WO2014184726A2 - Compounds and their use in therapy - Google Patents

Compounds and their use in therapy Download PDF

Info

Publication number
WO2014184726A2
WO2014184726A2 PCT/IB2014/061379 IB2014061379W WO2014184726A2 WO 2014184726 A2 WO2014184726 A2 WO 2014184726A2 IB 2014061379 W IB2014061379 W IB 2014061379W WO 2014184726 A2 WO2014184726 A2 WO 2014184726A2
Authority
WO
WIPO (PCT)
Prior art keywords
pharmaceutical formulation
formulation according
drug
lipid
phosphatidylcholine
Prior art date
Application number
PCT/IB2014/061379
Other languages
French (fr)
Other versions
WO2014184726A3 (en
Inventor
Albert VAN HELL
Marcel Verheij
Siewert Jan MARRINK
Gerben Koning
Original Assignee
Stichting Het Nederlands Kanker Instituut - Antoni Van Leeuwenhoek Ziekenhuis
Erasmus University Medical Centre
Rijksuniversiteit Groningen
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Stichting Het Nederlands Kanker Instituut - Antoni Van Leeuwenhoek Ziekenhuis, Erasmus University Medical Centre, Rijksuniversiteit Groningen filed Critical Stichting Het Nederlands Kanker Instituut - Antoni Van Leeuwenhoek Ziekenhuis
Publication of WO2014184726A2 publication Critical patent/WO2014184726A2/en
Publication of WO2014184726A3 publication Critical patent/WO2014184726A3/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/538—1,4-Oxazines, e.g. morpholine ortho- or peri-condensed with carbocyclic ring systems
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention pertains generally to the field of therapy and medical treatment, and more specifically to certain compounds (for convenience, collectively referred to herein as "short-chain phosphatidylcholine lipids" and “SCPC lipids”), which, inter alia, are useful in methods of improving drug delivery, bioavailability, and efficacy.
  • the present invention also pertains to pharmaceutical formulations, and more specifically to co-formulations of known drugs (e.g., amphiphilic drugs) (e.g., anthracyclines)
  • SCPC lipids e.g., doxorubicin
  • SCPC lipids e.g., doxorubicin
  • anthracycline doxorubicin has been in clinical use for several decades, and is still among the most widely used chemotherapeutic agents for treatment of a variety of neoplasms (see, e.g., Weiss, 1992; Zagotto et al., 2001 ; Lothstein et al., 2001).
  • Doxorubicin has several cytotoxic actions. It binds to DNA and inhibits both DNA and RNA synthesis, but its main cytotoxic action appears to be mediated through an effect on topoisomerase II (a DNA gyrase), the activity of which is markedly increased in proliferating cells.
  • topoisomerase II a DNA gyrase
  • the significance of the enzyme lies in the fact that during replication of the DNA helix, reversible swivelling needs to take place around the replication fork in order to prevent the daughter DNA molecule becoming inextricably entangled during mitotic segregation.
  • the swivel is produced by topoisomerase II, which nicks both DNA strands and subsequently reseals the breaks.
  • Doxorubicin intercalates in the DNA and its effect is, in essence, to stabilise the DNA-topoisomerase II complex after the strands have been nicked, thus causing the process to seize up at this point.
  • doxorubicin Compared to systemic application of doxorubicin in its free form, liposomal doxorubicin exhibits significant advantages, as for example reduced acute and chronic toxicities.
  • This coating prevents opsonization and reduces the uptake by macrophages from the reticulo-endothelial system, in turn resulting in prolonged circulation times, as compared to free doxorubicin or to non-coated liposomes (see, e.g., Vaage et al., 1992; Robert and Gianni, 1993; Gabizon et al., 1996; Uster ef a/., 1996).
  • PEG-liposome-encapsulated doxorubicin (commercially available as Caelyx® and Doxil®) is now in routine clinical use, and innovations such as the coupling of targeting-enhancing features (e.g., tumor cell specific antibodies or ligands) will further enhance its therapeutic value (see, e.g., Park et al., 2002; Pan et al., 2003; Koning et al., 1999; Koning et al., 2003).
  • Both Caelyx® and Doxil® consist of: doxorubicin hydrochloride (2 mg/mL); N-(carbonyl- methoxypolyethylene glycol 2000)-1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (MPEG2000-DSPE) (3.19 mg/mL); fully hydrogenated soy
  • phosphatidylcholine (9.58 mg/mL); cholesterol (3.19 mg/mL); ammonium sulfate ( ⁇ 2 mg/mL); histidine (as a buffer); hydrochloric acid and/or sodium hydroxide (for pH control); sucrose (to maintain isotonicity); and water-for-injection.
  • doxorubicin Given the intracellular localization of its molecular targets, sufficient cellular uptake of doxorubicin is required for its action (see, e.g., Speth et al., 1988; Lothstein et al., 2001). However, since doxorubicin does not possess the optimal degree of lipophilicity for efficient plasma membrane traversal, this might be a limiting factor for its efficacy (see, e.g., Heijn et al., 1999; Washington et al., 2001).
  • the inventors have demonstrated that the cellular uptake of free doxorubicin, and with that its cytotoxic action, is greatly enhanced by co-administration of a short-chain phosphatidylcholine lipid (also referred to herein as an SCPC lipid), as described herein.
  • a short-chain phosphatidylcholine lipid also referred to herein as an SCPC lipid
  • the inventors have demonstrated that the cytotoxic action of a number of drugs is increased, and in many cases, greatly increased, by co-administration of an SCPC lipid, as described herein.
  • SCPC lipid spontaneously inserts into lipid bilayers, and exchanges easily between membranes. Furthermore, it may also be expected that the effects demonstrated for doxorubicin will be observed for other drugs, especially other amphiphilic drugs.
  • the improved formulation offers many advantages.
  • the improved drug uptake permits the use of formulations with lower drug content to achieve the same result, thereby reducing undesired side effects, e.g., myocardial toxicity for doxorubicin.
  • One aspect of the present invention pertains to a pharmaceutical formulation comprising
  • a drug as described herein
  • a short-chain phosphatidylcholine lipid also referred to herein as an SCPC lipid
  • the pharmaceutical formulation is a pharmaceutical formulation comprising liposomes, said liposomes comprising said drug and said short-chain phosphatidylcholine lipid.
  • the pharmaceutical formulation is a pharmaceutical formulation comprising liposomes, said liposomes further comprise one or more or all of: additional phospholipid; cholesterol (CH); and a vesicle-forming lipid which is derivatized with a polymer chain.
  • Another aspect of the present invention pertains to a method of preparing a
  • composition comprising combining (i) a drug, as described herein, and
  • an SCPC lipid as described herein.
  • Another aspect of the present invention pertains to a pharmaceutical formulation comprising (i) a drug, as described herein, and (ii) an SCPC lipid, as described herein, for use in a method of treatment of the human or animal body by therapy.
  • Another aspect of the present invention pertains to a pharmaceutical formulation comprising (i) a drug, as described herein, and (ii) an SCPC lipid, as described herein, for use in a method of treatment of a disorder (e.g., a disease) as described herein.
  • a pharmaceutical formulation comprising (i) a drug, as described herein, and (ii) an SCPC lipid, as described herein, for use in a method of treatment of a disorder (e.g., a disease) as described herein.
  • Another aspect of the present invention pertains to a method of treatment, for example, of a disorder (e.g., a disease) as described herein, comprising administering to a patient in need of treatment a therapeutically effective amount of a pharmaceutical formulation comprising (i) a drug, as described herein, and (ii) an SCPC lipid, as described herein.
  • a pharmaceutical formulation comprising (i) a drug, as described herein, and (ii) an SCPC lipid, as described herein.
  • Another aspect of the present invention pertains to use of (i) a drug, as described herein, and (ii) an SCPC lipid, as described herein, in the manufacture of a medicament for the treatment of a disorder (e.g., a disease) as described herein.
  • a disorder e.g., a disease
  • Another aspect of the present invention pertains to a use of an SCPC lipid, as described herein, to increase the bioavailability of a drug, as described herein.
  • Another aspect of the present invention pertains to a use of an SCPC lipid, as described herein, to increase cellular uptake of a drug, as described herein.
  • Another aspect of the present invention pertains to a method of increasing the bioavailability of a drug, as described herein, in a patient, which method comprises the step of co-administering said drug and an SCPC lipid, as described herein.
  • Another aspect of the present invention pertains to a method of increasing the cellular uptake of a drug, as described herein, into a cell, which method comprises the step of contacting said cell with said drug and an SCPC lipid, as described herein.
  • a drug as described herein
  • SCPC lipid as described herein.
  • Figure 1 is a graph of doxorubicin content (expressed as % of control) as a function of concentration ( ⁇ ) of short-chain phosphatidyl-choline lipid, for five lipids: dinonaoyl-PC (C9), diundecanoyl-PC (C1 1), dilauroyl-PC (C12), ditridecanoyl-PC (C13), and dimyristoyl-PC (C14), as well as the comparison lipid, C8-GlcCer (N-octanoyl- glucosylceramide).
  • Figure 2 is a graph of cell viability (expressed as % of control) as a function of concentration ( ⁇ ) of short-chain phosphatidyl-choline lipid, for five lipids: dinonaoyl-PC (C9), diundecanoyl-PC (C1 1), dilauroyl-PC (C12), ditridecanoyl-PC (C13), and dimyristoyl-PC (C14), as well as the comparison lipid, C8-GlcCer (N-octanoyl- glucosylceramide).
  • dinonaoyl-PC C9
  • diundecanoyl-PC C1 1
  • dilauroyl-PC C12
  • ditridecanoyl-PC C13
  • dimyristoyl-PC C14
  • C8-GlcCer N-octanoyl- glucosylceramide
  • Figure 3 is a graph of cell viability (expressed as % of control) as a function of logarithm of concentration of dactinomycin, for dactinomycin alone (circles) and for dactinomycin with an SCPC lipid (triangles), specifically, dinonaoyl-PC (C9), at a concentration of 20 ⁇ .
  • an SCPC lipid increases bioavailability and/or cellular uptake of a drug (e.g., an amphiphilic drug, such as doxorubicin). Consequently, formulations comprising a drug and an SCPC lipid, as described herein, have important advantages, for example, a decreased dosage to achieve the same therapeutic effect.
  • a drug e.g., an amphiphilic drug, such as doxorubicin
  • one aspect of the present invention pertains to a pharmaceutical formulation comprising (i) a drug, as described herein, and (ii) a short-chain phosphatidylcholine lipid (also referred to herein as an SCPC lipid), as described herein.
  • a pharmaceutical formulation comprising (i) a drug, as described herein, and (ii) a short-chain phosphatidylcholine lipid (also referred to herein as an SCPC lipid), as described herein.
  • SCPC lipid short-chain phosphatidylcholine lipid
  • the drug is an amphiphilic drug.
  • amphiphilic is used herein in the conventional sense to mean a compound (e.g., a drug) that is both (i) hydrophilic and (ii) hydrophobic (e.g., lipophilic).
  • the delivery enhancement is not limited to a particular class of drugs (e.g., anthracyclines) but is in fact applicable to a range of drugs that are amphiphilic, for example, as characterised by their lipophilicity.
  • the amphiphilic drug has a log(lipophilicity) value of 0.4 to 16.
  • the lower limit is 0.5; the lower limit is 0.6; the lower limit is 0.8.
  • the upper limit is 12; the upper limit is 10; the upper limit is 8.
  • the range is 0.8 to 8.
  • lipophilicity is defined as the 1-octanol/water partitioning ratio.
  • the partitioning ratio is determined for 10-50 mM test compound (e.g., drug) using a two phase system consisting of 1 ml_ 1-octanol and 1 ml_ water. After addition of the test compound, tubes are vortexed and centrifuged (3000 rpm, 5 minutes, 20°C). Aliquots of both the organic and the aqueous phases are taken for fluorimetric quantification. Native fluorescence intensities are measured, for example, by a Perkin-Elmer Victor Wallac II fluorescence microplate reader, using lex 485 nm and lem 535 nm filters. All values are corrected for background fluorescence. As a measure of lipophilicity, the 1-octanol/water partitioning ratio is calculated and expressed on a log scale (see, for example,
  • the drug is an amphiphilic anti-proliferative drug.
  • the drug is an amphiphilic anti-cancer drug.
  • the drug is an amphiphilic anti-cancer drug from natural sources.
  • Anthracyclines are a class of drugs that originate from natural sources (specifically, derived from microorganisms), and are believed to act by DNA intercalation. Included among anthracyclines are anthracycline analogs.
  • amphiphilic anthracyclines examples include doxorubicin, daunorubicin, epirubicin, and aclarubicin.
  • amphiphilic anthracycline analogs include idarubicin, valrubicin, and mitoxantrone.
  • the drug is an anthracycline.
  • the drug is an amphiphilic anthracycline.
  • the drug is an anti-proliferative anthracycline.
  • the drug is an amphiphilic anti-proliferative anthracycline.
  • the drug is an anti-cancer anthracycline.
  • the drug is an amphiphilic anti-cancer anthracycline.
  • the drug is doxorubicin, daunorubicin, epirubicin, aclarubicin, idarubicin, valrubicin, or mitoxantrone; or a salt (e.g., an acid addition salt) thereof.
  • the drug is doxorubicin or a salt (e.g., an acid addition salt) thereof.
  • the drug is doxorubicin or doxorubicin hydrochloride.
  • Alkaloids are a class of drugs that originate from natural sources (specifically, derived from plants), and are believed to act by inhibiting topoisomerase I . Included among alkaloids are alkaloid analogs.
  • amphiphilic alkaloid An example of an amphiphilic alkaloid is camptothecin.
  • amphiphilic alkaloid analogs are topotecan, irinotecan, vincristine, vinorelbine, and vinblastine.
  • the drug is an alkaloid.
  • the drug is an amphiphilic alkaloid.
  • the drug is an anti-proliferative alkaloid.
  • the drug is an amphiphilic anti-proliferative alkaloid.
  • the drug is an anti-cancer alkaloid. ln one embodiment, the drug is an amphiphilic anti-cancer alkaloid.
  • the drug is camptothecin, topotecan, irinotecan, vincristine, vinorelbine, or vinblastine; or a salt (e.g., an acid addition salt) thereof.
  • SCPC lipids were shown to have a substantial potentiating effect for other drugs, many of which are drugs which have a similar amphiphilicity.
  • the drug is PI K-75, GSK1059615, ZSTK474, GDC-0941 , AZD6482, Danusertib, PHA-793887, PI K-93, MK-2206 dihydrochloride, PI K-90, BEZ235, PI-103, Bl 2536, AZD7762, NVP-ADW742, Temsirolimus, Crizotinib, JNJ-7706621 , Pelitinib, Sunitinib Malate, XL-184, Hesperadin, Ponatinib, or Regorafenib; or a salt (e.g., an acid addition salt) thereof.
  • a salt e.g., an acid addition salt
  • drugs examples include dactinomycin, plicamycin, teniposide, raltitrexed, irinotecan, rapamycin, gemcitabine (e.g. , gemcitabine hydrochloride), melphalan, acrichine, tamoxifen (e.g., tamoxifen citrate), vinorelbine (e.g. , vinorelbine tartrate), vincristine (e.g. , vincristine sulfate), vinblastine (e.g. , vinblastine sulfate), bortezomib, nilotinib, ABT737, erPC, and perifosine.
  • gemcitabine e.g. , gemcitabine hydrochloride
  • melphalan e.g., gemcitabine hydrochloride
  • melphalan e.g., gemcitabine hydrochloride
  • melphalan e.g., gemcita
  • the drug is dactinomycin, plicamycin, teniposide, raltitrexed, irinotecan, rapamycin, gemcitabine (e.g. , gemcitabine hydrochloride), melphalan, acrichine, tamoxifen (e.g., tamoxifen citrate), vinorelbine (e.g. , vinorelbine tartrate), vincristine (e.g. , vincristine sulfate), vinblastine (e.g. , vinblastine sulfate), bortezomib, nilotinib, ABT737, erPC, or perifosine; or a salt (e.g., an acid addition salt) thereof.
  • gemcitabine e.g. , gemcitabine hydrochloride
  • melphalan e.g., gemcitabine hydrochloride
  • melphalan e.g., gemcitabine hydro
  • Short-Chain Phosphatidylcholine (SCPC) Lipids are related to choline,
  • the short-chain phosphatidylcholine (SCPC) lipid is a compound of the following formula:
  • each of -R FA and -R FA2 is independently:
  • each of -R FA and -R FA2 is independently a linear or branched saturated alkyl group having from 7 to 13 carbon atoms. In one embodiment, each of -R FA and -R FA2 is independently a linear saturated alkyl group having from 7 to 13 carbon atoms.
  • each of -R FA and -R FA2 is independently a linear or branched saturated alkyl group having from 8 to 12 carbon atoms. ln one embodiment, each of -R and -R is independently a linear saturated alkyl group having from 8 to 12 carbon atoms.
  • each of -R FA and -R FA2 is independently a linear or branched saturated alkyl group having from 8 to 10 carbon atoms.
  • each of -R FA and -R FA2 is independently a linear saturated alkyl group having from 8 to 10 carbon atoms. In one embodiment, each of -R FA and -R FA2 is independently: -(CH 2 ) 6 CI-l3, -(CH 2 ) 7 CH 3 , -(CH 2 )gCH 3 , -(CH 2 )ioCH 3 , -(CH 2 )iiCH 3 , or -(CH 2 )i2CH 3 .
  • each of -R FA and -R FA2 is independently: -(CH 2 ) 7 CH 3 , -(CH 2 ) 8 CH 3 , -(CH 2 ) 9 CH 3 , -(CH 2 ) 10 CH 3 , or -(CH 2 )nCH 3 .
  • each of -R FA and -R FA2 is independently: -(CH 2 ) 7 CI-l3, -(CH 2 ) 8 CH 3 , or
  • -R FA and -R FA2 are the same.
  • -R FA and -R FA2 are different.
  • -R FA and -R FA2 are both -(CH 2 ) 7 CH 3 ; as in, for example,
  • dipelargonoyl phosphatidylcholine (denoted herein as "dipelargonoyl-PC" or
  • -R and -R are both -(CH 2 ) 8 CH 3 ; as in, for example,
  • di-caproyl phosphatidylcholine (denoted herein as “dicaproyl-PC” or “C10-PC”), shown below:
  • -R and -R are both -(CH 2 ) 9 CH 3 ; as in, for example,
  • di-undecanoyl phosphatidylcholine (denoted herein as “diundecanoyl-PC” or “C11-PC”), shown below:
  • -R and -R are both -(CH 2 )ioCH 3 ; as in, for example,
  • di-lauroyl phosphatidylcholine (denoted herein as “dilauroyl-PC” or “C12-PC”), shown below:
  • -R and -R are both -(CH 2 )iiCH 3 ; as in, for example,
  • di-tridecanoyl phosphatidylcholine (denoted herein as “ditridecanoyl-PC” or “C13-PC”), shown below:
  • each of -R and -R is independently a linear or branched alkenyl group having from 7 to 13 carbon atoms and from 1 to 3 carbon-carbon double bonds.
  • each of -R FA and -R FA2 is independently a linear alkenyl group having from 7 to 13 carbon atoms and 1 or 2 carbon-carbon double bonds.
  • each of -R FA and -R FA2 is independently a linear alkenyl group having from 7 to 13 carbon atoms and 1 carbon-carbon double bond.
  • one aspect of the present invention pertains to a pharmaceutical formulation comprising (i) a drug, as described herein, and (ii) a short-chain
  • phosphatidylcholine lipid also referred to herein as an SCPC lipid, as described herein.
  • the pharmaceutical formulation may further comprise one or more (e.g., 1 , 2, 3, 4, etc.) additional pharmaceutically acceptable ingredients well-known to those skilled in the art, for example, one or more (e.g., 1 , 2, 3, 4, etc.) pharmaceutically acceptable carriers, diluents, or excipients; or one or more (e.g., 1 , 2, 3, 4, etc.) pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, or sweetening agents.
  • the pharmaceutical formulation further comprises a pharmaceutically acceptable carrier, diluent, or excipient.
  • pharmaceutically acceptable pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Each carrier, diluent, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts. See, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA),
  • the pharmaceutical formulation may further comprise one or more (e.g., 1 , 2, 3, 4, etc.) additional active agents, for example, other therapeutic or prophylactic agents.
  • the pharmaceutical formulation further comprises an additional active agent, for example, an additional therapeutic or prophylactic agent.
  • one aspect of the present invention pertains to a pharmaceutical formulation comprising (i) a drug, as described herein, and (ii) a short-chain
  • phosphatidylcholine lipid also referred to herein as an SCPC lipid, as described herein.
  • the pharmaceutical formulations may be prepared by any methods well-known in the art of pharmacy. Such methods include the step of bringing into association (i) a drug, as described herein, and (ii) an SCPC lipid, as described herein, optionally with one or more pharmaceutically acceptable carriers, diluents, or excipients, as described herein. In general, the pharmaceutical formulations are prepared by uniformly and intimately bringing into association the ingredients, and then shaping the product, if necessary. The pharmaceutical formulation may be prepared to provide for rapid or slow release;
  • immediate, delayed, timed, or sustained release or a combination thereof.
  • Another aspect of the present invention pertains to a method of preparing a
  • each unit may contain a
  • compositions may suitably be in the form of liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions
  • elixirs e.g., oil-in-water, water-in-oil
  • elixirs e.g., syrups, electuaries, mouthwashes, drops
  • tablets including, e.g., coated tablets
  • granules, powders, losenges pastilles
  • capsules including, e.g., hard and soft gelatin capsules
  • cachets pills, ampoules, boluses, suppositories, pessaries, tinctures, gels, pastes, ointments, creams, lotions, oils, foams, sprays, mists, or aerosols.
  • the pharmaceutical formulations may suitably be provided as a patch, adhesive plaster, bandage, dressing, or the like which is impregnated with one or more compounds and optionally one or more other pharmaceutically acceptable ingredients, including, for example, penetration, permeation, and absorption enhancers.
  • Formulations may also suitably be provided in the form of a depot or reservoir.
  • Formulations suitable for oral administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions ⁇ e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, tablets, granules, powders, capsules, cachets, pills, ampoules, boluses.
  • Formulations suitable for buccal administration include mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs.
  • Losenges typically comprise the compound in a flavored basis, usually sucrose and acacia or tragacanth.
  • Pastilles typically comprise the compound in an inert matrix, such as gelatin and glycerin, or sucrose and acacia.
  • Mouthwashes typically comprise the compound in a suitable liquid carrier.
  • Formulations suitable for sublingual administration include tablets, losenges, pastilles, capsules, and pills.
  • Formulations suitable for oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil- in-water, water-in-oil), mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs.
  • solutions e.g., aqueous, non-aqueous
  • suspensions e.g., aqueous, non-aqueous
  • emulsions e.g., oil- in-water, water-in-oil
  • mouthwashes e.g., gluges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs.
  • Formulations suitable for non-oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions
  • suppositories e.g., oil-in-water, water-in-oil
  • suppositories pessaries, gels, pastes, ointments, creams, lotions, oils, as well as patches, adhesive plasters, depots, and reservoirs.
  • Formulations suitable for transdermal administration include gels, pastes, ointments, creams, lotions, and oils, as well as patches, adhesive plasters, bandages, dressings, depots, and reservoirs.
  • Tablets may be made by conventional means, e.g., compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the compound in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g., povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica); disintegrants (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose); surface-active or dispersing or wetting agents (e.g., sodium lauryl sulfate); preservatives (e.g., methyl p-hydroxybenzoate, propyl
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile.
  • Tablets may optionally be provided with a coating, for example, to affect release, for example an enteric coating, to provide release in parts of the gut other than the stomach.
  • Ointments are typically prepared from the compound and a paraffinic or a water-miscible ointment base.
  • Creams are typically prepared from the compound and an oil-in-water cream base.
  • the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1 ,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof.
  • the topical formulations may desirably include a compound which enhances absorption or penetration of the compound through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.
  • Emulsions are typically prepared from the compound and an oily phase, which may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil.
  • an emulsifier also known as an emulgent
  • a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat.
  • the emulsifier(s) with or without stabiliser(s) make up the so-called emulsifying wax
  • the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
  • Suitable emulgents and emulsion stabilisers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.
  • suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the compound in most oils likely to be used in pharmaceutical emulsion formulations may be very low.
  • the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers.
  • Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
  • Formulations suitable for intranasal administration, where the carrier is a liquid include, for example, nasal spray, nasal drops, or by aerosol administration by nebuliser, include aqueous or oily solutions of the compound.
  • Formulations suitable for intranasal administration, where the carrier is a solid include, for example, those presented as a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • Formulations suitable for pulmonary administration include those presented as an aerosol spray from a pressurised pack, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichoro-tetrafluoroethane, carbon dioxide, or other suitable gases.
  • a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichoro-tetrafluoroethane, carbon dioxide, or other suitable gases.
  • Formulations suitable for ocular administration include eye drops wherein the compound is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the compound.
  • Formulations suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols, for example, cocoa butter or a salicylate; or as a solution or suspension for treatment by enema.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the compound, such carriers as are known in the art to be appropriate.
  • Formulations suitable for parenteral administration include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the compound is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate).
  • sterile liquids e.g., solutions, suspensions
  • Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient.
  • excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like.
  • suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection.
  • the concentration of the compound in the liquid is from about 1 ng/mL to about 10 ⁇ / ⁇ , for example from about 10 ng/mL to about 1 ⁇ ...
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
  • the formulation may be in the form of liposomes or other micro particulates (e.g., non-vesicular structures, e.g., micelles, etc.).
  • the pharmaceutical formulation is suitable for parenteral
  • the pharmaceutical formulation may be in the form of micelles.
  • the pharmaceutical formulation is micellar; that is, the pharmaceutical formulation is a micellar pharmaceutical formulation.
  • micellar is intended to indicate that the drug, as described herein, and the short-chain phosphatidylcholine lipid, as described herein, are present in the form of a micelle (i.e., are micelle-entrapped).
  • the micelles may further comprise one or more additional components, such as those described below for liposomes.
  • additional components such as those described below for liposomes.
  • the pharmaceutical formulation may be in the form of liposomes or other
  • the pharmaceutical formulation is liposomal; that is, the pharmaceutical formulation is a liposomal pharmaceutical formulation.
  • liposomal is intended to indicate that the drug, as described herein, and the short-chain phosphatidylcholine lipid, as described herein, are present in the form of a liposome (i.e., are liposome-entrapped).
  • Each of the components i.e., the drug and the SCPC lipid
  • the SCPC lipid is predominantly membrane-entrapped, while the drug is predominantly partitioned into the aqueous compartment.
  • the pharmaceutical formulation comprises liposomes, said liposomes comprising (i) a drug, as described herein, and (ii) a short-chain phosphatidylcholine lipid (also referred to herein as an SCPC lipid), as described herein.
  • the pharmaceutical formulation is suitable for parenteral administration.
  • the pharmaceutical formulation is an aqueous, isotonic,
  • the pharmaceutical formulation is suitable for parenteral administration.
  • the liposomes are long-circulating liposomes (e.g., with a half-life that is from 0.2 to 5 times the half-life of Caelyx®.
  • Liposomes also called lipid vesicles
  • Liposomes are aqueous compartments enclosed by a lipid bilayer (as in monolamellar liposomes) or many concentric bilayers (as in multilamellar liposomes).
  • Lipids may be formed, for example, by suspending a suitable lipid (a vesicle- forming lipid), such as phosphatidyl choline, in an aqueous medium, and then sonicating (i.e., agitating by high frequency sound waves) to give a dispersion of closed vesicles.
  • a suitable lipid such as phosphatidyl choline
  • sonicating i.e., agitating by high frequency sound waves
  • the liposomes are quite uniform in size, nearly spherical, and have a diameter of about 100 nm. Larger vesicles (on the order of 1 ⁇ ) can be prepared by slowly evaporating the organic solvent from a suspension of phospholipid in a mixed solvent system.
  • Suitable vesicle-forming lipids include amphipathic lipids having hydrophobic and polar head group moieties, and which (a) can form (spontaneously) into bilayer vesicles in water, as exemplified by phospholipids, or (b) are stably incorporated into lipid bilayers, with its hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane, and its polar head group moiety oriented toward the exterior, polar surface of the membrane.
  • Vesicle-forming lipids of this type are preferably ones having two hydrocarbon chains, typically acyl chains, and a polar head group. Examples of this class are the
  • phospholipids such as phosphatidylcholines (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), and sphingomyelin (SM), where the two hydrocarbon chains are typically between about 14- 22 carbon atoms in length, and have varying degrees of unsaturation.
  • PC phosphatidylcholines
  • PE phosphatidylethanolamine
  • PA phosphatidic acid
  • PG phosphatidylglycerol
  • PI phosphatidylinositol
  • SM sphingomyelin
  • phosphatidylcholines include hydrogenated soy phosphatidylcholine (HSPC); dipalmitoyl-phosphatidylcholine (DPPC); DSPC (distearoyl phosphatidylcholine); and distearyl-phosphatidylethanolamine (DSPE).
  • HSPC hydrogenated soy phosphatidylcholine
  • DPPC dipalmitoyl-phosphatidylcholine
  • DSPC disearoyl phosphatidylcholine
  • DSPE distearyl-phosphatidylethanolamine
  • liposomes comprise 20-99% by weight of vesicle-forming lipids. In some cases, where other lipids are also present, the upper limit is less, e.g., 20-70%, etc.
  • liposomes may include (non-vesicle forming) lipids that stabilize a vesicle or liposome composed predominantly of phospholipids.
  • the most frequently employed lipid from this group is cholesterol (CH), typically at 25-40 mol%. Below about 20 mol% cholesterol in a bilayer, separate domains exist containing cholesterol and phospholipids and pure phospholipid. These bilayers show an increased permeability to water. At mole percentages above 50% cholesterol, starts to destabilize the bilayer.
  • other lipids such as sitosterol, may be used in addition to, or as an alternative to, cholesterol.
  • liposomes may include (vesicle forming) lipids (such as those described above) which have been derivatized with a polymer chain.
  • vesicle-forming lipids with diacyl chains such as phospholipids
  • phospholipids are preferred.
  • One exemplary class of phospholipids are the phosphatidylethanolamines (PE), which have a reactive amino group that is convenient for coupling to activated polymers.
  • PE phosphatidylethanolamines
  • An exemplary PE is distearyl PE (DSPE).
  • a preferred polymer for derivatiziation is polyethyleneglycol (PEG), typically with a molecular weight of 1 ,000-10,000 Da, more typically 2,000-5,000 Da.
  • the PEG chains provide a surface coating of hydrophilic chains which is sufficient to extend the blood circulation time of the liposomes, as compared to liposomes without the derivatized lipid.
  • hydrophilic polymers which may be suitable include polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polylactic acid, polyglycolic acid, derivatized celluloses (such as hydroxymethylcellulose and
  • the polymer-derivatized lipid if present, is typically present in an amount of 1-20 mol% (e.g., 1-10 mol%, 1-5 mol%).
  • a preferred vesicle-forming lipid that is derivatized with a polymer chain is: N-(carbonyl-methoxypolyethylene glycol 2000)-1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine sodium salt (MPEG2000-DSPE).
  • MPEG2000-DSPE N-(carbonyl-methoxypolyethylene glycol 2000)-1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine sodium salt
  • This lipid is used in commercial STEALTH® liposomes, which are used in liposome formulations of doxorubicin such as Caelyx® and Doxil®.
  • the commercial product Daunoxome® liposomal daunorubicin
  • DSPC distearoyl phosphatidylcholine
  • Liposomes may additionally comprise (vesicle-forming) lipids which have been modified for coupling antibody molecules to the liposome outer surface.
  • these lipids may be derivatized so as to have a pendant hydrophilic polymer spacer chain that is end-functionalized for coupling to an antibody.
  • the functionalized end group may be, for example, a maleimide group (which can be used for selective coupling to antibody sulfhydryl groups), a bromoacetamide or disulfide group (which can be used for coupling to antibody sulfhydryl groups), an activated ester or aldehyde group (which can be sued for coupling to antibody amine groups), or a hydrazide (which can be used to coupling to compounds containing aldehyde groups).
  • a maleimide group which can be used for selective coupling to antibody sulfhydryl groups
  • a bromoacetamide or disulfide group which can be used for coupling to antibody sulfhydryl groups
  • an activated ester or aldehyde group which can be sued for coupling to antibody amine groups
  • a hydrazide which can be used to coupling to compounds containing aldehyde groups.
  • a preferred polymer for the spacer chain is polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • Other hydrophilic polymers which may be suitable for the spacer chain include end-derivatized polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polylactic acid, polyglycolic acid, derivatized celluloses (such as hydroxymethylcellulose and
  • the polymer spacer chain is preferably shorter than the polymer chain described for the liposome surface polymer coating layer.
  • the spacer chain is generally of 100-5,000 Da, preferably 600-4,000 Da.
  • Liposomes may additionally comprise vesicle-forming lipids which have been modified by a biotin molecule.
  • the liposome has a biotinylated surface which can be used to link other compounds (for example, a biotinylated antibody, via avidin).
  • Biotinylated lipids such as biotinylated phosphatidylethanolamines, are commercially available.
  • the liposome formulation may include targeting-enhancing features, such as targeting ligands, for example, monoclonal antibodies, peptides, antibody fragments, (recombinant) proteins, growth factors, folate, carbohydrates, etc.
  • targeting ligands for example, monoclonal antibodies, peptides, antibody fragments, (recombinant) proteins, growth factors, folate, carbohydrates, etc.
  • Liposomes may be prepared by a variety of well-known techniques.
  • Multilamellar vesicles can be formed by simple lipid-film hydration techniques. In this procedure, a mixture of liposome-forming lipids dissolved in a suitable organic solvent is evaporated in a vessel to form a thin film, which is then covered by an aqueous medium. The lipid film hydrates to form MLVs, typically with sizes of 0.1-10 ⁇ .
  • liposomes may be prepared by vortexing dried lipid films in a buffered aqueous solution.
  • the drug is incorporated into liposomes by adding it to the vesicle-forming lipids prior to liposome formation, as described below, in order to entrap the drug in the formed liposome.
  • the drug may be hydrophobic, it may be added directly to the hydrophobic mixture.
  • the drug may be hydrophilic, it may be added to the aqueous medium which covers the thin film of evaporated lipids.
  • the drug may be incorporated into preformed liposomes, for example, by active transport mechanisms.
  • the drug may be taken up into liposomes in response to a potassium or hydrogen or ammonium sulfate or metal ion concentration differential.
  • One effective sizing method for REVs and MLVs involves extruding an aqueous suspension of the liposomes through a series of polycarbonate membranes having a selected uniform pore size in the range of 0.03 to 0.2 ⁇ , typically 0.05, 0.08, 0.1 , or 0.2 ⁇ .
  • the pore size of the membrane corresponds roughly to the largest sizes of liposomes produced by extrusion through that membrane, particularly where the preparation is extruded two or more times through the same membrane. Homogenization methods are also useful for down-sizing liposomes to sizes of 100 nm or less.
  • the pharmaceutical formulation comprises liposomes, said liposomes comprising (i) a drug, as described herein, and (ii) a short- chain phosphatidylcholine lipid (also referred to herein as an SCPC lipid), as described herein.
  • the liposomes further comprise additional phospholipid (i.e., in addition to SCPC lipid).
  • the liposomes further comprises cholesterol (CH). In one embodiment, the liposomes further comprise additional phospholipid and cholesterol (CH).
  • the additional phospholipid comprises additional phosphatidylcholine (PC) (i.e., in addition to SCPC lipid), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), and/or sphingomyelin (SM).
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PA phosphatidic acid
  • PG phosphatidylglycerol
  • PI phosphatidylinositol
  • SM sphingomyelin
  • the additional phospholipid comprises additional phosphatidylcholine (PC) and phosphatidylethanolamine (PE). In one embodiment, the additional phospholipid comprises additional phosphatidylcholine (PC).
  • PC phosphatidylcholine
  • PC phosphatidylcholine
  • the additional phosphatidylcholine (PC) is HSPC.
  • the additional phosphatidylcholine (PC) is DPPC.
  • the additional phosphatidylcholine (PC) is DOPC.
  • the additional phosphatidylcholine (PC) is DSPC.
  • the additional phospholipid comprises phosphatidylethanolamine (PE).
  • the phosphatidylethanolamine (PE) is DSPE.
  • the phosphatidylethanolamine (PE) is DOPE.
  • the phosphatidylethanolamine (PE) is DPPE.
  • the liposomes further comprise a vesicle-forming lipid which is derivatized with a polymer chain.
  • the liposomes comprise a drug (as described herein), an SCPC lipid (as described herein), a phospholipid (as described herein), cholesterol (CH), and a vesicle-forming lipid which is derivatized with a polymer chain (as described herein).
  • the vesicle-forming lipid derivatized with a polymer chain is a phospholipid which is derivatized with polyethyleneglycol (PEG).
  • PEG polyethyleneglycol
  • the vesicle-forming lipid derivatized with a polymer chain is phosphatidylethanolamine (PE) which is derivatized with polyethyleneglycol (PEG). In one embodiment, the vesicle-forming lipid derivatized with a polymer chain is
  • N-(carbonyl-methoxypolyethylene glycol)-1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine sodium salt MPEG-DSPE
  • the vesicle-forming lipid derivatized with a polymer chain is
  • N-(carbonyl-methoxypolyethylene glycol 2000)-1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine sodium salt MPEG2000-DSPE.
  • the liposomes comprise a drug (as described herein), an SCPC lipid (as described herein), HSPC, phosphatidylethanolamine (PE) (as described herein), and cholesterol (CH).
  • the liposomes comprise a drug (as described herein), an SCPC lipid (as described herein), HSPC, N-(carbonyl-methoxypolyethylene glycol 2000)- 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (MPEG2000-DSPE), and cholesterol (CH).
  • the amount of SCPC lipid is 0.5-50 mol%; 1-25 mol%; 1-20 mol%;
  • the amount of cholesterol (CH), if present, is 20-50 mol%;
  • the amount of phospholipid, if present, and excluding phospholipid which is derivatized with a polymer chain, if present, is 45-70 mol%; 35-75 mol%;
  • the amount of vesicle-forming lipid which is derivatized with a polymer chain if present, is 1-15 mol%; 1-10 mol%; 1-7 mol%; 1-5 mol%; 2-10 mol%;
  • the molar ratio of SCPC lipid to phospholipid, if present, and excluding phospholipid which is derivatized with a polymer chain, if present, is from 0.05 to 1 ; from 0.1 to 0.5; or from 0.2 to 0.4.
  • the molar ratio of cholesterol (CH), if present, to the amount of phospholipid, if present, and excluding phospholipid which is derivatized with a polymer chain, if present, is from 0.1 to 0.5; or from 0.2 to 0.4.
  • the molar ratio of vesicle-forming lipid which is derivatized with a polymer chain, if present, to phospholipid, if present, and excluding phospholipid which is derivatized with a polymer chain, if present, is from 0.1 to 0.5; or from 0.2 to 0.4. In one embodiment, the molar ratio of cholesterol (CH), if present, to vesicle-forming lipid which is derivatized with a polymer chain, if present, is from 0.8 to 1.2.
  • the liposomes comprise 0.05-0.50 ⁇ drug per ⁇ phospholipid. In one embodiment, the amount is 0.10-0.40 ⁇ / ⁇ ; 0.10-0.35 ⁇ / ⁇ ;
  • the pharmaceutical formulation comprises liposomes, as described herein, and has a drug concentration of 0.1-10 mg/mL; 0.5-5 mg/mL; 1-3 mg/mL; or 2 mg/mL.
