewith. |
| US8414934B2 (en) * | 2008-02-08 | 2013-04-09 | John I. Haas, Inc. | Compositions and methods for arachnid control |
| WO2009124176A1 (en) * | 2008-04-02 | 2009-10-08 | Metaprotemics, Llc | Substituted 1,3-cyclopentadione attenuated endothelial inflammation and endothelial-monocyte interactions |
| CA2897371C (en) | 2013-01-07 | 2022-08-30 | John I. Haas, Inc. | Compositions and methods for controlling a honey bee parasitic mite infestation |
| CA3043388A1 (en) | 2018-05-14 | 2019-11-14 | John I. Hass, Inc. | Compositions and methods for controlling a honey bee parasitic mite infestation |
| CN113476431B (en) * | 2021-08-13 | 2023-07-11 | 广州英赛特生物技术有限公司 | Application of cyclotrisone derivative in preparation of animal medicines |
Family Cites Families (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4668504A (en) * | 1970-11-18 | 1987-05-26 | Merck & Co., Inc. | Use of substituted propenoates to prevent nephrotoxicity of certain antibiotics |
| DE2626348C3 (en) * | 1976-06-11 | 1980-01-31 | Siemens Ag, 1000 Berlin Und 8000 Muenchen | Implantable dosing device |
| US4383529A (en) * | 1980-11-03 | 1983-05-17 | Wescor, Inc. | Iontophoretic electrode device, method and gel insert |
| US4450270A (en) * | 1981-10-02 | 1984-05-22 | Eli Lilly And Company | Dioximino cephalosporin antibiotics |
| US4590296A (en) * | 1984-01-25 | 1986-05-20 | Miller Brewing Company | Process for separation of beta-acids from extract containing alpha-acids and beta-acids |
| US4783453A (en) * | 1985-06-10 | 1988-11-08 | Merck & Co., Inc. | 2-aza-substituted 1-carbadethiapen-2-em-3-carboxylic acids |
| US4931279A (en) * | 1985-08-16 | 1990-06-05 | Bausch & Lomb Incorporated | Sustained release formulation containing an ion-exchange resin |
| US4921857A (en) * | 1987-09-18 | 1990-05-01 | Merck & Co., Inc. | 4-Oxo-4h-quinolizine-3-carboxylic acids and derivatives thereof |
| US4918240A (en) * | 1988-08-15 | 1990-04-17 | Kalamazoo Holdings, Inc. | Purification of beta acids for hydrogenolysis and such purified beta acids |
| US5082975A (en) * | 1988-08-15 | 1992-01-21 | Kalamazoo Holdings, Inc. | Synthesis of hexahydrolupulone, novel forms thereof, and its use as a selective inhibitor of cell growth and multiplication |
| US5262151A (en) * | 1991-11-25 | 1993-11-16 | Montgomery Robert E | Stabilized enzymatic antimicrobial compositions |
| US5286506A (en) * | 1992-10-29 | 1994-02-15 | Bio-Technical Resources | Inhibition of food pathogens by hop acids |
| US5370863A (en) * | 1992-12-16 | 1994-12-06 | Miller Brewing Company | Oral care compositions containing hop acids and method |
| US5827895A (en) * | 1996-02-27 | 1998-10-27 | Regents Of The University Of Minnesota | Hexahydrolupulones useful as anticancer agents |
-
1996
- 1996-09-20 US US08/717,120 patent/US5827895A/en not_active Expired - Lifetime
-
1997
- 1997-02-27 JP JP9531114A patent/JP2000505474A/en active Pending
- 1997-02-27 WO PCT/US1997/003073 patent/WO1997031630A1/en not_active Application Discontinuation
- 1997-02-27 CA CA002247370A patent/CA2247370A1/en not_active Abandoned
- 1997-02-27 EP EP97908754A patent/EP0885003A1/en not_active Withdrawn
-
1999
- 1999-07-28 US US09/125,928 patent/US6313178B1/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| WO1997031630A1 (en) | 1997-09-04 |
| EP0885003A1 (en) | 1998-12-23 |
| JP2000505474A (en) | 2000-05-09 |
| US6313178B1 (en) | 2001-11-06 |
| US5827895A (en) | 1998-10-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2247370A1 (en) | Use of hexahydrolupulones as antibacterial and anticancer agents | |
| TWI823845B (en) | Combination therapy for the treatment of cancer | |
| Huang et al. | Mitochondria as a novel target for cancer chemoprevention: emergence of mitochondrial-targeting agents | |
| EP2654739B1 (en) | Percutaneous absorption preparation containing rivastigmine | |
| CN108699024B (en) | Benzothienyl selective estrogen receptor down-regulator compounds | |
| TW200412349A (en) | Eponemycin and epoxomicin analogs and uses thereof | |
| JP2018514511A (en) | Compositions and methods for the treatment of breast disorders and estrogen-related disorders | |
| KR102543015B1 (en) | Compounds, compositions and methods for the prevention and/or treatment of cancer | |
| AU2016298175B2 (en) | Compositions and methods of treating cancer | |
| EP2433636A1 (en) | Treatment of Malignant Diseases | |
| JP2004515453A (en) | Compositions and methods for protecting cells while subjecting cancer to chemotherapy and radiation therapy | |
| KR20010074814A (en) | Use of estrogenic compounds as anti-fungal agents | |
| US20220000794A1 (en) | Transdermal pharmaceutical formulations for the treatment of multiple sclerosis | |
| EP1589973B1 (en) | Formulation and methods for the treatment of thrombocythemia | |
| TW201609094A (en) | Novel methods for treating cancer | |
| US10610563B2 (en) | Use of ubiquitin-proteasome system inhibitors for treatment of tumors associated with neurofibromatosis type-2 | |
| WO1996025154A1 (en) | Transdermal formulation | |
| WO1996025042A1 (en) | Transdermal formulation | |
| US6545057B2 (en) | Tricyclic antidepressants and their analogues as long-acting local anesthetics and analgesics | |
| WO2008076229A1 (en) | Atomoxetine formulations and associated methods | |
| JP7376105B2 (en) | Topical compositions and treatment methods | |
| US7074961B2 (en) | Antidepressants and their analogues as long-acting local anesthetics and analgesics | |
| WO1998011883A1 (en) | Use of hexahydrolupulones as antibacterial agents | |
| KR20200051690A (en) | Topical composition | |
| WO2017188350A1 (en) | Method for inhibiting tumor growth |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |