WO2017123616A1 - Inhibiting b-cell lymphoma 2 (bcl-2) and related proteins - Google Patents
Inhibiting b-cell lymphoma 2 (bcl-2) and related proteins Download PDFInfo
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- WO2017123616A1 WO2017123616A1 PCT/US2017/012992 US2017012992W WO2017123616A1 WO 2017123616 A1 WO2017123616 A1 WO 2017123616A1 US 2017012992 W US2017012992 W US 2017012992W WO 2017123616 A1 WO2017123616 A1 WO 2017123616A1
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- 0 CN(C)CC[C@](CSc1ccccc1)Nc(ccc(S(NC(c(cc1)ccc1N1CCN(Cc(cccc2)c2-c(cc2)ccc2Cl)CC1)=O)(=*)=O)c1)c1[N+]([O-])=O Chemical compound CN(C)CC[C@](CSc1ccccc1)Nc(ccc(S(NC(c(cc1)ccc1N1CCN(Cc(cccc2)c2-c(cc2)ccc2Cl)CC1)=O)(=*)=O)c1)c1[N+]([O-])=O 0.000 description 7
- JYYDKYGCFHRGSD-GOSISDBHSA-N CN(C)C1CCN(CC[C@H](CSc2ccccc2)Nc(ccc(S(N)(=O)=O)c2)c2[N+]([O-])=O)CC1 Chemical compound CN(C)C1CCN(CC[C@H](CSc2ccccc2)Nc(ccc(S(N)(=O)=O)c2)c2[N+]([O-])=O)CC1 JYYDKYGCFHRGSD-GOSISDBHSA-N 0.000 description 1
- XWZDVIYUEKDTSF-LJQANCHMSA-N CN(C)CCC(CC1)CCN1C(C[C@H](CO)NC(OCc1ccccc1)=O)=O Chemical compound CN(C)CCC(CC1)CCN1C(C[C@H](CO)NC(OCc1ccccc1)=O)=O XWZDVIYUEKDTSF-LJQANCHMSA-N 0.000 description 1
- HURXYPHGZAASER-QGZVFWFLSA-N CN(C)CCC(CC1)CCN1C(C[C@H](CSc1ccccc1)N)=O Chemical compound CN(C)CCC(CC1)CCN1C(C[C@H](CSc1ccccc1)N)=O HURXYPHGZAASER-QGZVFWFLSA-N 0.000 description 1
- XKJOURNXDJBPNM-XMMPIXPASA-N CN(C)CCC(CC1)CCN1C(C[C@H](CSc1ccccc1)NC(OCc1ccccc1)=O)=O Chemical compound CN(C)CCC(CC1)CCN1C(C[C@H](CSc1ccccc1)NC(OCc1ccccc1)=O)=O XKJOURNXDJBPNM-XMMPIXPASA-N 0.000 description 1
- SFSBVVJIMBCCIF-GOSISDBHSA-N CN(C)CCC1CCN(CC[C@H](CSc2ccccc2)N)CC1 Chemical compound CN(C)CCC1CCN(CC[C@H](CSc2ccccc2)N)CC1 SFSBVVJIMBCCIF-GOSISDBHSA-N 0.000 description 1
- RSFAUZNVDWGQBZ-UHFFFAOYSA-N CN(C)CCC1CCNCC1 Chemical compound CN(C)CCC1CCNCC1 RSFAUZNVDWGQBZ-UHFFFAOYSA-N 0.000 description 1
- QYPFFDQKVDAWLL-CYBMUJFWSA-N CN(CC1)CCN1C(C[C@H](CSc1ccccc1)N)=O Chemical compound CN(CC1)CCN1C(C[C@H](CSc1ccccc1)N)=O QYPFFDQKVDAWLL-CYBMUJFWSA-N 0.000 description 1
- NNGKPWCSKAJAQE-HXUWFJFHSA-N CN(CC1)CCN1C(C[C@H](CSc1ccccc1)NC(OCc1ccccc1)=O)=O Chemical compound CN(CC1)CCN1C(C[C@H](CSc1ccccc1)NC(OCc1ccccc1)=O)=O NNGKPWCSKAJAQE-HXUWFJFHSA-N 0.000 description 1
- NOLZZFKDQCGKIK-CYBMUJFWSA-N C[C@@](C1)(COC1=O)NC(OCc1ccccc1)=O Chemical compound C[C@@](C1)(COC1=O)NC(OCc1ccccc1)=O NOLZZFKDQCGKIK-CYBMUJFWSA-N 0.000 description 1
- FAYVDRRKPVJSPE-UHFFFAOYSA-N NS(c(cc1)cc([N+]([O-])=O)c1F)(=O)=O Chemical compound NS(c(cc1)cc([N+]([O-])=O)c1F)(=O)=O FAYVDRRKPVJSPE-UHFFFAOYSA-N 0.000 description 1
Classifications
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- 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
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- This disclosure relates to compounds and related methods of inhibiting B-cell lymphoma 2 (Bcl-2) and related proteins, including methods and compounds useful for the treatment of cancer.
- Bcl-2 B-cell lymphoma 2
- Apoptosis is recognized as an essential biological process for tissue homeostasis of all living species. In mammals in particular, it has been shown to regulate early embryonic development. Later in life, cell death is a default mechanism by which potentially dangerous cells (e.g., cells carrying cancerous defects) are removed.
- Bcl-2 B-cell lymphoma 2 family of proteins, which are key regulators of the mitochondrial (also called "intrinsic") pathway of apoptosis. See, Danial, N. N. and Korsmeyer, S. J. Cell (2004) 116, 205-219.
- Bcl-2 protein family which are central regulators of programmed cell death.
- pro-apoptosis proteins Box, Bad, Bid, Bim, Bik, Puma, Noxa, etc.
- anti -apoptosis proteins Bcl-2 family: Bcl-2, BC1-XL, Mcl-1, etc.
- Bcl-2 is an excellent target for hematological cancers while Bcl-X L is a target for solid tumors. It was also speculated that observed thrombocytopenia was caused by inhibiting Bcl-X L (Cell, 2007, 128, 1 173-1 186).
- the Bcl-2 family members that inhibit apoptosis are over-expressed in cancers and contribute to tumorigenesis. Bcl-2 expression has been strongly correlated with resistance to cancer therapy and decreased survival.
- the Bcl-2 family of proteins can be further classified into three subfamilies depending on how many of the homology domains each protein contains and on its biological activity (i.e., whether it has pro- or anti-apoptotic function).
- the first subgroup contains proteins having all 4 homology domains, i.e., BH1, BH2, BH3 and BH4. Their general effect is anti-apoptotic, that is to preserve a cell from starting a cell death process. Proteins such as, for example, Bcl-2, BC1-XL, and Mcl-1 are members of this first subgroup.
- Bcl-2 family e.g., inhibitors of both Bcl-2 and BC1-XL
- combination therapies e.g., in combination with chemotherapy and/or radiation therapy.
- therapeutically effective Bcl-2 family inhibitor compositions without dose-limiting thrombocytopenia (e.g., acceptably high platelet counts after administration to a subject), without inducing tumor lysis syndrome or other unacceptable side effects.
- Applicants have discovered novel chemical compounds useful for inhibiting the B- cell lymphoma 2 (Bcl-2) family of proteins and the treatment of cancer, and liposome formulations of certain inhibitors of Bcl-2 having desirable properties (e.g., extended half-life in blood circulation and efficacy in treating tumors), including novel compounds that inhibit both Bcl-2 and BC1-XL.
- the inventions are based in part on the discovery of certain novel compounds for inhibiting the B-cell lymphoma 2 (Bcl-2) family of proteins, as well as extended plasma half-lives and enhanced antitumor efficacy of certain liposomal
- Bcl-2 inhibitor compounds can inhibit both Bcl-2 and Bcl- XL with an activity measured by an IC5 0 value of less than about 100 nM at Bcl-2 and less then about 100 nM at Bcl-X L , as measured by the target activity assay of Example 1 ("Bcl- 2/BCI-XL inhibitors").
- the activity of the BC1-2 BC1-XL inhibitor compounds have a ratio of Bcl-2 to Bcl-XL activity of between about 0.1-1.0, as measured by the assay of Example 1.
- novel BC1-2/BC1-XL inhibitor compounds are provided.
- the Bcl-2/Bcl-X L inhibitor compounds can have a chemical structure of formula (I) or formula (II) as disclosed herein and an IC 50 value of less than about 100 nM against both Bcl-2 and Bcl- XL as measured by the assay of Example 1.
- Representative examples of the BC1-2 BC1-XL inhibitor compounds include compounds disclosed in Example 8.
- the novel BC1-2/BC1-XL inhibitor compounds have the chemical structure of formula (I), or pharmaceutically acceptable salts thereof, wherein R is a moiety comprising an amine moiety with a pK a of greater than 7.0 (preferably greater than 8.0, and most preferably at least about 9.5) selected to provide the compound of formula (I) with an IC5 0 value of less than about 100 nM for Bcl-2 and an IC5 0 value of less than about 100 nM for BC1-XL, as measured by the assay of Example 1 :
- the compound of formula (I) can include R as N or CH, and Ri as a moiety comprising an alkyl-substituted amine, preferably a tertiary alkyl-substituted amine.
- R can be N or CH and R x can be -N(R a )(R b ) or -(A)-N(R a )(R b ) where -(A)- is a (C C 4 ) aliphatic moiety (e.g., methylene or ethylene), and R a and R b are independently lower alkyl (e.g., C1-C4 alkyl, preferably methyl or ethyl), one of R a or R b is hydrogen and the other is C1-C4 alkyl, or R a and R together form a cyclic ring (e.g., a nitrogen-containing heterocyclic ring).
- R x can be -N(R a )
- R a and R together form a heterocyclic ring comprising one or more heteroatoms.
- Ri can be a heterocycloalkyl moiety comprising an alkyl-substituted (e.g., methyl-substituted) tertiary nitrogen.
- the compounds of formula (I) include Compounds 1-4, and
- Additional novel Bcl-2 inhibitor compounds have the chemical structure of formula (II) or pharmaceutically acceptable salts thereof, wherein R ' and R together form a moiety comprising an amine with a pK a of greater than 7.0 (preferably greater than 8.0, and most preferably at least about 9.5), selected to provide the compound of formula (I) with an IC5 0 value of less than about 100 nM for Bcl-2 and an IC 50 value of less than about 100 nM for BC1-XL, as measured by the assay of Example 1 :
- the compound of formula (II) can include R ' as N or CH, and R as a moiety comprising a tertiary alkyl-substituted amine.
- Ri can be -N(R a' )(R b ' ) or -(A )- N(R a' )(R b ' ) where -(A ' )- is a (C 1 -C4) aliphatic moiety (e.g., methylene or ethylene), and Ra ' and Rb' are independently lower alkyl (e.g., C 1 -C4 alkyl, preferably methyl or ethyl), one of Ra ' or Rb ' is hydrogen and the other is C 1 -C4 alkyl, or Ra ' and Rb ' together form a cyclic ring (e.g., a nitrogen-containing heterocyclic ring).
- Ri ' can be a heterocycloalkyl moiety comprising an alkyl-substituted (e.g., methyl-substituted) tertiary nitrogen.
- R can be heteroaryl such as pyrrolidine or piperidine when R ' is N.
- the compounds of formula (II) include Compounds 5-8, and
- Compound 9 and Compound 10 can be in the free base form, or in the form of a pharmaceutically acceptable salt.
- liposome formulations of Bcl-2/Bcl-X L inhibitor compounds are provided.
- the liposome formulation can encapsulate the BC1-2/BC1-XL inhibitor compounds, including the compounds of the first embodiment, in liposome vesicles.
- the liposome formulation can comprise liposomes that encapsulate at least 95%, preferably at least 99%, of the BC1-2/BC1-XL inhibitor compound in the liposome formulation.
- Non-encapsulated BC1-2 BC1-XL inhibitor compound can be removed from the liposome formulation after the BC1-2/BC1-XL inhibitor compound is encapsulated within the liposome.
- the liposome is formulated to extend the circulation time of the Bcl-2/Bcl-X L inhibitor and/or reduce the release of the Bcl-2/Bcl-X L inhibitor compounds from the liposome in plasma.
- the liposome is contacted with albumin (e.g., BSA) under conditions effective to increase the concentration of the Bcl- 2/Bcl-X L inhibitor compound in mouse plasma over at least 4 hours, and preferably over at least 48 hours, as described in FIG. 3 and Example 5.
- albumin e.g., BSA
- the Bcl-2/Bcl-X L inhibitor is formulated in a liposome in order to decrease undesirable thrombocytopenic effect of the inhibitor.
- Novel liposome formulations comprising one or more compounds of formula (I) and/or formula (II) encapsulated in a liposome are useful for inhibiting Bcl-2 and BC1-XL, and the treatment of cancer.
- Particularly preferred examples include liposomes comprising compounds selected from the group consisting of compounds 1, 2, 3, 4, 5, 6, 7, or 8.
- the liposome formulation can be treated with serum albumin (e.g., BSA) after encapsulation of the novel compounds of formula (I) and/or formula (II).
- the liposome formulation comprises a compound of formula (II) encapsulated in a liposome that is treated with serum albumin after encapsulation of the compound of formula (II) and prior to administration to a patient.
- a novel liposome formulation comprises Compound 6 in an albumin-treated liposome.
- the liposome can comprise one or more serum albumin protein on the outside of the liposome surface.
- the liposome formulation can include a compound of formula (I) and/or formula (II) encapsulated in a unilamellar vesicle formed from one or more liposome-forming lipids (e.g., distearoyl phosphatidylcholine (DSPC)), cholesterol and a hydrophilic polymer-conjugated lipid (e.g., methoxy-poly(ethylene glycol)- 1,2-distearyl-sn-glyceryl (PEG2000-DSG)).
- a liposome-forming lipids e.g., distearoyl phosphatidylcholine (DSPC)
- DSPC distearoyl phosphatidylcholine
- PEG2000-DSG hydrophilic polymer-conjugated lipid
- the liposome-forming lipid preferably comprises one or more phospholipids and, optionally, a sterol, such as cholesterol, with the ratio of the phospholipid(s) and the cholesterol selected to provide a desired amount of liposome membrane rigidity while maintaining a sufficiently reduced amount of leakage of the compound of formula (I) from the liposome.
- the type and amount of polymer-conjugated lipid can be selected to provide desirable levels of protein binding, liposome stability and circulation time in the blood stream.
- the liposome vesicle comprises DSPC and cholesterol in a 3 :2 molar ratio.
- the liposome can comprise a vesicle consisting of DSPC, cholesterol and PEG2000-DSG in a 3 :2:0.3 molar ratio.
- the Bcl-2 inhibitor compound(s) e.g., a compound of formula (I) or formula (II)
- a composition comprising a liposome having a vesicle formed from DSPC, cholesterol and PEG2000-DSG in a 3 :2:0.3 molar ratio, encapsulating Compound 6.
- the liposome formulation can include a compound of formula (I) and/or formula (II) encapsulated in a unilamellar vesicle formed from one or more liposome-forming lipids, a hydrophilic polymer-conjugated lipid (e.g., methoxy-poly(ethylene glycol)-oxycarbonyl-l,2- distearoyl-sn-phosphatidylethanolamine (PEG2000DSPE), and containing essentially no cholesterol.
- the liposome-forming lipid preferably comprises a neutral phospholipid (e.g., distearoyl phosphatidylcholine (DSPC)), an anionic lipid (e.g.,
- the liposome vesicle comprises DSPC and DSPG in a 2: 1 molar ratio.
- the liposome can comprise a vesicle consisting of DSPC, DSPG, and PEG2000-DSPE or PEG2000-DSG in a 2: 1 :0.2 molar ratio.
- the liposome can optionally comprise a lipid fluorescent label, such as DiIC18(3)-DS (Life Technologies, USA), in the amount of 0..05- 0.3 mol.% relative to phospholipid.
- the Bcl-2 inhibitor compound(s) can be entrapped within the liposome by a triethylammonium sucrose octasulfate gradient or an ammonium sulfate gradient at the drug/phospholipid ratio of less than 300 g/mol, preferably less than 250 g/mol, and more preferably at 100-200 g/mol.
- compositions comprising a liposome having a vesicle formed from DSPC, DSPG, PEG2000-DSPE, and DiIC18(3)-DS in a 2: 1 :0.2:0.002 molar ratio, encapsulating Compound 2 in the form of a sulfate or sucrose octasulfate salt at the drug phospholipid ratio of 150 g/mol.
- FIG. 1A is a graph showing the cellular activity of certain Bcl-2 inhibitor compounds, including compounds of formula (I), against certain cancer cell lines, obtained using the assay of Example 1.
- FIG. IB is a graph showing the cellular activity of certain Bcl-2 inhibitor compounds, including compounds of formula (II), against certain cancer cell lines, obtained using the assay of Example 1.
- FIG. 1C is a table showing the cellular activity of certain Bcl-2 inhibitor compounds, including compounds of formula (I) and formula (II), against certain cancer cell lines, obtained using the assay of Example 1.
- FIGs. 2A, 2B, 2C, and 2D are each graphs showing the dose-response of docetaxel in a MDA-MB231 breast carcinoma cells when combined with certain Bcl-2 inhibitor compounds tested at three different concentrations.
- FIG. 3 is a graph showing the drug pharmacokinetics of free Compound 6 (not in a liposome), a first liposome formulation of compound Compound 6 (not pre-treated with albumin) and a second liposome formulation of compound Compound 6 (pre-treated with albumin).
- FIG. 4 is a bar graph showing the platelet (PLT) count (in 1000's of PLT per microliter) in mice after administration of the first liposome formulation of compound Compound 6 (not pre-treated with albumin) and a second liposome formulation of compound Compound 6 (pre-treated with albumin), compared to normal PLT levels (dashed line).
- PLT platelet
- FIGs. 5A, 5B, and 5C are graphs showing the effect of dose escalation of the first liposome formulation of Compound 6 (not pre-treated with albumin) and the second liposome formulation of Compound 6 (pre-treated with albumin) on body weight measured in mice.
- FIGs. 6A, 6B, and 6C are graphs showing the effect of dose escalation of the first liposome formulation of Compound 6 (not pre-treated with albumin) and the second liposome formulation of Compound 6 (pre-treated with albumin) on platelet count in mice.
- FIGs. 7A, 7B, and 7C are graphs showing the effect of dose escalation of the formula (I) compound, Compound 2, vs the formula (II) compound, Compound 6, at 1 mg/kg (FIG. 7A), 2 mg/kg (FIG. 7B) and 5 mg/kg (FIG. 7C) on platelet count in mice.
- FIGs. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18 each show a chemical reaction scheme useful in the synthesis of the disclosed Bcl-2 inhibitors, as described in the Examples herein.
- FIG. 18A, 18B, 18C, and 18D are graphs showing the fluorescent peptide titrations at multiple protein concentrations, used in developing the assay of Example 1.
- FIGs. 19A, 19B, 19C, and 19D are graphs showing protein titrations at two f-peptide concentrations, used in developing the assay of Example 1.
- FIGs. 20A, 20B, 20C, and 20D are graphs showing extended range protein titrations at single f-peptide concentration, used in developing the assay of Example 1.
- FIGs. 21A, 21B, 21C, and 21D are graphs showing the signal stability over time, used in developing the assay of Example 1.
- FIGs. 22A, 22B, 22C, 22D, 22E, and 22F are graphs showing data from -60 min incubation was used for EC5 0 determinations, used in developing the assay of Example 1.
- FIG. 23 is a bar graph showing the platelet count (in 1000's of platelets (PTL) per microliter) in mice after intravenous administration of saline vehicle (Naive Control), free Compound 6, or liposomally formulated Compound 6 at 10 mg/kg, compared to normal platelet levels (dashed line).
- Novel BC1-2/BC1-XL inhibitor compounds can be obtained by methods described in the Examples.
- the term "Bcl-2 Bcl-X L inhibitor compound” refers to a compound having an IC 50 value of less than about 100 nM with both Bcl-2 and BC1-XL in the assay of Example 1.