  • the liposomes may additionally comprise other pharmaceutically acceptable ingredients, such as ammonium sulfate, histidine (as buffer), hydrochloric acid and/or sodium hydroxide (for pH control), sucrose (to maintain isotonicity), and water-for-injection.
  • the liposomes have a mean diameter of:
  • the pharmaceutical formulation is a (concentrated) liposomal pharmaceutical formulation (suitable for parenteral infusion) having a doxorubicin concentration of 2 mg/mL.
  • the liposomes are Caelyx® or Doxil® liposomes which have been treated with a short-chain phosphatidylcholine lipid, as described herein.
  • the pharmaceutical formulation comprises liposomes, said liposomes comprising (i) a drug, as described herein, and (ii) a short- chain phosphatidylcholine lipid (also referred to herein as an SCPC lipid), as described herein.
  • these liposomes may optionally further comprise one or more or all of: additional phospholipid (as described herein); cholesterol; and a vesicle- forming lipid which is derivatized with a polymer chain (as described herein).
  • liposomes may be prepared, for example, by:
  • lipid mixture comprising an SCPC lipid, and optionally, one or more or all of: additional phospholipid (as described herein); cholesterol; and a vesicle-forming lipid which is derivatized with a polymer chain (as described herein);
  • these liposomes may be prepared, for example, by:
  • lipid mixture comprising an SCPC lipid, and optionally, one or more or all of: additional phospholipid (as described herein); cholesterol; and a vesicle-forming lipid which is derivatized with a polymer chain (as described herein);
  • short-chain phosphatidyl-choline lipids described herein may be obtained from commercial sources.
  • Other short-chain phosphatidyl-choline lipids may be prepared using conventional methods known in the art, or by adapting conventional methods known in the art in conventional ways.
  • short-chain phosphatidyl-choline lipids may be prepared using conventional methods, for example, as described in Eibl, Chemistry and Physics of Lipids, 1980, from simple starting materials, such as glycero-3-phosphocholine, which may be prepared using conventional methods, for example, as described in Brockerhoff and Yurkowski, Can. J. Biochem.. 1965, Vol. 43, p. 1777.
  • glycero-3-phosphocholine may be acylated with excess fatty acid anhydride in the presence of the tetraethylammonium salt of the fatty acid (for example, at a molar ratio of glycero-3-phosphocholine : fatty acid anhydride : salt of 1 : 6 : 6).
  • the cadmium complex of glycero-3-phosphocholine is prepared and dissolved in dimethylsulfoxide.
  • Fatty acid anhydrides dissolved in benzene are then added, supplemented with 4-pyrrolidinepyridine (for example, at a molar ratio of glycero-3- phosphocholine : fatty acid anhydride : base of 1 : 8 : 2).
  • 4-pyrrolidinepyridine for example, at a molar ratio of glycero-3- phosphocholine : fatty acid anhydride : base of 1 : 8 : 2.
  • the reaction is completed after 2-5 hours at 45°C.
  • a short-chain phosphatidylcholine lipid also referred to herein as an SCPC lipid
  • SCPC lipid greatly increases the bioavailability of a drug and/or the cellular uptake of a drug, as described herein.
  • another aspect of the present invention pertains to a use of an SCPC lipid, as described herein, to increase the bioavailability of a drug, as described herein.
  • Another aspect of the present invention pertains to a use of an SCPC lipid, as described herein, to increase cellular uptake of a drug, as described herein.
  • Another aspect of the present invention pertains to a method of increasing the bioavailability of a drug, as described herein, in a patient, which method comprises the step of co-administering said drug and an SCPC lipid, as described herein.
  • Another aspect of the present invention pertains to a method of increasing the cellular uptake of a drug, as described herein, into a cell, which method comprises the step of contacting said cell with said drug and an SCPC lipid, as described herein.
  • the pharmaceutical formulations described herein are useful, for example, in methods of treatment of a disorder (e.g., a disease), according to the nature of the drug.
  • a disorder e.g., a disease
  • the pharmaceutical formulations are useful in methods of treatment of a proliferative condition, e.g., cancer.
  • Another aspect of the present invention pertains to a pharmaceutical formulation, as described herein, for use in a method of treatment of the human or animal body by therapy, for example, for use a method of treatment of a disorder (e.g., a disease) as described herein.
  • a pharmaceutical formulation as described herein, for use in a method of treatment of the human or animal body by therapy, for example, for use a method of treatment of a disorder (e.g., a disease) as described herein.
  • the medicament comprises the drug and the SCPC lipid.
  • Another aspect of the present invention pertains to a method of treatment, for example, of a disorder (e.g., a disease) as described herein, comprising administering to a patient in need of treatment a therapeutically effective amount of a pharmaceutical formulation, as described herein.
  • a disorder e.g., a disease
  • a pharmaceutical formulation as described herein.
  • the treatment is treatment of a proliferative condition.
  • proliferative condition means an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth.
  • the treatment is treatment of cancer.
  • the nature of the cancer will be determined by the nature of the drug. For example, where the drug is known to treat small cell lung cancer, the pharmaceutical formulations are useful in methods of treatment of small cell lung cancer.
  • the cancer is lung cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, oesophagus cancer, stomach cancer, bowel cancer, small bowel cancer, large bowel cancer, colon cancer, rectal cancer, colorectal cancer, thyroid gland cancer, adrenal gland cancer, breast cancer, gynaecological cancer, ovarian cancer, genito-urinary cancer, endometrial cancer, prostate cancer, testicular cancer, liver cancer, biliary tract cancer, kidney cancer, renal cell carcinoma, bladder cancer, pancreatic cancer, brain cancer, neuroblastoma, glioma, sarcoma, osteosarcoma, bone cancer, nasopharyngeal cancer (e.g., head cancer, neck cancer), skin cancer, squamous cancer, Kaposi's sarcoma, melanoma, malignant melanoma, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma,
  • the anti-cancer effect may arise through one or more mechanisms, including but not limited to, the regulation of cell proliferation, the inhibition of cell cycle progression, the inhibition of angiogenesis (the formation of new blood vessels), the inhibition of metastasis (the spread of a tumour from its origin), the inhibition of cell migration (the spread of cancer cells to other parts of the body), the inhibition of invasion (the spread of tumour cells into neighbouring normal structures), or the promotion of cell apoptosis (programmed cell death).
  • the treatment is treatment of condition (e.g., a proliferative condition, e.g., a cancer) that is treated by (e.g., treatable by) (e.g., known to be treated by) (e.g., known to be treatable by) doxorubicin.
  • condition e.g., a proliferative condition, e.g., a cancer
  • the treatment is treatment of cancer that is treated by (e.g., treatable by)
  • doxorubicin (e.g., known to be treated by) (e.g., known to be treatable by) doxorubicin.
  • the treatment is treatment of ovarian cancer, for example, metastatic carcinoma of the ovary, for example, metastatic carcinoma of the ovary in patients with disease that is refractory to both paclitaxel- and platinum-based chemotherapy regimens.
  • ovarian cancer for example, metastatic carcinoma of the ovary, for example, metastatic carcinoma of the ovary in patients with disease that is refractory to both paclitaxel- and platinum-based chemotherapy regimens.
  • the treatment is treatment of Kaposi's syndrome, for example, AIDS-related Kaposi's syndrome, for example, AIDS-related Kaposi's syndrome in patients with disease that has progressedon prior combination chemotherapy or in patients who are intolerant to such therapy.
  • Kaposi's syndrome for example, AIDS-related Kaposi's syndrome
  • AIDS-related Kaposi's syndrome for example, AIDS-related Kaposi's syndrome in patients with disease that has progressedon prior combination chemotherapy or in patients who are intolerant to such therapy.
  • treatment pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, alleviation of symptoms of the condition, amelioration of the condition, and cure of the condition.
  • Treatment as a prophylactic measure i.e., prophylaxis
  • prophylaxis is also included.
  • treatment for example, use with patients who have not yet developed the condition, but who are at risk of developing the condition, is encompassed by the term "treatment.”
  • treatment of cancer includes the prophylaxis of cancer, reducing the incidence of cancer, reducing the cancer of dementia, alleviating the symptoms of cancer, etc.
  • therapeutically-effective amount pertains to that amount of a compound, or a material, composition or dosage form comprising a compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.
  • treatment includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously.
  • the pharmaceutical formulations described herein may also be used in combination therapies, e.g., in conjunction with other agents, for example, anti-cancer agents, etc.
  • treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g., drugs, antibodies (e.g., as in immunotherapy), prodrugs (e.g., as in photodynamic therapy, GDEPT, ADEPT, etc.); surgery; radiation therapy; photodynamic therapy; gene therapy; and controlled diets.
  • the particular combination would be at the discretion of the physician who would select dosages using his common general knowledge and dosing regimens known to a skilled practitioner.
  • the agents i.e., the pharmaceutical formulation described herein, plus one or more other agents
  • the agents can be administered at closely spaced intervals (e.g., over a period of 5-10 minutes) or at longer intervals (e.g., 1 , 2, 3, 4 or more hours apart, or even longer periods apart where required), the precise dosage regimen being commensurate with the properties of the therapeutic agent(s).
  • agents i.e., the pharmaceutical formulation described here, plus one or more other agents
  • the agents may be formulated together in a single dosage form, or alternatively, the individual agents may be formulated separately and presented together in the form of a kit, optionally with instructions for their use.
  • the pharmaceutical formulations, as described herein, may also be used as cell culture additives.
  • the pharmaceutical formulations, as described herein, may also be used as part of an in vitro assay, for example, in order to determine whether a candidate host is likely to benefit from treatment with the pharmaceutical formulation.
  • the pharmaceutical formulations, as described herein, may also be used as a standard, for example, in an assay, in order to identify, characterise, and/or evaluate other pharmaceutical formulations.
  • kits comprising (a) a pharmaceutical formulation, as described herein, preferably provided in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, written instructions on how to administer the pharmaceutical formulation, etc.
  • kits comprising (a) a drug, as described herein, (b) a short-chain phosphatidylcholine lipid (also referred to herein as an SCPC lipid), as described herein (c) optionally, one or more or all of: phospholipid (as described herein); cholesterol; and a vesicle-forming lipid which is derivatized with a polymer chain (as described herein); all preferably provided in a suitable container and/or with suitable packaging; and (d) instructions for use, for example, written instructions on how to prepare (and optionally administer) a liposomal pharmaceutical formulation, as described herein.
  • a drug as described herein
  • SCPC lipid short-chain phosphatidylcholine lipid
  • SCPC lipid short-chain phosphatidylcholine lipid
  • c optionally, one or more or all of: phospholipid (as described herein); cholesterol; and a vesicle-forming lipid which is derivatized with a
  • kits comprising (a) a drug, as described herein, preferably provided in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, written instructions on how to prepare (and optionally administer) a pharmaceutical formulation, as described herein.
  • the kit may optionally including appropriate reagents (e.g., buffers, solvents) and devices (e.g., tubes, syringes) for assembly and use (e.g., administration).
  • the written instructions may also include a list of indications for which the active ingredient is a suitable treatment.
  • the pharmaceutical formulation may be administered to a subject by any convenient route of administration, whether systemically/peripherally or topically (i.e., at the site of desired action).
  • Routes of administration include, but are not limited to, oral ⁇ e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal ⁇ e.g., by nasal spray, drops or from an atomiser or dry powder delivery device); ocular ⁇ e.g., by eyedrops); pulmonary ⁇ e.g., by inhalation or insufflation therapy using, e.g., an aerosol, e.g., through the mouth or nose); rectal ⁇ e.g., by suppository or enema); vaginal ⁇ e.g., by pessary); parenteral, for example, by injection ⁇
  • the pharmaceutical formulation is administered
  • the subject/patient may be a chordate, a vertebrate, a mammal, a placental mammal, a marsupial ⁇ e.g., kangaroo, wombat), a rodent ⁇ e.g., a guinea pig, a hamster, a rat, a mouse), murine ⁇ e.g., a mouse), a lagomorph ⁇ e.g., a rabbit), avian ⁇ e.g., a bird), canine ⁇ e.g., a dog), feline ⁇ e.g., a cat), equine ⁇ e.g., a horse), porcine ⁇ e.g., a pig), ovine ⁇ e.g., a sheep), bovine ⁇ e.g., a cow), a primate, simian ⁇ e.g., a monkey or ape), a monkey
  • the subject/patient may be any of its forms of development, for example, a foetus.
  • the subject/patient is a human. Dosage It will be appreciated by one of skill in the art that appropriate dosages of the
  • the pharmaceutical formulation can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects.
  • the selected dosage level will depend on a variety of factors including, but not limited to, the drug, the route of administration, the time of administration, the rate of excretion of the drug, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the patient.
  • the amount of pharmaceutical formulation and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side- effects.
  • Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the pharmaceutical formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.
  • doxorubicin 60-75 mg/m 2
  • epirubicin 60-120 mg/m 2
  • daunorubicin 25-45 mg/m 2 .
  • Dosages above about 550 mg/m 2 doxorubicin may lead to irreversible myocardial toxicity leading to congestive heart failure often unresponsive to cardiac support therapy.
  • Liposomal doxorubicin e.g., Doxil®, Caelyx®
  • doxorubicin concentration of about 2 mg/mL
  • Short-chain phosphatidyl-choline lipids were dissolved in ethanol at a concentration of 10 mM.
  • Bovine aortic endothelial cells (BAEC) were cultured in flat-bottom 96-well plates in 10% serum-containing medium, in a water-saturated atmosphere of 5% C0 2 at 37°C. After reaching confluency, the cells were serum starved for at least 4 hours.
  • Cells were then pre-incubated with short-chain phosphatidyl-choline lipids (in ethanol) at a concentration of 2.5, 5, 10, 25, 50, 100, or 250 ⁇ for 15 minutes, followed by incubation with 50 ⁇ doxorubicin (from an aqueous stock solution at a concentration of 2 mg/mL) for 1 hour (while the short-chain phosphatidyl-choline lipids remained present).
  • Figure 1 is a graph of doxorubicin content (expressed as % of control) as a function of concentration ( ⁇ ) of short-chain phosphatidyl-choline lipid, for five lipids: dinonaoyl-PC (C9), diundecanoyl-PC (C1 1), dilauroyl-PC (C12), ditridecanoyl-PC (C13), and dimyristoyl-PC (C14), as well as the comparison lipid, C8-GlcCer (N-octanoyl- glucosylceramide).
  • Short-chain phosphatidyl-choline lipids were dissolved in ethanol at a concentration of 10 mM.
  • Bovine aortic endothelial cells (BAEC) were cultured in flat-bottom 96-well plates in 10% serum-containing medium, in a water-saturated atmosphere of 5% C0 2 at 37°C. After reaching confluency, the cells were serum starved for at least 4 hours. Cells were then incubated with short-chain phosphatidyl-choline lipids (in ethanol) for 24 hours. Cells were incubated with ethanol alone as a routine check, to check that cell viability is not decreased by more than 10% as compared to the untreated wells.
  • the medium was replaced with serum-supplemented medium, and cell viability was determined 48 hours after washing by adding 50 ⁇ g of the mitochondrial dehydrogenase substrate 2,3-bis(2- methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt (XTT) and 38 ng N-methyl dibenzopyrazine methyl sulfate (PMS) to each well. After incubation at 37°C for 2 hours, absorbance was read using a Victor Wallac microplate reader
  • Figure 2 is a graph of cell viability (expressed as % of control) as a function of concentration ( ⁇ ) of short-chain phosphatidyl-choline lipid, for five lipids: dinonaoyl-PC (C9), diundecanoyl-PC (C1 1), dilauroyl-PC (C12), ditridecanoyl-PC (C13), and dimyristoyl-PC (C14), as well as the comparison lipid, C8-GlcCer (N-octanoyl- glucosylceramide).
  • dinonaoyl-PC C9
  • diundecanoyl-PC C1 1
  • dilauroyl-PC C12
  • ditridecanoyl-PC C13
  • dimyristoyl-PC C14
  • C8-GlcCer N-octanoyl- glucosylceramide
  • the NCI-88 approved oncology set library was screened on A431 vulva carcinoma and WEP mouse mammary carcinoma cells.
  • the Selleck Kinase inhibitor library was screened on HCT-119 colon carcinoma cells.
  • Cells were cultured in serum containing medium, in a water-saturated atmosphere of 5% C0 2 at 37°C.
  • Short-chain phosphatidylcholine (SCPC) lipids were dissolved in ethanol at a concentration of 10 mM. SCPC lipids were applied at a final concentration of 20 ⁇ . In this way, the SCPC lipids were allowed to insert into the plasma membrane of the cells. From the libraries, test drugs were applied at concentrations of 0.001 , 0.01 , 0.1 , 1.0, and 10 ⁇ .
  • SCPC lipids and library test drugs were added to serum-free culture medium conditions. After an incubation period of 4 hours, cells were washed with phosphate buffered saline (PBS) three times. Then, serum-containing (10%) medium was applied again, followed by a 48 hour period of cell expansion.
  • PBS phosphate buffered saline
  • SCPC lipids and library test drugs were directly applied to cells in culture medium containing 2% fetal calf serum (FCS), followed by a 48 hours cell expansion period (the SCPC lipids and library test drugs were not removed before the expansion). SCPC lipids were always added prior to the library test drugs, and preferably 15 minutes before the library test drugs.
  • SCPC lipids without library test drugs, were applied as a negative control. Phenyl arsene oxide was applied as a positive control. After incubation with the SCPC lipids and library test drugs, and a period of cell expansion, cell viability was measured by a Promega Cell Titerdian metabolic assay. Read-out was performed on a PerkinElmer Envision Multilabel Reader.
  • the screenings were performed in 384 well-plate format, twice for each cell line, and with the library test drugs added in duplicate on each plate. After read-out, data were analyzed by automated curve fitting. The generated IC 2 s, IC 50 and IC 75 values, plus a relative IC 50 value (relative to top and bottom plateaus of the sigmoidal curve) were used for further analysis. In cases where sufficient data points for a library test drug (i.e., >50% of these four values per data set) were available, the effect of the SCPC lipid was considered eligible for evaluation. The data were ranked on their maximal IC shift, which is a logarithmic value.
  • the results are summarized in the following table, which presents data for library test drugs which, in combination with an SCPC lipid, caused a shift of IC value, so as to lower the test compound's IC value by more than one-half (0.5) of a logi 0 -value.
  • the table also includes the molecular target which the test compound inhibits, so as to decrease cell viability.
  • the data show that the SCPC increased the potencies of the library test drugs by a factor of at least about 3 (i.e., 10 0 5 ) and in many cases, by a factor of least about 10 (i.e., 10 °), and in at least one case, by a factor of about 200 (i.e., 10 2 3 ).
  • Figure 3 is a graph of cell viability (expressed as % of control) as a function of logarithm of concentration of dactinomycin, for dactinomycin alone (circles) and for dactinomycin with an SCPC lipid (triangles), specifically, dinonaoyl-PC (C9), at a concentration of 20 ⁇ .
  • IC 50 was about -6.
  • IC 50 was about -7.4.
  • the shift in log 0 (IC 50 ) caused by the SCPC lipid was about 1.4 log 0 units, or a factor of about 24.
  • immunoliposomes enhanced efficacy attributable to targeted delivery

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Dispersion Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Dermatology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention pertains generally to the field of therapy and medical treatment, and more specifically to certain compounds of the following formula (for convenience, collectively referred to herein as "short-chain phosphatidylcholine lipids" and "SCPC lipids"), which, inter alia, are useful in methods of improving drug delivery, bioavailability, and efficacy. The present invention also pertains to pharmaceutical formulations, and more specifically to co-formulations of known drugs (e.g., amphiphilic drugs) (e.g., anthracyclines) (e.g., doxorubicin) with these SCPC lipids, and their use, for example, in therapy.

Description

COMPOUNDS AND THEIR USE IN THERAPY
RELATED APPLICATION This application is related to United Kingdom patent application number 1308753.1 filed 15 May 2013, the contents of which are incorporated herein by reference in their entirety.
TECHNICAL FIELD
The present invention pertains generally to the field of therapy and medical treatment, and more specifically to certain compounds (for convenience, collectively referred to herein as "short-chain phosphatidylcholine lipids" and "SCPC lipids"), which, inter alia, are useful in methods of improving drug delivery, bioavailability, and efficacy. The present invention also pertains to pharmaceutical formulations, and more specifically to co-formulations of known drugs (e.g., amphiphilic drugs) (e.g., anthracyclines)
(e.g., doxorubicin) with these SCPC lipids, and their use, for example, in therapy.
BACKGROUND
A number of patents and publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.
Throughout this specification, including the claims which follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and
"comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like. Ranges are often expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent "about," it will be understood that the particular value forms another embodiment.
This disclosure includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Anthracvclines: Doxorubicin
The anthracycline doxorubicin has been in clinical use for several decades, and is still among the most widely used chemotherapeutic agents for treatment of a variety of neoplasms (see, e.g., Weiss, 1992; Zagotto et al., 2001 ; Lothstein et al., 2001).
Doxorubicin has several cytotoxic actions. It binds to DNA and inhibits both DNA and RNA synthesis, but its main cytotoxic action appears to be mediated through an effect on topoisomerase II (a DNA gyrase), the activity of which is markedly increased in proliferating cells. The significance of the enzyme lies in the fact that during replication of the DNA helix, reversible swivelling needs to take place around the replication fork in order to prevent the daughter DNA molecule becoming inextricably entangled during mitotic segregation. The swivel is produced by topoisomerase II, which nicks both DNA strands and subsequently reseals the breaks. Doxorubicin intercalates in the DNA and its effect is, in essence, to stabilise the DNA-topoisomerase II complex after the strands have been nicked, thus causing the process to seize up at this point.
Despite many years of research in developing new and better anthracyclines, little or no change in the molecular structure of doxorubicin made it to the clinics. However, with the development of liposomal formulations, its delivery form underwent a major improvement (see, e.g., Tardi et al., 1996; Gabizon, 2001). Compared to systemic application of doxorubicin in its free form, liposomal doxorubicin exhibits significant advantages, as for example reduced acute and chronic toxicities. Improved loading procedures, resulting in high doxorubicin packing efficiencies, further increased the therapeutic index of encapsulated doxorubicin (see, e.g., Horowitz et al., 1992; Haran et al., 1993). Another major step forward was the development of polyethyleneglycol (PEG)-coated liposomes. This coating prevents opsonization and reduces the uptake by macrophages from the reticulo-endothelial system, in turn resulting in prolonged circulation times, as compared to free doxorubicin or to non-coated liposomes (see, e.g., Vaage et al., 1992; Robert and Gianni, 1993; Gabizon et al., 1996; Uster ef a/., 1996). PEG-liposome-encapsulated doxorubicin (commercially available as Caelyx® and Doxil®) is now in routine clinical use, and innovations such as the coupling of targeting-enhancing features (e.g., tumor cell specific antibodies or ligands) will further enhance its therapeutic value (see, e.g., Park et al., 2002; Pan et al., 2003; Koning et al., 1999; Koning et al., 2003).
Both Caelyx® and Doxil® consist of: doxorubicin hydrochloride (2 mg/mL); N-(carbonyl- methoxypolyethylene glycol 2000)-1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (MPEG2000-DSPE) (3.19 mg/mL); fully hydrogenated soy
phosphatidylcholine (HSPC) (9.58 mg/mL); cholesterol (3.19 mg/mL); ammonium sulfate (~2 mg/mL); histidine (as a buffer); hydrochloric acid and/or sodium hydroxide (for pH control); sucrose (to maintain isotonicity); and water-for-injection.
The endothelial lining of healthy blood vessels effectively prevents escape of liposomes from the circulation. In contrast, angiogenesis-associated vascular abnormalities of many solid tumors, do allow extravasation of long-circulating PEG-liposomes into the tumor stroma (see, e.g., Yuan et al., 1994). Despite this tumor-specific accumulation, liposomes are, however, not taken up by tumor cells. Instead, doxorubicin is gradually released into the interstitial space (see, e.g., Horowitz et al., 1992; Harasym et al., 1997). Given the intracellular localization of its molecular targets, sufficient cellular uptake of doxorubicin is required for its action (see, e.g., Speth et al., 1988; Lothstein et al., 2001). However, since doxorubicin does not possess the optimal degree of lipophilicity for efficient plasma membrane traversal, this might be a limiting factor for its efficacy (see, e.g., Heijn et al., 1999; Washington et al., 2001). The inventors have demonstrated that the cellular uptake of free doxorubicin, and with that its cytotoxic action, is greatly enhanced by co-administration of a short-chain phosphatidylcholine lipid (also referred to herein as an SCPC lipid), as described herein.
Furthermore, the inventors have demonstrated that the cytotoxic action of a number of drugs is increased, and in many cases, greatly increased, by co-administration of an SCPC lipid, as described herein.
It is believed that the SCPC lipid spontaneously inserts into lipid bilayers, and exchanges easily between membranes. Furthermore, it may also be expected that the effects demonstrated for doxorubicin will be observed for other drugs, especially other amphiphilic drugs.
This important and beneficial effect, that is, the ability of an SCPC lipid to increase bioavailability and/or cellular uptake of a drug, is surprising and unexpected. The improved formulation offers many advantages. For example, the improved drug uptake permits the use of formulations with lower drug content to achieve the same result, thereby reducing undesired side effects, e.g., myocardial toxicity for doxorubicin.
SUMMARY OF THE I NVENTION
One aspect of the present invention pertains to a pharmaceutical formulation comprising
(i) a drug, as described herein, and (ii) a short-chain phosphatidylcholine lipid (also referred to herein as an SCPC lipid), as described herein.
In one embodiment, the pharmaceutical formulation is a pharmaceutical formulation comprising liposomes, said liposomes comprising said drug and said short-chain phosphatidylcholine lipid.
In one embodiment, the pharmaceutical formulation is a pharmaceutical formulation comprising liposomes, said liposomes further comprise one or more or all of: additional phospholipid; cholesterol (CH); and a vesicle-forming lipid which is derivatized with a polymer chain.
Another aspect of the present invention pertains to a method of preparing a
pharmaceutical formulation comprising combining (i) a drug, as described herein, and
(ii) an SCPC lipid, as described herein. Another aspect of the present invention pertains to a pharmaceutical formulation comprising (i) a drug, as described herein, and (ii) an SCPC lipid, as described herein, for use in a method of treatment of the human or animal body by therapy.
Another aspect of the present invention pertains to a pharmaceutical formulation comprising (i) a drug, as described herein, and (ii) an SCPC lipid, as described herein, for use in a method of treatment of a disorder (e.g., a disease) as described herein.
Another aspect of the present invention pertains to a method of treatment, for example, of a disorder (e.g., a disease) as described herein, comprising administering to a patient in need of treatment a therapeutically effective amount of a pharmaceutical formulation comprising (i) a drug, as described herein, and (ii) an SCPC lipid, as described herein.
Another aspect of the present invention pertains to use of (i) a drug, as described herein, and (ii) an SCPC lipid, as described herein, in the manufacture of a medicament for the treatment of a disorder (e.g., a disease) as described herein.
Another aspect of the present invention pertains to a use of an SCPC lipid, as described herein, to increase the bioavailability of a drug, as described herein. Another aspect of the present invention pertains to a use of an SCPC lipid, as described herein, to increase cellular uptake of a drug, as described herein. Another aspect of the present invention pertains to a method of increasing the bioavailability of a drug, as described herein, in a patient, which method comprises the step of co-administering said drug and an SCPC lipid, as described herein.
Another aspect of the present invention pertains to a method of increasing the cellular uptake of a drug, as described herein, into a cell, which method comprises the step of contacting said cell with said drug and an SCPC lipid, as described herein. As will be appreciated by one of skill in the art, features and preferred embodiments of one aspect of the invention will also pertain to other aspects of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph of doxorubicin content (expressed as % of control) as a function of concentration (μΜ) of short-chain phosphatidyl-choline lipid, for five lipids: dinonaoyl-PC (C9), diundecanoyl-PC (C1 1), dilauroyl-PC (C12), ditridecanoyl-PC (C13), and dimyristoyl-PC (C14), as well as the comparison lipid, C8-GlcCer (N-octanoyl- glucosylceramide).
Figure 2 is a graph of cell viability (expressed as % of control) as a function of concentration (μΜ) of short-chain phosphatidyl-choline lipid, for five lipids: dinonaoyl-PC (C9), diundecanoyl-PC (C1 1), dilauroyl-PC (C12), ditridecanoyl-PC (C13), and dimyristoyl-PC (C14), as well as the comparison lipid, C8-GlcCer (N-octanoyl- glucosylceramide). Figure 3 is a graph of cell viability (expressed as % of control) as a function of logarithm of concentration of dactinomycin, for dactinomycin alone (circles) and for dactinomycin with an SCPC lipid (triangles), specifically, dinonaoyl-PC (C9), at a concentration of 20 μΜ.
DETAILED DESCRIPTION OF THE INVENTION
The inventors have determined that an SCPC lipid, as described herein, increases bioavailability and/or cellular uptake of a drug (e.g., an amphiphilic drug, such as doxorubicin). Consequently, formulations comprising a drug and an SCPC lipid, as described herein, have important advantages, for example, a decreased dosage to achieve the same therapeutic effect.
Pharmaceutical Formulations
Thus, one aspect of the present invention pertains to a pharmaceutical formulation comprising (i) a drug, as described herein, and (ii) a short-chain phosphatidylcholine lipid (also referred to herein as an SCPC lipid), as described herein. Drugs - Amphiphilic Drugs
In one embodiment, the drug is an amphiphilic drug.
The term "amphiphilic" is used herein in the conventional sense to mean a compound (e.g., a drug) that is both (i) hydrophilic and (ii) hydrophobic (e.g., lipophilic).
The inventors have demonstrated that the delivery enhancement is not limited to a particular class of drugs (e.g., anthracyclines) but is in fact applicable to a range of drugs that are amphiphilic, for example, as characterised by their lipophilicity.
In one embodiment, the amphiphilic drug has a log(lipophilicity) value of 0.4 to 16.
In one embodiment, the lower limit is 0.5; the lower limit is 0.6; the lower limit is 0.8. In one embodiment, the upper limit is 12; the upper limit is 10; the upper limit is 8.
For example, in one embodiment, the range is 0.8 to 8.
In this context, lipophilicity is defined as the 1-octanol/water partitioning ratio. The partitioning ratio is determined for 10-50 mM test compound (e.g., drug) using a two phase system consisting of 1 ml_ 1-octanol and 1 ml_ water. After addition of the test compound, tubes are vortexed and centrifuged (3000 rpm, 5 minutes, 20°C). Aliquots of both the organic and the aqueous phases are taken for fluorimetric quantification. Native fluorescence intensities are measured, for example, by a Perkin-Elmer Victor Wallac II fluorescence microplate reader, using lex 485 nm and lem 535 nm filters. All values are corrected for background fluorescence. As a measure of lipophilicity, the 1-octanol/water partitioning ratio is calculated and expressed on a log scale (see, for example,
Washington et al., 2001). ln one embodiment, the drug is an amphiphilic anti-proliferative drug.
In one embodiment, the drug is an amphiphilic anti-cancer drug.
In one embodiment, the drug is an amphiphilic anti-cancer drug from natural sources. Drugs - Anthracyclines and Analogs
Anthracyclines are a class of drugs that originate from natural sources (specifically, derived from microorganisms), and are believed to act by DNA intercalation. Included among anthracyclines are anthracycline analogs.
Examples of amphiphilic anthracyclines include doxorubicin, daunorubicin, epirubicin, and aclarubicin. Examples of amphiphilic anthracycline analogs include idarubicin, valrubicin, and mitoxantrone. In one embodiment, the drug is an anthracycline.
In one embodiment, the drug is an amphiphilic anthracycline.
In one embodiment, the drug is an anti-proliferative anthracycline.
In one embodiment, the drug is an amphiphilic anti-proliferative anthracycline.
In one embodiment, the drug is an anti-cancer anthracycline.
In one embodiment, the drug is an amphiphilic anti-cancer anthracycline.
In one embodiment, the drug is doxorubicin, daunorubicin, epirubicin, aclarubicin, idarubicin, valrubicin, or mitoxantrone; or a salt (e.g., an acid addition salt) thereof. In one embodiment, the drug is doxorubicin or a salt (e.g., an acid addition salt) thereof. In one embodiment, the drug is doxorubicin or doxorubicin hydrochloride.
Drugs - Alkaloids and Analogs Alkaloids are a class of drugs that originate from natural sources (specifically, derived from plants), and are believed to act by inhibiting topoisomerase I . Included among alkaloids are alkaloid analogs.
An example of an amphiphilic alkaloid is camptothecin. Examples of amphiphilic alkaloid analogs are topotecan, irinotecan, vincristine, vinorelbine, and vinblastine.
In one embodiment, the drug is an alkaloid.
In one embodiment, the drug is an amphiphilic alkaloid.
In one embodiment, the drug is an anti-proliferative alkaloid.
In one embodiment, the drug is an amphiphilic anti-proliferative alkaloid.
In one embodiment, the drug is an anti-cancer alkaloid. ln one embodiment, the drug is an amphiphilic anti-cancer alkaloid.
In one embodiment, the drug is camptothecin, topotecan, irinotecan, vincristine, vinorelbine, or vinblastine; or a salt (e.g., an acid addition salt) thereof.
Drugs - Additional Drugs
As demonstrated in the Biological Study for High Throughput Screening shown below, SCPC lipids were shown to have a substantial potentiating effect for other drugs, many of which are drugs which have a similar amphiphilicity.
Drug CAS No. Chemical Name
2-Methyl-5-nitro-1 -benzenesulfonic acid 2-[(6-
PIK-75 372196-67-3 bromoimidazo[1 ,2-a]pyridin-3-yl)methylene]-1 - methylhydrazide
(5Z)-5-[[4-(4-pyridinyl)-6-quinolinyl]methylene]-
GSK1059615 958852-01 -2
2,4-thiazolidinedione
2-(2-Difluoromethylbenzimidazol-1-yl)-4,6-
ZSTK474 4751 10-96-4
dimorpholino-1 ,3,5-triazine
2-(1 H-indazol-4-yl)-6-(4-methanesulfonyl-
GDC-0941 957054-33-0 piperazin-1 -ylmethyl)-4-morpholin-4-yl- thieno[3,2-d]pyrimidine
2-[[(1 R)-1 -[7-methyl-2-(4-morpholinyl)-4-oxo-
AZD6482 1 173900-33-8 4h-pyrido[1 ,2-a]pyrimidin-9- yl]ethyl]amino]benzoic acid
N-[5-((2R)-2-Methoxy-2-phenylethanoyl)-
Danusertib 827318-97-8 1 ,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl]-4-
(4-methylpiperazin-1 -yl)benzamide
3-Methyl-N-[1 ,4,5,6-tetrahydro-6,6-dimethyl-5-
PHA-793887 718630-59-2 [(1 -methyl-4-piperidinyl)carbonyl]pyrrolo[3,4- c]pyrazol-3-yl]butanamide
N-[5-[4-Chloro-3-[[(2-
PIK-93 593960-1 1 -3 hydroxyethyl)amino]sulfonyl]phenyl]-4-methyl- 2-thiazolyl]acetamide
8-[4-(1 -Aminocyclobutyl)phenyl]-9-phenyl-
MK-2206
1032350-13-2 1 ,2,4-triazolo[3,4-f][1 ,6]naphthyridin-3(2H)-one dihydrochloride
dihydrochloride
N-(2,3-Dihydro-7,8-dimethoxyimidazo[1 ,2-
PIK-90 677338-12-4
c]quinazolin-5-yl)-3-pyridinecarboxamide Drug CAS No. Chemical Name
2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-
BF7?35 915019-65-7 2,3-dihydro-1 H-imidazo[4,5-c]quinolin-1- yl]phenyl}propanenitrile
3-(4-(4-Morpholinyl)pyrido[3',2':4,5]furo[3,2-
PI-103 371935-79-4
d]pyrimidin-2-yl)phenol Hydrochloride
4-[[(7R)-8-Cyclopentyl-7-ethyl-5,6,7,8- tetrahydro-5-methyl-6-oxo-2-pteridinyl]amino]-
Bl 2536 755038-02-9
3-methoxy-N-(1-methyl-4- piperidinyl)benzamide
3-[(Aminocarbonyl)amino]-5-(3-fluorophenyl)-
AZD7762 860352-01-8
N-(3S)-3-piperidinyl-2-Thiophenecarboxamide
[5-(3-Benzyloxyphenyl)-7-[cis-3-[(pyrrolidin-1-
NVP-ADW742 475489-15-7 yl)methyl]cyclobutyl]-7H-pyrrolo[2,3- d]pyrimidin-4-yl]amine
(1 R,2R,4S)-4-{(2R)-2-
[(3S,6R E,9R, 1 Ofl, 12fl, 14S, 15E, 17E, 19E.21 S ,23S,26fl,27fl,34aS)-9,27-dihydroxy-10,21- dimethoxy-6,8, 12,14,20,26-hexamethyl- 1 ,5,1 1 ,28,29-pentaoxo-
Temsirolimus
162635-04-3 1 ,4,5,6,9, 10, 11 ,12, 13, 14,21 ,22,23,24,25,26,27, (Torisel)
28,29,31 , 32,33,34,34a-tetracosahydro-3H- 23,27-epoxypyrido[2, 1- c][1 ,4]oxazacyclohentriacontin-3-yl]propyl}-2- methoxycyclohexyl 3-hydroxy-2- (hydroxymethyl)-2-methylpropanoate
Crizotinib
3-[(1 /:?)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-
(Xalkori, 877399-52-5
5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine
PF-02341066)
4-[[5-Amino-1-(2,6-difluorobenzoyl)-1 H-1 ,2,4-
JNJ-7706621 443797-96-4
triazol-3-yl]amino]benzenesulfonamide
(E)-N-(4-(3-chloro-4-fluorophenylamino)-3-
Pelitinib
257933-82-7 cyano-7-ethoxyquinolin-6-yl)-4- (EKB-569)
(dimethylamino)but-2-enamide
Sunitinib Malate A/-(2-diethylaminoethyl)-5-[(Z)-(5-fluoro-2-oxo-
(Sutent, 341031-54-7 1 H-indol-3-ylidene)methyl]-2,4-dimethyl-1 H-
SU 11248) pyrrole-3-carboxamide
XL- 184 A/-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-
(Cabozantinib, 849217-68-1 A/-(4-fluorophenyl)cyclopropane-1 , 1-
Cometriq) dicarboxamide Drug CAS No. Chemical Name
A/-[(3Z)-2-Oxo-3-[phenyl-[4-(piperidin-1 -
Hesperadin 422513-13-1 ylmethyl)anilino]methylidene]-1 /- -indol-5- yl]ethanesulfonamide
Ponatinib 3-(2-lmidazo[1 ,2-b]pyridazin-3-ylethynyl)-4-
(Lclusig, 943319-70-8 methyl-A/-[4-[(4-methylpiperazin-1-yl)methyl]-3-
AP24534) (trifluoromethyl)phenyl]benzamide
4-[4-({[4-Chloro-3-
Regorafenib
(trifluoromethyl)phenyl]carbamoyl}amino)-3- (Stivarga, BAY 755037-03-7
fluorophenoxy]-A/-methylpyridine-2- 73-4506)
carboxamide hydrate
In one embodiment, the drug is PI K-75, GSK1059615, ZSTK474, GDC-0941 , AZD6482, Danusertib, PHA-793887, PI K-93, MK-2206 dihydrochloride, PI K-90, BEZ235, PI-103, Bl 2536, AZD7762, NVP-ADW742, Temsirolimus, Crizotinib, JNJ-7706621 , Pelitinib, Sunitinib Malate, XL-184, Hesperadin, Ponatinib, or Regorafenib; or a salt (e.g., an acid addition salt) thereof.
As demonstrated in the Biological Study for dactinomycin shown below, an SCPC lipid was shown to have a substantial potentiating effect for still other drugs.
Examples of such drugs include dactinomycin, plicamycin, teniposide, raltitrexed, irinotecan, rapamycin, gemcitabine (e.g. , gemcitabine hydrochloride), melphalan, acrichine, tamoxifen (e.g., tamoxifen citrate), vinorelbine (e.g. , vinorelbine tartrate), vincristine (e.g. , vincristine sulfate), vinblastine (e.g. , vinblastine sulfate), bortezomib, nilotinib, ABT737, erPC, and perifosine.