- the BC1-2/BC1-XL inhibitor compounds have a ratio of IC 50 values with Bcl-2 and Bcl-XL of between about 0.1 and 10.0 (including, for example, ratios between about 0.2 and 5.0, and ratios therebetween) measured by the assay of Example 1.
- novel compounds having the chemical structure of formula (la), or pharmaceutically acceptable salts thereof, wherein R is a moiety comprising an amine with a pK a of greater than 7.0 (prefera and most preferably at least about 9.5):
- the compound of formula (la) can include R as N or CH, and Ri as a moiety comprising a tertiary alkyl-substituted amine.
- R can be N or CH and Ri can be - N(R a )(R b ) or -(A)-N(R a )(R ) where -(A)- is a (C 1 -C4) aliphatic moiety (e.g., methylene or ethylene), and R a and R b are independently lower alkyl (e.g., C1-C4 alkyl, preferably methyl or ethyl), or one of R a or R is hydrogen and the other is C 1 -C 4 alkyl, or R a and R together form a cyclic ring (e.g., a nitrogen-containing heterocyclic ring).
- R a and R b together form a heterocyclic ring comprising one or more heteroatoms.
- Ri can be a heterocycloalkyl moiety comprising an alkyl-substituted (e.g., methyl- substituted) tertiary nitrogen.
- the compounds of formula (la) include
- the compounds have the chemical structure of formula (I), where R is a moiety comprising an amine with a pK a of greater than 7.0 (preferably greater than 8.0, and most preferably at least about 9.5) selected to provide the compound of formula (I) with an IC 50 value of less than about 100 nM for Bcl-2 and an IC 50 value of less than about 100 nM for BC1-XL, as measured by the assay of Example 1 :
- the activity of compounds of formula (I) at Bcl-2 ranged from over 5-times more potent than Comparator A (e.g., Compound 2 and Compound 3) to more than 10-times less potent than Comparator A (e.g., Compound 4).
- the activity of compounds of formula (I) at Bcl-X L ranged from more than 5-times more potent than Comparator A (e.g., Compound 2 and Compound 3) to more than 30-times less potent than Comparator A (e.g., Compound 4).
- Compounds of formula (I) include compounds having a ratio of activity at BC1-2 BC1-XL from about 70-105% of the comparable Bcl-2/Bcl-X L activity ratio for
- Comparator A e.g., Compound 1, Compound 2, and Compound 3
- the BC1-2/BC1-XL inhibitor compounds are compounds of formula (I) having IC 50 values with Bcl-2 measured by Example 1 of less than that measured for Comparator A (i.e., less than 2.46, including about 0.1-2.46 and about 0.4-2.46 and about 0.1- 0.5).
- the BC1-2 BC1-XL inhibitor compounds are compounds of formula (I) having IC5 0 values with BC1-XL measured by Example 1 of less than that measured for Comparator A (i.e., less than 47.88, including about 0.1-47.88 and about 0.25-47.88 and about 0.1-47.88).
- the Bcl-2 and Bcl-X L activities in Table 1 are selected examples of novel Bcl-2/ Bcl- XL active compounds.
- other novel compounds can be identified based on this information.
- the compounds of formula (I) include a substituted amine at the R-Ri position, including a primary or secondary substituted amine (e.g., a amino moiety substituted with one or more alkyl moieties), wherein the R-Ri moiety is selected to provide desired liposome loading characteristics (e.g., a substituted amino moiety having a pKa of greater than 7, 8 or 9, including a pKa value of 7-10, 8-10, or 9-10).
- the compounds can have the structure at R-R L in formula (I) having one or two tertiary alkyl substituted amine moieties, including R-Ri moieties selected from the group consisting of:
- novel compounds having the chemical structure of formula (Ila) or pharmaceutically acceptable salts thereof, whereing R ' and Ri ' together form a moiety comprising an amine with a pK a of greater than 7.0 (preferably greater than 8.0, and most preferably at least about
- the compound of formula (Ila) can include R ' as N or CH, and Ri ' as a moiety comprising a tertiary alkyl-substituted amine.