In one embodiment, the drug is dactinomycin, plicamycin, teniposide, raltitrexed, irinotecan, rapamycin, gemcitabine (e.g. , gemcitabine hydrochloride), melphalan, acrichine, tamoxifen (e.g., tamoxifen citrate), vinorelbine (e.g. , vinorelbine tartrate), vincristine (e.g. , vincristine sulfate), vinblastine (e.g. , vinblastine sulfate), bortezomib, nilotinib, ABT737, erPC, or perifosine; or a salt (e.g., an acid addition salt) thereof.
Short-Chain Phosphatidylcholine (SCPC) Lipids The short-chain phosphatidylcholine (SCPC) lipids are related to choline,
phosphocholine, and glycerol, all shown below.
Figure imgf000014_0001
More generally, phosphatidylcholines are lipids of the following formula, wherein R and R2 denote hydrocarbon chains, for example, derived from fatty acids R -C(=0)OH and R2-C(=0)OH, respectively.
Figure imgf000015_0001
Common "saturated" fatty acids are shown in the following table.
Common Name Chemical Formula Code
acetic acid CH3COOH 2:0
propionic acid CH3CH2COOH 3:0
butyric acid CH3(CH2)2COOH 4:0
valeric acid CH3(CH2)3COOH 5:0
caproic acid CH3(CH2)4COOH 6:0
enanthic acid CH3(CH2)5COOH 7:0
caprylic acid CH3(CH2)6COOH 8:0
pelargonic acid CH3(CH2)7COOH 9:0
capric acid CH3(CH2)8COOH 10:0
undecylic acid CH3(CH2)9COOH 11 :0
lauric acid CH3(CH2)10COOH 12:0
tridecylic acid CH3(CH2)nCOOH 13:0
myristic acid CH3(CH2)12COOH 14:0
pentadecanoic acid CH3(CH2)13COOH 15:0
palmitic acid CH3(CH2)14COOH 16:0
margaric acid CH3(CH2)15COOH 17:0
stearic acid CH3(CH2)16COOH 18:0
nondecylic acid CH3(CH2)17COOH 19:0
arachidic acid CH3(CH2)18COOH 20:0
heneicosylic acid CH3(CH2)19COOH 21 :0
behenic acid CH3(CH2)20COOH 22:0
tricosylic acid CH3(CH2)21COOH 23:0
lignoceric acid CH3(CH2)22COOH 24:0 Common Name Chemical Formula Code pentacosylic acid CH3(CH2)23COOH 25:0 cerotic acid CH3(CH2)24COOH 26:0
Common "mono-unsaturated" fatty acids are shown in the following table.
Figure imgf000016_0001
Common "poly-unsaturated" fatty acids are shown in the following table.
Common Name Chemical Formula Code Isomer
CH3(CH2)4-CH=CH-CH2- cis.cis- linoleic acid 18:2
CH=CH-(CH2)7COOH Δ9,Δ12
CH3(CH2)4-CH=CH-CH2- trans, trans- linoelaidic acid 18:2
CH=CH-(CH2)7COOH Δ9,Δ12
CH3CH2-CH=CH-CH2-CH=CH- cis,cis,cis- a-linolenic acid 18:3
CH2-CH=CH-(CH2)7COOH Δ9,Δ 2,Δ15
CH3(CH2)4-CH=CH-CH2- cis,cis,cis- γ-linolenic acid CH=CH-CH2-CH=CH- 18:3
Δ6,Δ9,Δ12
(CH2)4COOH
CH3CH2-CH=CH-CH2-CH=CH- cis, cis, cis, cis- stearidonic acid CH2-CH=CH-CH2-CH=CH- 18:4
Δ6,Δ9,Δ 2,Δ15
(CH2)4COOH
CH3(CH2)7-CH=CH-CH2- cis,cis,cis- mead acid CH=CH-CH2-CH=CH- 20:3
Δ5,Δ8,Δ11
(CH2)3COOH
CH3(CH2)4-CH=CH-CH2- cis, cis, cis, cis- arachidonic acid CH=CH-CH2-CH=CH-CH2- 20:4
Δ5,Δ8,Δ ,Δ14
CH=CH-(CH2)3COOH Common Name Chemical Formula Code Isomer
CH3CH2-CH=CH-CH2-CH=CH- eicosapentaenoic cis, cis, cis, cis, cis-
CH2-CH=CH-CH2-CH=CH-CH2- 20:5
acid Δ5,Δ8,Δ ,Δ 4,Δ17
CH=CH-(CH2)3COOH
CH3(CH2)4-CH=CH-CH2- cis, cis, cis, cis- adrenic acid CH=CH-CH2-CH=CH-CH2- 22:4
Δ7,Δ10,Δ13,Δ16
CH=CH-(CH2)5COOH
CH3CH2-CH=CH-CH2-CH=CH- docosahexaenoic CH2-CH=CH-CH2-CH=CH-CH2- cis, cis, cis, cis, cis, cis-
22:6
acid CH=CH-CH2-CH=CH- Δ4,Δ7,Δ 0,Δ 3,Δ 6,Δ19
(CH2)2COOH
In one embodiment, the short-chain phosphatidylcholine (SCPC) lipid is a compound of the following formula:
Figure imgf000017_0001
wherein each of -R and -R is independently:
a linear or branched saturated alkyl group having from 7 to 13 carbon atoms; or a linear or branched alkenyl group having from 7 to 13 carbon atoms and from 1 to 3 carbon-carbon double bonds.
In one embodiment, each of -RFA and -RFA2 is independently:
a linear saturated alkyl group having from 7 to 13 carbon atoms; or
a linear alkenyl group having from 7 to 13 carbon atoms and from 1 to 3 carbon- carbon double bonds.
In one embodiment, each of -RFA and -RFA2 is independently a linear or branched saturated alkyl group having from 7 to 13 carbon atoms. In one embodiment, each of -RFA and -RFA2 is independently a linear saturated alkyl group having from 7 to 13 carbon atoms.
In one embodiment, each of -RFA and -RFA2 is independently a linear or branched saturated alkyl group having from 8 to 12 carbon atoms. ln one embodiment, each of -R and -R is independently a linear saturated alkyl group having from 8 to 12 carbon atoms.
In one embodiment, each of -RFA and -RFA2 is independently a linear or branched saturated alkyl group having from 8 to 10 carbon atoms.
In one embodiment, each of -RFA and -RFA2 is independently a linear saturated alkyl group having from 8 to 10 carbon atoms. In one embodiment, each of -RFA and -RFA2 is independently: -(CH2)6CI-l3, -(CH2)7CH3,
Figure imgf000018_0001
-(CH2)gCH3, -(CH2)ioCH3, -(CH2)iiCH3, or -(CH2)i2CH3.
In one embodiment, each of -RFA and -RFA2 is independently: -(CH2)7CH3, -(CH2)8CH3, -(CH2)9CH3, -(CH2)10CH3, or -(CH2)nCH3.
In one embodiment, each of -RFA and -RFA2 is independently: -(CH2)7CI-l3, -(CH2)8CH3, or
Figure imgf000018_0002
In one embodiment, -RFA and -RFA2 are the same.
In one embodiment, -RFA and -RFA2 are different.
In one embodiment, -RFA and -RFA2 are both -(CH2)7CH3; as in, for example,
di-pelargonoyl phosphatidylcholine (denoted herein as "dipelargonoyl-PC" or
"dinonaoyl-PC" or "C9-PC"), shown below:
Figure imgf000018_0003
In one embodiment, -R and -R are both -(CH2)8CH3; as in, for example,
di-caproyl phosphatidylcholine (denoted herein as "dicaproyl-PC" or "C10-PC"), shown below:
Figure imgf000018_0004
ln one embodiment, -R and -R are both -(CH2)9CH3; as in, for example,
di-undecanoyl phosphatidylcholine (denoted herein as "diundecanoyl-PC" or "C11-PC"), shown below:
Figure imgf000019_0001
In one embodiment, -R and -R are both -(CH2)ioCH3; as in, for example,
di-lauroyl phosphatidylcholine (denoted herein as "dilauroyl-PC" or "C12-PC"), shown below:
Figure imgf000019_0002
In one embodiment, -R and -R are both -(CH2)iiCH3; as in, for example,
di-tridecanoyl phosphatidylcholine (denoted herein as "ditridecanoyl-PC" or "C13-PC"), shown below:
Figure imgf000019_0003
In one embodiment, each of -R and -R is independently a linear or branched alkenyl group having from 7 to 13 carbon atoms and from 1 to 3 carbon-carbon double bonds.
In one embodiment, each of -RFA and -RFA2 is independently a linear alkenyl group having from 7 to 13 carbon atoms and 1 or 2 carbon-carbon double bonds.
In one embodiment, each of -RFA and -RFA2 is independently a linear alkenyl group having from 7 to 13 carbon atoms and 1 carbon-carbon double bond. Pharmaceutical Formulations - Additional Ingredients
As discussed above, one aspect of the present invention pertains to a pharmaceutical formulation comprising (i) a drug, as described herein, and (ii) a short-chain
phosphatidylcholine lipid (also referred to herein as an SCPC lipid), as described herein.
The pharmaceutical formulation may further comprise one or more (e.g., 1 , 2, 3, 4, etc.) additional pharmaceutically acceptable ingredients well-known to those skilled in the art, for example, one or more (e.g., 1 , 2, 3, 4, etc.) pharmaceutically acceptable carriers, diluents, or excipients; or one or more (e.g., 1 , 2, 3, 4, etc.) pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, or sweetening agents. In one embodiment, the pharmaceutical formulation further comprises a pharmaceutically acceptable carrier, diluent, or excipient.
The term "pharmaceutically acceptable," as used herein, pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.
Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts. See, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA),
Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.
The pharmaceutical formulation may further comprise one or more (e.g., 1 , 2, 3, 4, etc.) additional active agents, for example, other therapeutic or prophylactic agents. In one embodiment, the pharmaceutical formulation further comprises an additional active agent, for example, an additional therapeutic or prophylactic agent. Pharmaceutical Formulations - Methods of Preparation
As discussed above, one aspect of the present invention pertains to a pharmaceutical formulation comprising (i) a drug, as described herein, and (ii) a short-chain
phosphatidylcholine lipid (also referred to herein as an SCPC lipid), as described herein.
The pharmaceutical formulations may be prepared by any methods well-known in the art of pharmacy. Such methods include the step of bringing into association (i) a drug, as described herein, and (ii) an SCPC lipid, as described herein, optionally with one or more pharmaceutically acceptable carriers, diluents, or excipients, as described herein. In general, the pharmaceutical formulations are prepared by uniformly and intimately bringing into association the ingredients, and then shaping the product, if necessary. The pharmaceutical formulation may be prepared to provide for rapid or slow release;
immediate, delayed, timed, or sustained release; or a combination thereof.
Another aspect of the present invention pertains to a method of preparing a
pharmaceutical formulation comprising combining (i) a drug, as described herein, and (ii) an SCPC lipid, as described herein. If formulated as discrete units (e.g., ampoules, etc.), each unit may contain a
predetermined amount (dosage) of the drug and SCPC lipid.
Pharmaceutical Formulations - Forms The pharmaceutical formulations may suitably be in the form of liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions
(e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, mouthwashes, drops, tablets (including, e.g., coated tablets), granules, powders, losenges, pastilles, capsules (including, e.g., hard and soft gelatin capsules), cachets, pills, ampoules, boluses, suppositories, pessaries, tinctures, gels, pastes, ointments, creams, lotions, oils, foams, sprays, mists, or aerosols.
The pharmaceutical formulations may suitably be provided as a patch, adhesive plaster, bandage, dressing, or the like which is impregnated with one or more compounds and optionally one or more other pharmaceutically acceptable ingredients, including, for example, penetration, permeation, and absorption enhancers. Formulations may also suitably be provided in the form of a depot or reservoir.
Formulations suitable for oral administration (e.g., by ingestion) include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions {e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, tablets, granules, powders, capsules, cachets, pills, ampoules, boluses.
Formulations suitable for buccal administration include mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs. Losenges typically comprise the compound in a flavored basis, usually sucrose and acacia or tragacanth. Pastilles typically comprise the compound in an inert matrix, such as gelatin and glycerin, or sucrose and acacia. Mouthwashes typically comprise the compound in a suitable liquid carrier.
Formulations suitable for sublingual administration include tablets, losenges, pastilles, capsules, and pills.
Formulations suitable for oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil- in-water, water-in-oil), mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs.
Formulations suitable for non-oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions
(e.g., oil-in-water, water-in-oil), suppositories, pessaries, gels, pastes, ointments, creams, lotions, oils, as well as patches, adhesive plasters, depots, and reservoirs.
Formulations suitable for transdermal administration include gels, pastes, ointments, creams, lotions, and oils, as well as patches, adhesive plasters, bandages, dressings, depots, and reservoirs.
Tablets may be made by conventional means, e.g., compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the compound in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g., povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica); disintegrants (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose); surface-active or dispersing or wetting agents (e.g., sodium lauryl sulfate); preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid); flavours, flavour enhancing agents, and sweeteners. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with a coating, for example, to affect release, for example an enteric coating, to provide release in parts of the gut other than the stomach. Ointments are typically prepared from the compound and a paraffinic or a water-miscible ointment base.
Creams are typically prepared from the compound and an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1 ,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the compound through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.
Emulsions are typically prepared from the compound and an oily phase, which may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabiliser(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
Suitable emulgents and emulsion stabilisers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the compound in most oils likely to be used in pharmaceutical emulsion formulations may be very low. Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used. Formulations suitable for intranasal administration, where the carrier is a liquid, include, for example, nasal spray, nasal drops, or by aerosol administration by nebuliser, include aqueous or oily solutions of the compound. Formulations suitable for intranasal administration, where the carrier is a solid, include, for example, those presented as a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
Formulations suitable for pulmonary administration (e.g., by inhalation or insufflation therapy) include those presented as an aerosol spray from a pressurised pack, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichoro-tetrafluoroethane, carbon dioxide, or other suitable gases.
Formulations suitable for ocular administration include eye drops wherein the compound is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the compound. Formulations suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols, for example, cocoa butter or a salicylate; or as a solution or suspension for treatment by enema. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the compound, such carriers as are known in the art to be appropriate.
Pharmaceutical Formulations - Forms Suitable for Parenteral Administration
Formulations suitable for parenteral administration (e.g., by injection), include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the compound is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate). Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection.
Typically, the concentration of the compound in the liquid is from about 1 ng/mL to about 10 μς/ηιί, for example from about 10 ng/mL to about 1 μςΛη... The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. The formulation may be in the form of liposomes or other micro particulates (e.g., non-vesicular structures, e.g., micelles, etc.).
In one embodiment, the pharmaceutical formulation is suitable for parenteral
administration.
Pharmaceutical Formulations - Micelles
The pharmaceutical formulation may be in the form of micelles. In this case, the pharmaceutical formulation is micellar; that is, the pharmaceutical formulation is a micellar pharmaceutical formulation.
The term "micellar", as used herein, is intended to indicate that the drug, as described herein, and the short-chain phosphatidylcholine lipid, as described herein, are present in the form of a micelle (i.e., are micelle-entrapped).
Optionally, the micelles may further comprise one or more additional components, such as those described below for liposomes. Pharmaceutical Formulations - Liposomes
The pharmaceutical formulation may be in the form of liposomes or other
micro particulates (e.g., non-vesicular structures, e.g., micelles, etc.). In this case, the pharmaceutical formulation is liposomal; that is, the pharmaceutical formulation is a liposomal pharmaceutical formulation.
The term "liposomal", as used herein, is intended to indicate that the drug, as described herein, and the short-chain phosphatidylcholine lipid, as described herein, are present in the form of a liposome (i.e., are liposome-entrapped). Each of the components (i.e., the drug and the SCPC lipid) may tend to partition into the aqueous compartment of liposome, or into the lipid bilayer phase of the liposome (membrane-entrapped). For example, it is believed that the SCPC lipid is predominantly membrane-entrapped, while the drug is predominantly partitioned into the aqueous compartment. ln one embodiment, the pharmaceutical formulation comprises liposomes, said liposomes comprising (i) a drug, as described herein, and (ii) a short-chain phosphatidylcholine lipid (also referred to herein as an SCPC lipid), as described herein. In one embodiment, the pharmaceutical formulation is suitable for parenteral administration.
In one embodiment, the pharmaceutical formulation is an aqueous, isotonic,
pyrogen-free, sterile suspension of liposomes, said liposomes comprising: (i) a drug, as described herein, and (ii) a short-chain phosphatidylcholine lipid (also referred to herein as an SCPC lipid), as described herein. In one embodiment, the pharmaceutical formulation is suitable for parenteral administration.
In one embodiment, the liposomes are long-circulating liposomes (e.g., with a half-life that is from 0.2 to 5 times the half-life of Caelyx®. Liposomes (also called lipid vesicles) are aqueous compartments enclosed by a lipid bilayer (as in monolamellar liposomes) or many concentric bilayers (as in multilamellar liposomes). Lipids may be formed, for example, by suspending a suitable lipid (a vesicle- forming lipid), such as phosphatidyl choline, in an aqueous medium, and then sonicating (i.e., agitating by high frequency sound waves) to give a dispersion of closed vesicles. When prepared in this way, the liposomes are quite uniform in size, nearly spherical, and have a diameter of about 100 nm. Larger vesicles (on the order of 1 μηι) can be prepared by slowly evaporating the organic solvent from a suspension of phospholipid in a mixed solvent system. Suitable vesicle-forming lipids include amphipathic lipids having hydrophobic and polar head group moieties, and which (a) can form (spontaneously) into bilayer vesicles in water, as exemplified by phospholipids, or (b) are stably incorporated into lipid bilayers, with its hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane, and its polar head group moiety oriented toward the exterior, polar surface of the membrane.
Vesicle-forming lipids of this type are preferably ones having two hydrocarbon chains, typically acyl chains, and a polar head group. Examples of this class are the
phospholipids, such as phosphatidylcholines (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), and sphingomyelin (SM), where the two hydrocarbon chains are typically between about 14- 22 carbon atoms in length, and have varying degrees of unsaturation. A wide range of such lipids (with various acyl chain lengths, and various degrees of saturation) can be obtained commercially, or prepared according to published methods. Some preferred phosphatidylcholines include hydrogenated soy phosphatidylcholine (HSPC); dipalmitoyl-phosphatidylcholine (DPPC); DSPC (distearoyl phosphatidylcholine); and distearyl-phosphatidylethanolamine (DSPE). Generally, liposomes comprise 20-99% by weight of vesicle-forming lipids. In some cases, where other lipids are also present, the upper limit is less, e.g., 20-70%, etc.
Additionally, liposomes may include (non-vesicle forming) lipids that stabilize a vesicle or liposome composed predominantly of phospholipids. The most frequently employed lipid from this group is cholesterol (CH), typically at 25-40 mol%. Below about 20 mol% cholesterol in a bilayer, separate domains exist containing cholesterol and phospholipids and pure phospholipid. These bilayers show an increased permeability to water. At mole percentages above 50% cholesterol, starts to destabilize the bilayer. In some cases, other lipids, such as sitosterol, may be used in addition to, or as an alternative to, cholesterol.
Additionally, liposomes may include (vesicle forming) lipids (such as those described above) which have been derivatized with a polymer chain. In this respect, vesicle-forming lipids with diacyl chains, such as phospholipids, are preferred. One exemplary class of phospholipids are the phosphatidylethanolamines (PE), which have a reactive amino group that is convenient for coupling to activated polymers. An exemplary PE is distearyl PE (DSPE). A preferred polymer for derivatiziation is polyethyleneglycol (PEG), typically with a molecular weight of 1 ,000-10,000 Da, more typically 2,000-5,000 Da. Once a liposome is formed, the PEG chains provide a surface coating of hydrophilic chains which is sufficient to extend the blood circulation time of the liposomes, as compared to liposomes without the derivatized lipid. Other hydrophilic polymers which may be suitable include polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polylactic acid, polyglycolic acid, derivatized celluloses (such as hydroxymethylcellulose and