- Ri ' can be -N(R a' )(R 3 ⁇ 4 ' ) or -(A ' )- N(R a' )(R ' ) where -(A ' )- is a (Ci-C 4 ) aliphatic moiety (e.g., methylene or ethylene), and Ra ' and Rb ' are independently lower alkyl (e.g., Ci-C 4 alkyl, preferably methyl or ethyl), one of Ra ' or Rb ' is hydrogen and the other is C1-C4 alkyl, or Ra ' and Rb ' together form a cyclic ring (e.g., a nitrogen-containing heterocyclic ring).
- Ri ' can be a heterocycloalkyl moiety comprising an alkyl-substituted (e.g., methyl-substituted) tertiary nitrogen.
- Ri ' can be heteroaryl such as pyrrolidine or piperidine when R ' is N.
- the compounds of formula (Ila) include Compounds 5a-8a, and pharmaceutically acceptable salts thereof:
- Additional novel Bcl-2 inhibitor compounds have the chemical structure of formula (II), where R ' is a moiety comprising an amine with a pK a of greater than 7.0 (preferably greater than 8.0, and most preferably at least about 9.5) selected to provide the compound of formula (I) with an IC 50 value of less than about 100 nM for Bcl-2 and an IC 50 value of less than about 100 nM for BC1-XL, as measured by the assay of Example 1 :
- R ' is N or CH
- Ri ' as a moiety comprising an alkyl-substituted amine, preferably a tertiary alkyl-substituted amine.
- R can be -N(R a )(R ) or -(A ' )-
- Ra ' or Rb ' is hydrogen and the other is C1-C4 alkyl, or R a' and R ' together form a cyclic ring
- the R ' -R moiety in formula (II) is selected to provide inhibitor activity measured as a value of less than about 100 nM at both Bcl-2 and Bcl-X L as measured in Example 1.
- the compound of formula (II) can include R ' as CH, and R as a moiety comprising a tertiary alkyl-substituted amine.
- R ' is CH and Ri ' can be -N(R a' )(R b ) or -(A ' )-N(R a' )(R 3 ⁇ 4 ) where -(A )- is a (C1-C4) aliphatic moiety (e.g., methylene or ethylene), and R a' and R b ' are independently lower alkyl (e.g., C1-C4 alkyl, preferably methyl or ethyl).
- R ' is N or CH and Ri ' can be a heterocycloalkyl moiety comprising an alkyl- substituted (e.g., methyl- substituted) tertiary nitrogen.
- the BC1-2/BC1-XL inhibitor compounds are compounds of formula (II) having IC 50 values with Bel -2 measured by Example 1 of less than that measured for Comparator B (i.e., less than 0.32, including about 0.1-0.32 and about 0.2-0.32.
- the BC1-2/BC1-XL inhibitor compounds are compounds of formula (I) having IC50 values with Bcl-X L measured by Example 1 of less than that measured for Comparator B (i.e., less than 0.23, including about 0.1-0.23 and about 0.18-0.23).
- novel Bcl-2/ BC1-XL active compounds activities in Table 2 are selected examples of novel Bcl-2/ BC1-XL active compounds.
- other novel compounds can be identified based on this information.
- the compounds of formula (II) include a substituted amine at the R'-Ri ' position, including a primary or secondary substituted amine (e.g., a amino moiety substituted with one or more alkyl moieties), wherein the R ' -R moiety is selected to provide desired liposome loading characteristics (e.g., a substituted amino moiety having a pK a of greater than 7, 8 or 9, including a pK a value of 7-10, 8-10, or 9-10) and inhibitor activity of less than about 100 nM at both Bcl-2 and Bcl-X L as measured in Example 1.
- desired liposome loading characteristics e.g., a substituted amino moiety having a pK a of greater than 7, 8 or 9, including a pK a value of 7-10, 8-10, or 9-10
- inhibitor activity e.g., a substituted amino moiety having a pK a of greater than 7, 8 or 9, including a pK a
- the compounds can have the structure at R ' -Ri ' in formula (II) having one or two alkyl substituted amine moieties (e.g., one or more tertiary alkyl substituted moieties), including R ' -Ri ' moieties selected from the group consisting of:
- the compounds of formula (II) include compounds 5-8:
- Compound 9a and Compound 10a can be in the free base form, or in the form of a pharmaceutically acceptable salt.