hydroxyethylcellulose), and amino acid-based polymers. The polymer-derivatized lipid, if present, is typically present in an amount of 1-20 mol% (e.g., 1-10 mol%, 1-5 mol%).
An example of a preferred vesicle-forming lipid that is derivatized with a polymer chain is: N-(carbonyl-methoxypolyethylene glycol 2000)-1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine sodium salt (MPEG2000-DSPE). This lipid is used in commercial STEALTH® liposomes, which are used in liposome formulations of doxorubicin such as Caelyx® and Doxil®.
Note such derivatized lipids are not always necessary or desired: the commercial product Daunoxome® (liposomal daunorubicin) is formulated in the form of relatively small (e.g., <100 nm) long-circulating liposomes using distearoyl phosphatidylcholine (DSPC). Liposomes may additionally comprise (vesicle-forming) lipids which have been modified for coupling antibody molecules to the liposome outer surface. For example, these lipids may be derivatized so as to have a pendant hydrophilic polymer spacer chain that is end-functionalized for coupling to an antibody. The functionalized end group may be, for example, a maleimide group (which can be used for selective coupling to antibody sulfhydryl groups), a bromoacetamide or disulfide group (which can be used for coupling to antibody sulfhydryl groups), an activated ester or aldehyde group (which can be sued for coupling to antibody amine groups), or a hydrazide (which can be used to coupling to compounds containing aldehyde groups).
Again, a preferred polymer for the spacer chain is polyethylene glycol (PEG). Other hydrophilic polymers which may be suitable for the spacer chain include end-derivatized polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polylactic acid, polyglycolic acid, derivatized celluloses (such as hydroxymethylcellulose and
hydroxyethylcellulose), and amino acid-based polymers. The polymer spacer chain is preferably shorter than the polymer chain described for the liposome surface polymer coating layer. For example, in a liposome composition containing a layer formed by PEG polymers of 2,000-5,000 Da, the spacer chain is generally of 100-5,000 Da, preferably 600-4,000 Da.
Liposomes may additionally comprise vesicle-forming lipids which have been modified by a biotin molecule. In this way, the liposome has a biotinylated surface which can be used to link other compounds (for example, a biotinylated antibody, via avidin). Biotinylated lipids, such as biotinylated phosphatidylethanolamines, are commercially available.
In addition, the liposome formulation may include targeting-enhancing features, such as targeting ligands, for example, monoclonal antibodies, peptides, antibody fragments, (recombinant) proteins, growth factors, folate, carbohydrates, etc.
Liposomes may be prepared by a variety of well-known techniques. Multilamellar vesicles (MLVs) can be formed by simple lipid-film hydration techniques. In this procedure, a mixture of liposome-forming lipids dissolved in a suitable organic solvent is evaporated in a vessel to form a thin film, which is then covered by an aqueous medium. The lipid film hydrates to form MLVs, typically with sizes of 0.1-10 μηι. In an alternative method, liposomes may be prepared by vortexing dried lipid films in a buffered aqueous solution.
Generally, the drug is incorporated into liposomes by adding it to the vesicle-forming lipids prior to liposome formation, as described below, in order to entrap the drug in the formed liposome. If the drug is hydrophobic, it may be added directly to the hydrophobic mixture. If the drug is hydrophilic, it may be added to the aqueous medium which covers the thin film of evaporated lipids. Alternatively, the drug may be incorporated into preformed liposomes, for example, by active transport mechanisms. For example, the drug may be taken up into liposomes in response to a potassium or hydrogen or ammonium sulfate or metal ion concentration differential. One effective sizing method for REVs and MLVs involves extruding an aqueous suspension of the liposomes through a series of polycarbonate membranes having a selected uniform pore size in the range of 0.03 to 0.2 μηι, typically 0.05, 0.08, 0.1 , or 0.2 μηι. The pore size of the membrane corresponds roughly to the largest sizes of liposomes produced by extrusion through that membrane, particularly where the preparation is extruded two or more times through the same membrane. Homogenization methods are also useful for down-sizing liposomes to sizes of 100 nm or less.
Some Preferred Liposomal Pharmaceutical Formulations As discussed above, in one embodiment, the pharmaceutical formulation comprises liposomes, said liposomes comprising (i) a drug, as described herein, and (ii) a short- chain phosphatidylcholine lipid (also referred to herein as an SCPC lipid), as described herein. In one embodiment, the liposomes further comprise additional phospholipid (i.e., in addition to SCPC lipid).
In one embodiment, the liposomes further comprises cholesterol (CH). In one embodiment, the liposomes further comprise additional phospholipid and cholesterol (CH).
In one embodiment, the additional phospholipid comprises additional phosphatidylcholine (PC) (i.e., in addition to SCPC lipid), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), and/or sphingomyelin (SM).
In one embodiment, the additional phospholipid comprises additional phosphatidylcholine (PC) and phosphatidylethanolamine (PE). In one embodiment, the additional phospholipid comprises additional phosphatidylcholine (PC). Some examples of well-known phosphatidylcholines (PC) are shown below.
Figure imgf000030_0001
In one embodiment, the additional phosphatidylcholine (PC) is HSPC.
In one embodiment, the additional phosphatidylcholine (PC) is DPPC.
In one embodiment, the additional phosphatidylcholine (PC) is DOPC.
In one embodiment, the additional phosphatidylcholine (PC) is DSPC. In one embodiment, the additional phospholipid comprises phosphatidylethanolamine (PE).
Some examples of well-known phosphatidylethanolamines (PE) are shown below. Abbreviation Name
DEPE 1 ,2-Dierucoyl-s glycero-3-phosphoethanolamine
DLPE 1 ,2-Dilauroyl-sn-glycero-3-phosphoethanolamine
DMPE 1 ,2-Dimyristoyl-s glycero-3-phosphoethanolamine
DOPE 1 ,2-Dioleoyl-s glycero-3-phosphoethanolamine
DPPE 1 ,2-Dipalmitoyl-s glycero-3-phosphoethanolamine
DSPE 1 ,2-Distearoyl-s glycero-3-phosphoethanolamine
POPE 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine
In one embodiment, the phosphatidylethanolamine (PE) is DSPE.
In one embodiment, the phosphatidylethanolamine (PE) is DOPE.
In one embodiment, the phosphatidylethanolamine (PE) is DPPE.
In one embodiment, the liposomes further comprise a vesicle-forming lipid which is derivatized with a polymer chain.
For example, in one embodiment, the liposomes comprise a drug (as described herein), an SCPC lipid (as described herein), a phospholipid (as described herein), cholesterol (CH), and a vesicle-forming lipid which is derivatized with a polymer chain (as described herein).
In one embodiment, the vesicle-forming lipid derivatized with a polymer chain is a phospholipid which is derivatized with polyethyleneglycol (PEG).
In one embodiment, the vesicle-forming lipid derivatized with a polymer chain is phosphatidylethanolamine (PE) which is derivatized with polyethyleneglycol (PEG). In one embodiment, the vesicle-forming lipid derivatized with a polymer chain is
N-(carbonyl-methoxypolyethylene glycol)-1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine sodium salt (MPEG-DSPE).
In one embodiment, the vesicle-forming lipid derivatized with a polymer chain is
N-(carbonyl-methoxypolyethylene glycol 2000)-1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine sodium salt (MPEG2000-DSPE).
For example, in one embodiment, the liposomes comprise a drug (as described herein), an SCPC lipid (as described herein), HSPC, phosphatidylethanolamine (PE) (as described herein), and cholesterol (CH). Similarly, in one embodiment, the liposomes comprise a drug (as described herein), an SCPC lipid (as described herein), HSPC, N-(carbonyl-methoxypolyethylene glycol 2000)- 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (MPEG2000-DSPE), and cholesterol (CH).
In one embodiment, the amount of SCPC lipid is 0.5-50 mol%; 1-25 mol%; 1-20 mol%;
1- 15 mol%; 1-10 mol%; 2-20 mol%; 2-15 mol%; 2-10 mol%; 3-20 mol%; 3-15 mol%; or 3-10 mol%, based on the total lipid content. In one embodiment, the amount of cholesterol (CH), if present, is 20-50 mol%;
25-50 mol%; 25-45 mol%; 25-40 mol%; 30-50 mol%; 30-45 mol%; 30-40 mol%;
35-45 mol%; or 40%, based on the total lipid content.
In one embodiment, the amount of phospholipid, if present, and excluding phospholipid which is derivatized with a polymer chain, if present, is 45-70 mol%; 35-75 mol%;
40-70 mol%; 45-65 mol%; 45-60 mol%; 50-65 mol%; or 55 mol%, based on the total lipid content.
In one embodiment, the amount of vesicle-forming lipid which is derivatized with a polymer chain, if present, is 1-15 mol%; 1-10 mol%; 1-7 mol%; 1-5 mol%; 2-10 mol%;
2- 7 mol%; 2-5 mol%; 3-10 mol%; 3-7 mol%; or 5 mol%, based on the total lipid content.
In one embodiment, the molar ratio of SCPC lipid to phospholipid, if present, and excluding phospholipid which is derivatized with a polymer chain, if present, is from 0.05 to 1 ; from 0.1 to 0.5; or from 0.2 to 0.4.
In one embodiment, the molar ratio of cholesterol (CH), if present, to the amount of phospholipid, if present, and excluding phospholipid which is derivatized with a polymer chain, if present, is from 0.1 to 0.5; or from 0.2 to 0.4.
In one embodiment, the molar ratio of vesicle-forming lipid which is derivatized with a polymer chain, if present, to phospholipid, if present, and excluding phospholipid which is derivatized with a polymer chain, if present, is from 0.1 to 0.5; or from 0.2 to 0.4. In one embodiment, the molar ratio of cholesterol (CH), if present, to vesicle-forming lipid which is derivatized with a polymer chain, if present, is from 0.8 to 1.2.
In one embodiment, the liposomes comprise 0.05-0.50 μηιοΙ drug per μηιοΙ phospholipid. In one embodiment, the amount is 0.10-0.40 μηιοΙ/μΓΤΐοΙ; 0.10-0.35 μηιοΙ/μΓΤΐοΙ;
0.10-0.30 μηιοΙ/μΓΤΐοΙ; 0.15-0.40 μηιοΙ/μΓΤΐοΙ; 0.15-0.35 μηιοΙ/μΓΤΐοΙ; 0.15-0.30 μηιοΙ/μΓΤΐοΙ; 0.20-0.40 μηιοΙ/μΓΤΐοΙ; 0.20-0.35 μηιοΙ/μΓΤΐοΙ; 0.20-0.30 μηιοΙ/μΓΤΐοΙ. In one embodiment, the pharmaceutical formulation comprises liposomes, as described herein, and has a drug concentration of 0.1-10 mg/mL; 0.5-5 mg/mL; 1-3 mg/mL; or 2 mg/mL.
The liposomes may additionally comprise other pharmaceutically acceptable ingredients, such as ammonium sulfate, histidine (as buffer), hydrochloric acid and/or sodium hydroxide (for pH control), sucrose (to maintain isotonicity), and water-for-injection. In one embodiment, the liposomes have a mean diameter of:
30 to 500 nm; 30 to 300 nm; 30 to 200 nm; 30 to 150 nm; 30 to 120 nm;
50 to 500 nm; 50 to 300 nm; 50 to 200 nm; 50 to 150 nm; 50 to 120 nm;
70 to 500 nm; 70 to 300 nm; 70 to 200 nm; 70 to 150 nm; 70 to 120 nm. In one embodiment, the pharmaceutical formulation is a (concentrated) liposomal pharmaceutical formulation (suitable for parenteral infusion) having a doxorubicin concentration of 2 mg/mL.
In one embodiment, the liposomes are Caelyx® or Doxil® liposomes which have been treated with a short-chain phosphatidylcholine lipid, as described herein.
As described above, in one embodiment, the pharmaceutical formulation comprises liposomes, said liposomes comprising (i) a drug, as described herein, and (ii) a short- chain phosphatidylcholine lipid (also referred to herein as an SCPC lipid), as described herein. Also as described above, these liposomes may optionally further comprise one or more or all of: additional phospholipid (as described herein); cholesterol; and a vesicle- forming lipid which is derivatized with a polymer chain (as described herein).
These liposomes may be prepared, for example, by:
(a) forming a lipid mixture comprising an SCPC lipid, and optionally, one or more or all of: additional phospholipid (as described herein); cholesterol; and a vesicle-forming lipid which is derivatized with a polymer chain (as described herein);
(b) forming liposomes from said lipid mixture; and
(c) adding the drug to the liposomes formed in (b).
Alternatively, these liposomes may be prepared, for example, by:
(a) forming a lipid mixture comprising an SCPC lipid, and optionally, one or more or all of: additional phospholipid (as described herein); cholesterol; and a vesicle-forming lipid which is derivatized with a polymer chain (as described herein);
(b) adding the drug to said lipid mixture; and
(c) forming liposomes from the mixture formed in (b). General Chemical Synthesis
Many of the short-chain phosphatidyl-choline lipids described herein may be obtained from commercial sources. Other short-chain phosphatidyl-choline lipids may be prepared using conventional methods known in the art, or by adapting conventional methods known in the art in conventional ways.
For example, short-chain phosphatidyl-choline lipids may be prepared using conventional methods, for example, as described in Eibl, Chemistry and Physics of Lipids, 1980, from simple starting materials, such as glycero-3-phosphocholine, which may be prepared using conventional methods, for example, as described in Brockerhoff and Yurkowski, Can. J. Biochem.. 1965, Vol. 43, p. 1777. For example, glycero-3-phosphocholine may be acylated with excess fatty acid anhydride in the presence of the tetraethylammonium salt of the fatty acid (for example, at a molar ratio of glycero-3-phosphocholine : fatty acid anhydride : salt of 1 : 6 : 6). In an alternative approach, the cadmium complex of glycero-3-phosphocholine is prepared and dissolved in dimethylsulfoxide. Fatty acid anhydrides dissolved in benzene are then added, supplemented with 4-pyrrolidinepyridine (for example, at a molar ratio of glycero-3- phosphocholine : fatty acid anhydride : base of 1 : 8 : 2). Typically, the reaction is completed after 2-5 hours at 45°C.
Scheme 1
RO
\
C H2-O H ( CO C H2-0— CO— R
/
RO
C H— O H C H— O— CO— R
C H2-0 PO— O— C H2-C H2— N+(CH3)3 C H2-0 PO— O— C H2-C H2— N+(CH3)3
O" O"
Alternatively, a method similar to that described in Patel et al., J. Lipid Res., 1979, Vol. 20, p. 674 may be used. In such a method, 1-hexadecyl-2-octadecenyl-sn-glycerol is condensed with phosphorus oxychloride in the presence of quinoline. Choline- toluenesulfonate (Tos) is then dissolved in pyridine and added to the reaction mixture, for example, at a molar ratio of dialkylglycerol : phosphorus oxychloride :
cholinetoluenesulfonate of 1 : 1.25 : 2. Typically, the reaction is completed after 5 hours at 20°C. The resulting sn-glycero-3-phosphocholine can be purified by chromatography. Scheme 2
C H2-OR.| Ri
H O— C H2— C H2— N (CH3)3 Jos"
C H— OR2 C H— O— CO— R2
2) H20
C H2-0— POCI2 O— C H2— C H2— N+(CH3)3
O I "
Use to Increase Bioavailability and/or Cellular Uptake
The inventors have demonstrated that a short-chain phosphatidylcholine lipid (also referred to herein as an SCPC lipid), as described herein, greatly increases the bioavailability of a drug and/or the cellular uptake of a drug, as described herein. Thus, another aspect of the present invention pertains to a use of an SCPC lipid, as described herein, to increase the bioavailability of a drug, as described herein.
Another aspect of the present invention pertains to a use of an SCPC lipid, as described herein, to increase cellular uptake of a drug, as described herein.
Another aspect of the present invention pertains to a method of increasing the bioavailability of a drug, as described herein, in a patient, which method comprises the step of co-administering said drug and an SCPC lipid, as described herein. Another aspect of the present invention pertains to a method of increasing the cellular uptake of a drug, as described herein, into a cell, which method comprises the step of contacting said cell with said drug and an SCPC lipid, as described herein.
Use in Medicine
The pharmaceutical formulations described herein are useful, for example, in methods of treatment of a disorder (e.g., a disease), according to the nature of the drug. For example, where the drug is an anti-proliferative agent, e.g., an anti-cancer drug, the pharmaceutical formulations are useful in methods of treatment of a proliferative condition, e.g., cancer.
Use in Methods of Therapy
Another aspect of the present invention pertains to a pharmaceutical formulation, as described herein, for use in a method of treatment of the human or animal body by therapy, for example, for use a method of treatment of a disorder (e.g., a disease) as described herein. Use in the Manufacture of Medicaments
Another aspect of the present invention pertains to use of (i) a drug, as described herein, and (ii) a short-chain phosphatidylcholine lipid (also referred to herein as an SCPC lipid), as described herein, in the manufacture of a pharmaceutical formulation, as described herein, for the treatment of a disorder (e.g., a disease), as described herein.
In one embodiment, the medicament comprises the drug and the SCPC lipid.
Methods of Treatment
Another aspect of the present invention pertains to a method of treatment, for example, of a disorder (e.g., a disease) as described herein, comprising administering to a patient in need of treatment a therapeutically effective amount of a pharmaceutical formulation, as described herein.
Conditions Treated - Proliferative Conditions In one embodiment, where the drug is an anti-proliferative agent, the treatment is treatment of a proliferative condition. The terms "proliferative condition", as used herein, means an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth. In one embodiment, where the drug is an anti-cancer agent, the treatment is treatment of cancer. The nature of the cancer will be determined by the nature of the drug. For example, where the drug is known to treat small cell lung cancer, the pharmaceutical formulations are useful in methods of treatment of small cell lung cancer. In one embodiment, the cancer is lung cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, oesophagus cancer, stomach cancer, bowel cancer, small bowel cancer, large bowel cancer, colon cancer, rectal cancer, colorectal cancer, thyroid gland cancer, adrenal gland cancer, breast cancer, gynaecological cancer, ovarian cancer, genito-urinary cancer, endometrial cancer, prostate cancer, testicular cancer, liver cancer, biliary tract cancer, kidney cancer, renal cell carcinoma, bladder cancer, pancreatic cancer, brain cancer, neuroblastoma, glioma, sarcoma, osteosarcoma, bone cancer, nasopharyngeal cancer (e.g., head cancer, neck cancer), skin cancer, squamous cancer, Kaposi's sarcoma, melanoma, malignant melanoma, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or leukemia. The anti-cancer effect may arise through one or more mechanisms, including but not limited to, the regulation of cell proliferation, the inhibition of cell cycle progression, the inhibition of angiogenesis (the formation of new blood vessels), the inhibition of metastasis (the spread of a tumour from its origin), the inhibition of cell migration (the spread of cancer cells to other parts of the body), the inhibition of invasion (the spread of tumour cells into neighbouring normal structures), or the promotion of cell apoptosis (programmed cell death).
In one embodiment, where the drug is doxorubicin or a salt (e.g., acid addition salt) thereof, the treatment is treatment of condition (e.g., a proliferative condition, e.g., a cancer) that is treated by (e.g., treatable by) (e.g., known to be treated by) (e.g., known to be treatable by) doxorubicin.
In one embodiment, where the drug is doxorubicin or a salt (e.g., acid addition salt) thereof, the treatment is treatment of cancer that is treated by (e.g., treatable by)
(e.g., known to be treated by) (e.g., known to be treatable by) doxorubicin.
In one embodiment, where the drug is doxorubicin or a salt (e.g., acid addition salt) thereof, the treatment is treatment of ovarian cancer, for example, metastatic carcinoma of the ovary, for example, metastatic carcinoma of the ovary in patients with disease that is refractory to both paclitaxel- and platinum-based chemotherapy regimens.
In one embodiment, where the drug is doxorubicin or a salt (e.g., acid addition salt) thereof, the treatment is treatment of Kaposi's syndrome, for example, AIDS-related Kaposi's syndrome, for example, AIDS-related Kaposi's syndrome in patients with disease that has progressedon prior combination chemotherapy or in patients who are intolerant to such therapy.
Treatment
The term "treatment," as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, alleviation of symptoms of the condition, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis) is also included. For example, use with patients who have not yet developed the condition, but who are at risk of developing the condition, is encompassed by the term "treatment." For example, treatment of cancer includes the prophylaxis of cancer, reducing the incidence of cancer, reducing the cancer of dementia, alleviating the symptoms of cancer, etc. The term "therapeutically-effective amount," as used herein, pertains to that amount of a compound, or a material, composition or dosage form comprising a compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen. Combination Therapies
The term "treatment" includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously. For example, the pharmaceutical formulations described herein may also be used in combination therapies, e.g., in conjunction with other agents, for example, anti-cancer agents, etc. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g., drugs, antibodies (e.g., as in immunotherapy), prodrugs (e.g., as in photodynamic therapy, GDEPT, ADEPT, etc.); surgery; radiation therapy; photodynamic therapy; gene therapy; and controlled diets.
The particular combination would be at the discretion of the physician who would select dosages using his common general knowledge and dosing regimens known to a skilled practitioner. The agents (i.e., the pharmaceutical formulation described herein, plus one or more other agents) may be administered simultaneously or sequentially, and may be administered in individually varying dose schedules and via different routes. For example, when administered sequentially, the agents can be administered at closely spaced intervals (e.g., over a period of 5-10 minutes) or at longer intervals (e.g., 1 , 2, 3, 4 or more hours apart, or even longer periods apart where required), the precise dosage regimen being commensurate with the properties of the therapeutic agent(s).
The agents (i.e., the pharmaceutical formulation described here, plus one or more other agents) may be formulated together in a single dosage form, or alternatively, the individual agents may be formulated separately and presented together in the form of a kit, optionally with instructions for their use.
Other Uses
The pharmaceutical formulations, as described herein, may also be used as cell culture additives. The pharmaceutical formulations, as described herein, may also be used as part of an in vitro assay, for example, in order to determine whether a candidate host is likely to benefit from treatment with the pharmaceutical formulation. The pharmaceutical formulations, as described herein, may also be used as a standard, for example, in an assay, in order to identify, characterise, and/or evaluate other pharmaceutical formulations.
Kits
Another aspect of the invention pertains to a kit comprising (a) a pharmaceutical formulation, as described herein, preferably provided in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, written instructions on how to administer the pharmaceutical formulation, etc.
Another aspect of the invention pertains to a kit comprising (a) a drug, as described herein, (b) a short-chain phosphatidylcholine lipid (also referred to herein as an SCPC lipid), as described herein (c) optionally, one or more or all of: phospholipid (as described herein); cholesterol; and a vesicle-forming lipid which is derivatized with a polymer chain (as described herein); all preferably provided in a suitable container and/or with suitable packaging; and (d) instructions for use, for example, written instructions on how to prepare (and optionally administer) a liposomal pharmaceutical formulation, as described herein. Another aspect of the invention pertains to a kit comprising (a) a drug, as described herein, preferably provided in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, written instructions on how to prepare (and optionally administer) a pharmaceutical formulation, as described herein. If appropriate, the kit may optionally including appropriate reagents (e.g., buffers, solvents) and devices (e.g., tubes, syringes) for assembly and use (e.g., administration).
The written instructions may also include a list of indications for which the active ingredient is a suitable treatment.
Routes of Administration
The pharmaceutical formulation may be administered to a subject by any convenient route of administration, whether systemically/peripherally or topically (i.e., at the site of desired action). Routes of administration include, but are not limited to, oral {e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal {e.g., by nasal spray, drops or from an atomiser or dry powder delivery device); ocular {e.g., by eyedrops); pulmonary {e.g., by inhalation or insufflation therapy using, e.g., an aerosol, e.g., through the mouth or nose); rectal {e.g., by suppository or enema); vaginal {e.g., by pessary); parenteral, for example, by injection {e.g., infusion), including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly.
In one preferred embodiment, the pharmaceutical formulation is administered
parenterally, for example, by intravenous infusion. The Subject/Patient
The subject/patient may be a chordate, a vertebrate, a mammal, a placental mammal, a marsupial {e.g., kangaroo, wombat), a rodent {e.g., a guinea pig, a hamster, a rat, a mouse), murine {e.g., a mouse), a lagomorph {e.g., a rabbit), avian {e.g., a bird), canine {e.g., a dog), feline {e.g., a cat), equine {e.g., a horse), porcine {e.g., a pig), ovine {e.g., a sheep), bovine {e.g., a cow), a primate, simian {e.g., a monkey or ape), a monkey
{e.g., marmoset, baboon), an ape {e.g., gorilla, chimpanzee, orangutang, gibbon), or a human. Furthermore, the subject/patient may be any of its forms of development, for example, a foetus.
In one preferred embodiment, the subject/patient is a human. Dosage It will be appreciated by one of skill in the art that appropriate dosages of the
pharmaceutical formulation can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the drug, the route of administration, the time of administration, the rate of excretion of the drug, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the patient. The amount of pharmaceutical formulation and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side- effects.
Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the pharmaceutical formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.
Appropriate dosages for known drugs are known in the art. Examples of suitable dosages include: doxorubicin: 60-75 mg/m2; epirubicin: 60-120 mg/m2; and daunorubicin: 25-45 mg/m2. Dosages above about 550 mg/m2 doxorubicin may lead to irreversible myocardial toxicity leading to congestive heart failure often unresponsive to cardiac support therapy.
Liposomal doxorubicin (e.g., Doxil®, Caelyx®) is typically provided with a doxorubicin concentration of about 2 mg/mL, and is administered by intravenous infusion at a dosage of about 50 mg/m2, intravenously, at a rate of about 1 mg/min (and so, for a period of 90 minutes).
Biological Study 1
Doxorubicin
Short-chain phosphatidyl-choline lipids were dissolved in ethanol at a concentration of 10 mM. Bovine aortic endothelial cells (BAEC) were cultured in flat-bottom 96-well plates in 10% serum-containing medium, in a water-saturated atmosphere of 5% C02 at 37°C. After reaching confluency, the cells were serum starved for at least 4 hours. Cells were then pre-incubated with short-chain phosphatidyl-choline lipids (in ethanol) at a concentration of 2.5, 5, 10, 25, 50, 100, or 250 μΜ for 15 minutes, followed by incubation with 50 μΜ doxorubicin (from an aqueous stock solution at a concentration of 2 mg/mL) for 1 hour (while the short-chain phosphatidyl-choline lipids remained present).
Afterwards, the cells were washed three times with phosphate buffered saline (PBS), and then lysed in 1 % (w/v) Triton X-100. Doxorubicin fluorescence was determined with a PerkinElmer Victor Wallac II microplate reader using 485 and 535 nm filters for excitation and emission, respectively. All values were corrected for background fluorescence and corrected for differences in protein content, as determined with a bicinchoninic acid assay (Smith et al., 1985, "Measurement of protein using bicinchoninic acid", Anal. Biochem., Vol. 150, No. 1 , pp. 76-85). Cellular doxorubicin content was quantified using dilution series of known amounts of doxorubicin.
The results are summarized in Figure 1.
Figure 1 is a graph of doxorubicin content (expressed as % of control) as a function of concentration (μΜ) of short-chain phosphatidyl-choline lipid, for five lipids: dinonaoyl-PC (C9), diundecanoyl-PC (C1 1), dilauroyl-PC (C12), ditridecanoyl-PC (C13), and dimyristoyl-PC (C14), as well as the comparison lipid, C8-GlcCer (N-octanoyl- glucosylceramide).
The data demonstrate that short-chain phosphatidyl-choline lipids with a fatty chain ranging from C9 to C13 substantially increase the cellular uptake of doxorubicin, as compared to a short-chain phosphatidyl-choline lipid with a C14 fatty chain. Notably, the C9 and C1 1 short-chain phosphatidyl-choline lipids greatly increased the cellular uptake of doxorubicin at concentrations above about 10 μΜ, and especially at concentrations above about 30 μΜ. Biological Study 2
Cell Viability
Short-chain phosphatidyl-choline lipids were dissolved in ethanol at a concentration of 10 mM. Bovine aortic endothelial cells (BAEC) were cultured in flat-bottom 96-well plates in 10% serum-containing medium, in a water-saturated atmosphere of 5% C02 at 37°C. After reaching confluency, the cells were serum starved for at least 4 hours. Cells were then incubated with short-chain phosphatidyl-choline lipids (in ethanol) for 24 hours. Cells were incubated with ethanol alone as a routine check, to check that cell viability is not decreased by more than 10% as compared to the untreated wells. The medium was replaced with serum-supplemented medium, and cell viability was determined 48 hours after washing by adding 50 μg of the mitochondrial dehydrogenase substrate 2,3-bis(2- methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt (XTT) and 38 ng N-methyl dibenzopyrazine methyl sulfate (PMS) to each well. After incubation at 37°C for 2 hours, absorbance was read using a Victor Wallac microplate reader
(PerkinElmer, Waltham, MA, USA) at 490 nm after a brief period (2 seconds) of automated shaking. 100% viability was set using wells containing untreated cells, and 0% viability using cells lysed in 2% (v/v) Triton X100. The results are summarized in Figure 2.
Figure 2 is a graph of cell viability (expressed as % of control) as a function of concentration (μΜ) of short-chain phosphatidyl-choline lipid, for five lipids: dinonaoyl-PC (C9), diundecanoyl-PC (C1 1), dilauroyl-PC (C12), ditridecanoyl-PC (C13), and dimyristoyl-PC (C14), as well as the comparison lipid, C8-GlcCer (N-octanoyl- glucosylceramide).
The data demonstrate that short-chain phosphatidyl-choline lipids with a fatty chain ranging from C9 to C14 do not adversely affect cell viability at lipid concentrations up to about 20 μΜ. Notably, the C9 and C11 short-chain phosphatidyl-choline lipids are well tolerated up to about 60 μΜ.
Biological Study 3
High Throughput Screening
The NCI-88 approved oncology set library was screened on A431 vulva carcinoma and WEP mouse mammary carcinoma cells. The Selleck Kinase inhibitor library was screened on HCT-119 colon carcinoma cells. Cells were cultured in serum containing medium, in a water-saturated atmosphere of 5% C02 at 37°C. Short-chain phosphatidylcholine (SCPC) lipids were dissolved in ethanol at a concentration of 10 mM. SCPC lipids were applied at a final concentration of 20 μΜ. In this way, the SCPC lipids were allowed to insert into the plasma membrane of the cells. From the libraries, test drugs were applied at concentrations of 0.001 , 0.01 , 0.1 , 1.0, and 10 μΜ. For the NCI-88 library, SCPC lipids and library test drugs were added to serum-free culture medium conditions. After an incubation period of 4 hours, cells were washed with phosphate buffered saline (PBS) three times. Then, serum-containing (10%) medium was applied again, followed by a 48 hour period of cell expansion. For the Selleck Kinase library, SCPC lipids and library test drugs were directly applied to cells in culture medium containing 2% fetal calf serum (FCS), followed by a 48 hours cell expansion period (the SCPC lipids and library test drugs were not removed before the expansion). SCPC lipids were always added prior to the library test drugs, and preferably 15 minutes before the library test drugs. SCPC lipids, without library test drugs, were applied as a negative control. Phenyl arsene oxide was applied as a positive control. After incubation with the SCPC lipids and library test drugs, and a period of cell expansion, cell viability was measured by a Promega Cell Titer bleu metabolic assay. Read-out was performed on a PerkinElmer Envision Multilabel Reader.
The screenings were performed in 384 well-plate format, twice for each cell line, and with the library test drugs added in duplicate on each plate. After read-out, data were analyzed by automated curve fitting. The generated IC2s, IC50 and IC75 values, plus a relative IC50 value (relative to top and bottom plateaus of the sigmoidal curve) were used for further analysis. In cases where sufficient data points for a library test drug (i.e., >50% of these four values per data set) were available, the effect of the SCPC lipid was considered eligible for evaluation. The data were ranked on their maximal IC shift, which is a logarithmic value. The results are summarized in the following table, which presents data for library test drugs which, in combination with an SCPC lipid, caused a shift of IC value, so as to lower the test compound's IC value by more than one-half (0.5) of a logi0-value. The table also includes the molecular target which the test compound inhibits, so as to decrease cell viability. The data show that the SCPC increased the potencies of the library test drugs by a factor of at least about 3 (i.e., 100 5) and in many cases, by a factor of least about 10 (i.e., 10 °), and in at least one case, by a factor of about 200 (i.e., 102 3).
Maximum
SCPC Lipid Drug Target
IC shift (log™)
C9-PC PIK-75 PI3K, DNA-PK 2.3
C9-PC GSK1059615 PI3K, mTOR 1.7
C9-PC ZSTK474 PI3K 1.5
C11-PC GDC-0941 PI3K 1.2
C11-PC AZD6482 PI3K 1.1
C11-PC Danusertib FGFR, Bcr-Abl, Src 1.1
C9-PC PHA-793887 CDK 1.0
C11-PC PIK-93 PI3K, VEGFR 1.0
MK-2206
C11-PC Akt 0.9
dihydrochloride
C11-PC PIK-90 PI3K 0.9
C11-PC AZD7762 Chk 0.8
C9-PC BEZ235 mTOR, PI3K 0.8
C9-PC Bl 2536 PLK 0.8
C11-PC NVP-ADW742 IGF-1 R 0.8
C11-PC PI-103 DNA-PK, PI3K, mTOR 0.8
C11-PC Temsirolimus (Torisel) mTOR 0.8
Crizotinib
C11-PC c-Met, ALK 0.7
(PF-02341066)
C9-PC JNJ-7706621 CDK, Aurora Kinase 0.7
C11-PC Pelitinib (EKB-569) EGFR 0.7
Sunitinib Malate
C11-PC VEGFR, PDGFR, c-Kit, Fit 0.7
(Sutent)
C11-PC XL-184 (Cabozantinib) VEGFR, c-Met, Fit, Tie-2 0.7
C9-PC Hesperadin Aurora Kinase 0.6
C11-PC Ponatinib Bcr-Abl, VEGFR, FGFR 0.5
C9-PC Regorafenib c-Kit, Raf, VEGFR 0.5 Biological Study 4
Dactinomycin
The effect of an SCPC lipid (dinonaoyl-PC) on the potency of dactinomycin was examined using the methods described above for High Throughput Screening (the NCI-88 approved oncology set library for WEP mouse mammary carcinoma cells).
The results are summarized in Figure 3. Figure 3 is a graph of cell viability (expressed as % of control) as a function of logarithm of concentration of dactinomycin, for dactinomycin alone (circles) and for dactinomycin with an SCPC lipid (triangles), specifically, dinonaoyl-PC (C9), at a concentration of 20 μΜ. For dacinomycin alone, the IC50 was about -6. For dactinomycin with SCPC lipid, the IC50 was about -7.4. The shift in log 0(IC50) caused by the SCPC lipid was about 1.4 log 0 units, or a factor of about 24.
* * *
The foregoing has described the principles, preferred embodiments, and modes of operation of the present invention. However, the invention should not be construed as limited to the particular embodiments discussed. Instead, the above-described embodiments should be regarded as illustrative rather than restrictive. It should be appreciated that variations may be made in those embodiments by workers skilled in the art without departing from the scope of the present invention.
REFERENCES
A number of publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference. Gabizon A, 2001 , "Pegylated liposomal doxorubicin: metamorphosis of an old drug into a new form of chemotherapy," Cancer Inv., Vol. 19, pp. 424-436.
Gabizon A, Chemla M, Tzemach D, Horowitz AT, Goren D, 1996, "Liposome longevity and stability in circulation: effects on the in vivo delivery to tumors and therapeutic efficacy of encapsulated anthracyclines," J. Drug Target, Vol. 3, pp. 391-398. Haran G, Cohen R, Bar LK, Barenholz Y, 1993, "Transmembrane ammonium sulphate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases," Biochim. Biophvs. Acta, Vol. 1 151 , pp. 201-215.
Harasym TO, Cullis PR, Bally MB, 1997, "I ntratumor distribution of doxorubicin following i.v. administration of drug encapsulated in egg phsophatidylcholine/ cholesterol liposomes," Cancer Chemother. Pharmacol., Vol. 40, pp. 309-317.
Heijn M, Roberge S, Jain RK, 1999, "Cellular membrane permeability of anthracyclines does not correlate with their delivery in a tissue-isolated tumor," Cancer Res., Vol. 59, pp. 4458-4463.
Horowitz AT, Barenholz Y, Gabizon A, 1992, "In vitro cytotoxicity of liposome- encapsulated doxrubicin: dependence on liposome composition and drug release," Biochim. Biophvs. Acta, Vol. 1 109, pp. 203-209.
Jeckel D, Wieland F, 1993, "Truncated ceramide analogs as probes for sphingolipid biosynthesis and transport," Adv. Lipid Res., Vol. 26, pp. 143-160.
Koning GA, Gorter A, Scherphof GL, Kamps JAAM, 1999, "Antiproliferative effect of immunoliposomes containing 5-fluorodeoxyuridine-dipalmitate on colon cancer cells," Br. J. Cancer. Vol. 80, pp. 1718-1725.
Koning GA, Morselt HWM, Gorter A, Allen TM, Zaiipsky S, Scherphof GL, Kamps JAAM, 2003, "Interaction of differently designed immunoliposomes with colon cancer cells and kupffer Cells. An in vitro comparison," Pharm. Res., Vol. 20, pp. 1249-1258.
Koning, G.A., Veldman, R.J., van Hell, A., Zerp, S., Storm, G., van Blitterswijk, W.J., Verheij, M., 2003, "Short-chain liposomal sphingolipids potentiate in vitro doxorubicin cytotoxicity by enhancing its cellualr influx," Conference Abstract for Liposome Advances: Progress in Drug and Vaccine Delivery, 15-19 December 2003, London, UK. Lothstein L, Israel M, Sweatman TW, 2001 , "A nth racy din drug targeting: cytoplasmic versus nuclear - a fork in the road," Drug Res. Updates, Vol. 4, pp. 169-177.
Pan XQ, Wang H, Lee RJ, 2003, "Antitumor activity of folate receptor-targeted liposomal doxorubcin in a KB oral carcinoma murine xenograft model," Pharm. Res., Vol. 20, pp. 417-422.
Park JW, Hong K, Kirpotin DB, Colbern G, Shalaby R, Baselga J, Shao Y, Nielsen UB, Marks JD, Moore D, Papahadjopoulos D, Benz CC, 2002, "Anti-HER2
immunoliposomes: enhanced efficacy attributable to targeted delivery," Clin.
Cancer Res.. Vol. 8, pp. 1172-1181.
Robert J, Gianni L, 1993, "Pharmacokinetics and metabolism of anthracyclines," in
Cancer Surveys 17 (Workman P and Graham MA, eds), pp. 219-252.
Speth PA, Raijmakers RA, Boezeman JB, Linssen PC, de Wtte TJ, Wessels HM,
Haanen C, 1988, "In vivo cellular adriamycin concentrations related to growth inhibition of normal and leukemic human bone marrow cells," Eur. J. Cancer Clin. Oncol.. Vol. 24, pp. 667-674.
Tardi PG, Boman NL, Cullis PR, 1996, "Liposomal doxorubicin," J. Drug Target, Vol. 4, pp. 129-140.
Uster PS, Allen TM, Daniel BE, Mendez CJ, Newman MS, Zhu GZ, 1996, "Insertion of poly(ethylene glycol) derivatized phospholipid into pre-fromed liposomes results in prolonged in vivo circulation time," FEBS Lett.. Vol. 386, pp. 243-246.
Vaage J, Mayhew E, Lasic D, Martin F, 1992, "Therapy of primary and metastatic mouse mammary carcinomas with doxorubicin encapsulated in long circulating
liposomes." Int. J .Cancer. Vol. 51 , pp. 942-948.
Washington N, Washington C, Wlson CG, 2001 , "Cell membranes, epithelial barriers and drug absorption," in Physiological Pharmaceutics: Barriers to Drug Absorption, pp. 1-18, (publishers: Taylor and Francis: London).
Weiss RB, 1992, "The anthracyclines: will we ever find a better doxorubicin?",
Sem. Oncol.. Vol. 19, pp. 670-686.
Yuan F, Leunig M, Huang SK, Berk DA, Papahadjopoulos D, Jain RK, 1994,
"Microvascular permeability and interstitial penetration of sterically stabilized
(stealth) liposomes in a human tumor xenograft," Cancer Res., Vol. 54, pp. 3352-3356.
Zagotto G, Gatto B, Moro S, Siss C, Palumbo M, 2001 , "Anthracyclins: recent
developments in their separation and quantitation," J. Chromat. B, Vol. 764, pp. 161-171.

Claims

1. A pharmaceutical formulation comprising:
(i) a drug;
(ii) a short-chain hosphatidylcholine (SCPC) lipid of the following formula:
Figure imgf000049_0001
wherein each of -R and -R is independently:
a linear or branched saturated alkyl group having from 7 to 13 carbon atoms; or
a linear or branched alkenyl group having from 7 to 13 carbon atoms and from 1 to 3 carbon-carbon double bonds.
A pharmaceutical formulation according to claim 1 , wherein the drug is an amphiphilic drug.
A pharmaceutical formulation according to claim 1 or 2, wherein the drug is an anthracycline.
A pharmaceutical formulation according to claim 1 , wherein the drug is
doxorubicin, daunorubicin, epirubicin, aclarubicin, idarubicin, valrubicin, or mitoxantrone; or a salt thereof.
A pharmaceutical formulation according to claim 1 , wherein the drug is
doxorubicin or doxorubicin hydrochloride.
A pharmaceutical formulation according to claim 1 or 2, wherein the drug is an alkaloid.
A pharmaceutical formulation according to claim 1 , wherein the drug is
camptothecin, topotecan, irinotecan, vincristine, vinorelbine, or vinblastine; or a salt thereof.
A pharmaceutical formulation according to claim 1 , wherein the drug is PIK-75, GSK1059615, ZSTK474, GDC-0941 , AZD6482, Danusertib, PHA-793887, PIK-93, MK-2206 dihydrochloride, PIK-90, BEZ235, PI-103, Bl 2536, AZD7762, NVP-ADW742, Temsirolimus, Crizotinib, JNJ-7706621 , Pelitinib, Sunitinib Malate, XL-184, Hesperadin, Ponatinib, or Regorafenib; or a salt thereof.
9. A pharmaceutical formulation according to claim 1 , wherein the drug is dactinomycin, plicamycin, teniposide, raltitrexed, irinotecan, rapamycin, gemcitabine (e.g., gemcitabine hydrochloride), melphalan, acrichine, tamoxifen (e.g., tamoxifen citrate), vinorelbine (e.g., vinorelbine tartrate), vincristine (e.g., vincristine sulfate), vinblastine (e.g., vinblastine sulfate), bortezomib, nilotinib, ABT737, erPC, or perifosine; or a salt thereof.
10. A pharmaceutical formulation according to any one of claims 1 to 9, wherein each of -RFA and -RFA2 is independently a linear or branched saturated alkyl group having from 7 to 13 carbon atoms.
11. A pharmaceutical formulation according to any one of claims 1 to 9, wherein each of -RFA and -RFA2 is independently -(CH2)6CH3, -(CH2)7CH3, -(CH2)8CH3, -(CH2)9CH3, -(CH2)10CH3, -(CH2)nCH3, or -(CH2)12CH3.
12. A pharmaceutical formulation according to any one of claims 1 to 9, wherein each of -RFA and -RFA2 is independently -(CH2)7CH3, -(CH2)8CH3, -(CH2)9CH3, -(CH2)10CH3, or -(CH2)nCH3.
13. A pharmaceutical formulation according to any one of claims 1 to 9, wherein each of -RFA and -RFA2 is independently -(CH2)7CH3, -(CH2)8CH3, or -(CH2)9CH3.
14. A pharmaceutical formulation according to any one of claims 1 to 13, wherein -RFA and -RFA2 are the same.
15. A pharmaceutical formulation according to any one of claims 1 to 13, wherein -RFA and -RFA2 are different.
16. A pharmaceutical formulation according to any one of claims 1 to 9, wherein -RFA and -RFA2 are both -(CH2)7CH3.
17. A pharmaceutical formulation according to any one of claims 1 to 9, wherein -RFA and -RFA2 are both -(CH2)8CH3.
18. A pharmaceutical formulation according to any one of claims 1 to 9, wherein -RFA and -RFA2 are both -(CH2)9CH3.
19. A pharmaceutical formulation according to any one of claims 1 to 9, wherein -RFA and -RFA2 are both -(CH2)10CH3.
A pharmaceutical formulation according to any one of claims 1 to 9, wherein -R and -RFA2 are both -(CH2)nCH3.
A pharmaceutical formulation according to any one of claims 1 to 20, which is a pharmaceutical formulation comprising liposomes, said liposomes comprising said drug and said short-chain phosphatidylcholine lipid.
A pharmaceutical formulation according to claim 21 , wherein the liposomes further comprise additional phospholipid.
A pharmaceutical formulation according to claim 21 or 22, wherein the liposomes further comprise cholesterol (CH).
A pharmaceutical formulation according to claim 22 or 23, wherein the additional phospholipid, if present, comprises additional phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), and/or sphingomyelin (SM).
A pharmaceutical formulation according to claim 22 or 23, wherein the additional phospholipid, if present, comprises additional phosphatidylcholine (PC) and/or phosphatidylethanolamine (PE).
A pharmaceutical formulation according to claim 22 or 23, wherein the additional phospholipid comprises additional phosphatidylcholine (PC) and
phosphatidylethanolamine (PE).
A pharmaceutical formulation according to claim 22 or 23, wherein the additional phospholipid comprises additional phosphatidylcholine (PC).
A pharmaceutical formulation according to claim 22 or 23, wherein the additional phospholipid comprises phosphatidylethanolamine (PE).
A pharmaceutical formulation according to any one of claims 22 to 28, wherein the additional phosphatidylcholine (PC), if present, is HSPC.
A pharmaceutical formulation according to any one of claims 22 to 28, wherein the additional phosphatidylcholine (PC), if present, is DPPC.
A pharmaceutical formulation according to any one of claims 22 to 28, wherein the additional phosphatidylcholine (PC), if present, is DOPC.
A pharmaceutical formulation according to any one of claims 22 to 28, wherein the additional phosphatidylcholine (PC), if present, is DSPC.
A pharmaceutical formulation according to any one of claims 22 to 32, wherein the phosphatidylethanolamine (PE), if present, is DSPE.
A pharmaceutical formulation according to any one of claims 22 to 32, wherein the phosphatidylethanolamine (PE), if present, is DOPE.
A pharmaceutical formulation according to any one of claims 22 to 32, wherein the phosphatidylethanolamine (PE), if present, is DPPE.
A pharmaceutical formulation according to any one of claims 21 to 34, wherein the liposomes further comprise a vesicle-forming lipid which is derivatized with a polymer chain.
A pharmaceutical formulation according to any one of claims 21 to 34, wherein the liposomes further comprise a phospholipid which is derivatized with
polyethyleneglycol (PEG).
A pharmaceutical formulation according to any one of claims 21 to 34, wherein the liposomes further comprise phosphatidylethanolamine (PE) which is derivatized with polyethyleneglycol (PEG).
A pharmaceutical formulation according to any one of claims 21 to 34, wherein the liposomes further comprise N-(carbonyl-methoxypolyethylene glycol)-1 ,2- distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (MPEG-DSPE).
A pharmaceutical formulation according to any one of claims 21 to 34, wherein the liposomes further comprise N-(carbonyl-methoxypolyethylene glycol 2000)-1 ,2- distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (MPEG2000-DSPE).
A method of preparing a pharmaceutical formulation according to any one of claims 1 to 20, comprising combining said drug and said short-chain
phosphatidylcholine lipid.
A pharmaceutical formulation according to any one of claims 1 to 40, for use in a method of treatment of the human or animal body by therapy.
43. A pharmaceutical formulation according to any one of claims 1 to 40, wherein for use in a method of treatment of a proliferative condition.
44. A pharmaceutical formulation for use according to claim 43, wherein the proliferative condition is cancer.
45. A pharmaceutical formulation for use according to claim 43, wherein the
proliferative condition is ovarian cancer or Kaposi's syndrome.
46. A method of treatment of a proliferative condition, comprising administering to a patient in need of treatment a therapeutically effective amount of a pharmaceutical formulation according to any one of claims 1 to 40.
47. A method according to claim 46, wherein the proliferative condition is cancer.
48. A method according to claim 46, wherein the proliferative condition is ovarian
cancer or Kaposi's syndrome.
49. Use of:
(i) a drug; and
(ii) a short-chain hosphatidylcholine lipid of the following formula:
Figure imgf000053_0001
wherein each of -R and -R is independently:
a linear or branched saturated alkyl group having from 7 to 13 carbon atoms; or
a linear or branched alkenyl group having from 7 to 13 carbon atoms and from 1 to 3 carbon-carbon double bonds;
in the manufacture of a medicament for the treatment of a proliferative condition.
50 A method according to claim 49, wherein the proliferative condition is cancer.
51 A method according to claim 49, wherein the proliferative condition is ovarian
cancer or Kaposi's syndrome. Use of a short-chain hosphatidylcholine lipid of the following formula:
Figure imgf000054_0001
wherein each of -R and -R is independently:
a linear or branched saturated alkyl group having from 7 to 13 carbon atoms; or
a linear or branched alkenyl group having from 7 to 13 carbon atoms and from 1 to 3 carbon-carbon double bonds;
to increase the bioavailability of a drug.
Use of a short-chain phosphatidylcholine lipid of the following formula:
Figure imgf000054_0002
wherein each of -R and -R is independently:
a linear or branched saturated alkyl group having from 7 to 13 carbon atoms; or
a linear or branched alkenyl group having from 7 to 13 carbon atoms and from 1 to 3 carbon-carbon double bonds;
to increase the cellular uptake of a drug.
A method of increasing the bioavailability of a drug in a patient, which method comprises the step of co-administering said drug and a short-chain
phosphatidylcholine li id of the following formula:
wherein each
Figure imgf000054_0003
a linear or branched saturated alkyl group having from 7 to 13 carbon atoms; or
a linear or branched alkenyl group having from 7 to 13 carbon atoms and from 1 to 3 carbon-carbon double bonds. A method of increasing the cellular uptake of a drug into a cell, which method comprises the step of contacting said cell with said drug and a short-chain phosphatidylcholine li id of the following formula:
Figure imgf000055_0001
wherein each of -R and -R is independently:
a linear or branched saturated alkyl group having from 7 to 13 carbon atoms; or
a linear or branched alkenyl group having from 7 to 13 carbon atoms and from 1 to 3 carbon-carbon double bonds.
PCT/IB2014/061379 2013-05-15 2014-05-12 Compounds and their use in therapy WO2014184726A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB1308753.1 2013-05-15
GBGB1308753.1A GB201308753D0 (en) 2013-05-15 2013-05-15 Compounds and their use in therapy

Publications (2)

Publication Number Publication Date
WO2014184726A2 true WO2014184726A2 (en) 2014-11-20
WO2014184726A3 WO2014184726A3 (en) 2015-02-19

Family

ID=48700839

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2014/061379 WO2014184726A2 (en) 2013-05-15 2014-05-12 Compounds and their use in therapy

Country Status (2)

Country Link
GB (1) GB201308753D0 (en)
WO (1) WO2014184726A2 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11712418B2 (en) 2017-05-26 2023-08-01 Bruin Biosciences, Inc. Chemoembolization agents

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108888773B (en) * 2018-09-17 2021-06-29 中国医学科学院生物医学工程研究所 Self-assembled spherical medicine nano preparation and preparation method and application thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK36492D0 (en) * 1992-03-19 1992-03-19 Novo Nordisk As PREPARATION
CN102716085B (en) * 2012-06-29 2013-08-21 海南灵康制药有限公司 Topotecan hydrochloride liposome injection

Non-Patent Citations (29)

* Cited by examiner, † Cited by third party
Title
"Handbook of Pharmaceutical Additives", 2001, SYNAPSE INFORMATION RESOURCES, INC.
"Handbook of Pharmaceutical Excipients", 1994
"Remington's Pharmaceutical Sciences", 2000, LIPPINCOTT, WILLIAMS & WILKINS
BROCKERHOFF; YURKOWSKI, CAN. J. BIOCHEM.,, vol. 43, 1965, pages 1777
EIBL, CHEMISTRY AND PHYSICS OF LIPIDS, 1980
GABIZON A: "Pegylated liposomal doxorubicin: metamorphosis of an old drug into a new form of chemotherapy", CANCER INV., vol. 19, 2001, pages 424 - 436, XP009094919, DOI: doi:10.1081/CNV-100103136
GABIZON A; CHEMLA M; TZEMACH D; HOROWITZ AT; GOREN D: "Liposome longevity and stability in circulation: effects on the in vivo delivery to tumors and therapeutic efficacy of encapsulated anthracyclines", J. DRUG TARGET., vol. 3, 1996, pages 391 - 398
HARAN G; COHEN R; BAR LK; BARENHOLZ Y: "Transmembrane ammonium sulphate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases", BIOCHIM. BIOPHYS. ACTA, vol. 1151, 1993, pages 201 - 215, XP023352276, DOI: doi:10.1016/0005-2736(93)90105-9
HARASYM TO; CULLIS PR; BALLY MB: "Intratumor distribution of doxorubicin following i.v. administration of drug encapsulated in egg phsophatidylcholine/ cholesterol liposomes", CANCER CHEMOTHER. PHARMACOL., vol. 40, 1997, pages 309 - 317
HEIJN M; ROBERGE S; JAIN RK: "Cellular membrane permeability of anthracyclines does not correlate with their delivery in a tissue-isolated tumor", CANCER RES., vol. 59, 1999, pages 4458 - 4463
HOROWITZ AT; BARENHOLZ Y; GABIZON A: "In vitro cytotoxicity of liposome-encapsulated doxrubicin: dependence on liposome composition and drug release", BIOCHIM. BIOPHYS. ACTA, vol. 1109, 1992, pages 203 - 209, XP023575252, DOI: doi:10.1016/0005-2736(92)90084-Y
JECKEL D; WIELAND F: "Truncated ceramide analogs as probes for sphingolipid biosynthesis and transport", ADV. LIPID RES., vol. 26, 1993, pages 143 - 160
KONING GA; GORTER A; SCHERPHOF GL; KAMPS JAAM: "Antiproliferative effect of immunoliposomes containing 5-fluorodeoxyuridine-dipalmitate on colon cancer cells", BR. J. CANCER, vol. 80, 1999, pages 1718 - 1725
KONING GA; MORSELT HWM; GORTER A; ALLEN TM; ZALIPSKY S; SCHERPHOF GL; KAMPS JAAM: "Interaction of differently designed immunoliposomes with colon cancer cells and kupffer Cells. An in vitro comparison", PHARM. RES., vol. 20, 2003, pages 1249 - 1258
KONING, G.A.; VELDMAN, R.J.; VAN HELL, A.; ZERP, S.; STORM, G.; VAN BLITTERSWIJK, W.J.; VERHEIJ, M.: "Short-chain liposomal sphingolipids potentiate in vitro doxorubicin cytotoxicity by enhancing its cellualr influx", CONFERENCE ABSTRACT FOR LIPOSOME ADVANCES: PROGRESS IN DRUG AND VACCINE DELIVERY, 2003
LOTHSTEIN L; ISRAEL M; SWEATMAN TW: "Anthracyclin drug targeting: cytoplasmic versus nuclear - a fork in the road", DRUG RES. UPDATES, vol. 4, 2001, pages 169 - 177
PAN XQ; WANG H; LEE RJ: "Antitumor activity of folate receptor-targeted liposomal doxorubcin in a KB oral carcinoma murine xenograft model", PHARM. RES., vol. 20, 2003, pages 417 - 422, XP019370632, DOI: doi:10.1023/A:1022656105022
PARK JW; HONG K; KIRPOTIN DB; COLBERN G; SHALABY R; BASELGA J; SHAO Y; NIELSEN UB; MARKS JD; MOORE D: "Anti-HER2 immunoliposomes: enhanced efficacy attributable to targeted delivery", CLIN. CANCER RES., vol. 8, 2002, pages 1172 - 1181, XP002470761
PATEL ET AL., J. LIPID RES., vol. 20, 1979, pages 674
ROBERT J; GIANNI L: "Cancer Surveys", vol. 17, 1993, article "Pharmacokinetics and metabolism of anthracyclines", pages: 219 - 252
SMITH ET AL.: "Measurement of protein using bicinchoninic acid", ANAL. BIOCHEM., vol. 150, no. 1, 1985, pages 76 - 85, XP024823132, DOI: doi:10.1016/0003-2697(85)90442-7
SPETH PA; RAIJMAKERS RA; BOEZEMAN JB; LINSSEN PC; DE WITTE TJ; WESSELS HM; HAANEN C: "In vivo cellular adriamycin concentrations related to growth inhibition of normal and leukemic human bone marrow cells", EUR. J. CANCER CLIN. ONCOL., vol. 24, 1988, pages 667 - 674, XP026207296, DOI: doi:10.1016/0277-5379(88)90297-0
TARDI PG; BOMAN NL; CULLIS PR: "Liposomal doxorubicin", J. DRUG TARGET,, vol. 4, 1996, pages 129 - 140
USTER PS; ALLEN TM; DANIEL BE; MENDEZ CJ; NEWMAN MS; ZHU GZ: "Insertion of poly(ethylene glycol) derivatized phospholipid into pre-fromed liposomes results in prolonged in vivo circulation time", FEBS LETT., vol. 386, 1996, pages 243 - 246, XP002236375, DOI: doi:10.1016/0014-5793(96)00452-8
VAAGE J; MAYHEW E; LASIC D; MARTIN F: "Therapy of primary and metastatic mouse mammary carcinomas with doxorubicin encapsulated in long circulating liposomes", INT. J .CANCER, vol. 51, 1992, pages 942 - 948, XP000979548, DOI: doi:10.1002/ijc.2910510618
WASHINGTON N; WASHINGTON C; WILSON CG: "Physiological Pharmaceutics: Barriers to Drug Absorption", 2001, TAYLOR AND FRANCIS, article "Cell membranes, epithelial barriers and drug absorption", pages: 1 - 18
WEISS RB: "The anthracyclines: will we ever find a better doxorubicin?", SEM. ONCOL., vol. 19, 1992, pages 670 - 686
YUAN F; LEUNIG M; HUANG SK; BERK DA; PAPAHADJOPOULOS D; JAIN RK: "Microvascular permeability and interstitial penetration of sterically stabilized (stealth) liposomes in a human tumor xenograft", CANCER RES., vol. 54, 1994, pages 3352 - 3356
ZAGOTTO G; GATTO B; MORO S; SISS C; PALUMBO M: "Anthracyclins: recent developments in their separation and quantitation", J. CHROMAT. B, vol. 764, 2001, pages 161 - 171, XP004322151, DOI: doi:10.1016/S0378-4347(01)00346-2

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11712418B2 (en) 2017-05-26 2023-08-01 Bruin Biosciences, Inc. Chemoembolization agents

Also Published As

Publication number Publication date
GB201308753D0 (en) 2013-06-26
WO2014184726A3 (en) 2015-02-19

Similar Documents

Publication Publication Date Title
US9707204B2 (en) Treatment of breast cancer
JP5981214B2 (en) Compositions and methods for the treatment of lymphoma
ES2382337T3 (en) Compositions and procedures to treat leukemia
US20210213051A1 (en) Combined pharmaceutical formulation comprising drug-containing liposome composition and platinum preparation
KR20210008527A (en) Composition comprising bisfluoroalkyl-1,4-benzodiazepinone compound and method of use thereof
AU2019271044A1 (en) Compositions comprising bisfluoroalkyl-l,4-benzodiazepinone compounds for treating adenoid cystic carcinoma
WO2014184726A2 (en) Compounds and their use in therapy
US20220313608A1 (en) Treatment of breast cancer
JP2023502253A (en) Lung-targeted anticancer therapy with liposomal anamycin
WO2019168177A1 (en) Cancer prophylactic or therapeutic agent
WO2023122577A2 (en) Methods of treating cancer using liposomal particles comprising anticancer agents and pharmaceutical compositions related thereto
JP2023174607A5 (en)
CN117897139A (en) Liposome formulations of BCL inhibitors

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14727936

Country of ref document: EP

Kind code of ref document: A2

122 Ep: pct application non-entry in european phase

Ref document number: 14727936

Country of ref document: EP

Kind code of ref document: A2