- Compound 9 and Compound 10 can be in the free base form, or in the form of a pharmaceutically acceptable salt.
- liposome formulations of Bcl-2/Bcl-X L inhibitor compounds are provided.
- the liposome formulation can encapsulate the BC1-2/BC1-XL inhibitor compounds of the first embodiment in liposome vesicles.
- Representative examples of liposome formulations of certain BC1-2/BC1-XL inhibitor compounds are provided in Example 4.
- the liposome formulation can comprise liposomes that encapsulate at least 95%, preferably at least 99%, of the BC1-2/BC1-XL inhibitor compound in the liposome formulation.
- Non-encapsulated Bcl-2/Bcl-X L inhibitor compound can be removed from the liposome formulation after the BC1-2/BC1-XL inhibitor compound is encapsulated within the liposome.
- the liposome is formulated to reduce the release of the BC1-2 BC1-XL inhibitor compounds from the liposome in plasma.
- the liposome is contacted with albumin (e.g., BSA) under conditions effective to increase the concentration of the Bcl-2/Bcl- XL inhibitor compound in mouse plasma over at least 4 hours, and preferably over at least 48 hours, as described in FIG. 3 and Example 5.
- albumin e.g., BSA
- the liposome formulations can be selected to reduce incidence of thrombocytopenia (e.g., compositions tested in the data presented in FIGs. 4, 6A, 6B, 6C, 7A, 7B, 7C, and 23).
- the liposome formulations can be selected to provide extended blood circulation times, for example as measured in a mouse model in the data shown in FIG. 3.
- the liposome average size can be selected to lie below 0.2 micron (200 nm) to provide for convenient sterilization of the liposome composition by microfiltration.
- the liposome size can be less than 170 nm, less than 150 nm, preferably less than 130 nm, or more preferably in the range of 70-120 nm, to provide for good blood circulation properties and uptake by tumors.
- the liposome formulations provide a level of PLT count that is normal or within a medically acceptable range within a therapeutically relevant time period (e.g., any time post injection, within the duration of a chemotherapy treatment cycle, within 14-28 days after injection, or the like).
- the liposome formulations provide a clearance rate of the Bcl-2/Bcl-X L inhibitor compounds characterized by the plasma half- life of greater than about 5 hours (e.g., 10-100 hours) after administration (e.g., intravenous administration).
- the blood circulation half-life of the BC1-2/BC1-XL inhibitor compounds in the liposome formulation is preferably at least about 5 hours, including blood circulation half- lives of about up to about 10 hours and 11 -15 hours.
- FIG. 3 provides a representative example of measurement of the Bcl-2/Bcl-X L inhibitor compound concentration in plasma of a mouse for two liposome formulations.
- the liposome formulation is contacted with albumin prior to administration under conditions effective to enhance the amount of the Bcl-2/Bcl-X L inhibitor compound present in the plasma after administration of the liposome (e.g., to reduce the reduction in the Bcl-2/Bcl-X L inhibitor compound concentration in the plasma within about 4 hours after administration of the liposome).
- Liposomes typically comprise vesicles containing one or more lipid bilayers enclosing an aqueous interior. Liposome compositions usually include liposomes in a medium, such as an aqueous fluid exterior to the liposome.
- Liposome lipids can include amphiphilic lipid components that, upon contact with aqueous medium, spontaneously form bilayer membranes, such as phospholipids, for example, phosphatidylcholines. Liposomes also can include membrane-rigidifying components, such as sterols, for example, cholesterol. In some cases, liposomes also include lipids conjugated to hydrophilic polymers, such as,
- PEG polyethyleneglycol
- a variety of liposomal properties or formulation methods play important roles in determining the degree of stability, and hence the rate of drug release from the liposomal carrier.
- distearoylphosphatidylcholine or hydrogenated soy phosphatidylcholine in liposomal formulations of amphipathic drugs improves stability considerably when compared to liposomes containing unsaturated phospholipids.
- the inclusion of cholesterol reduces destabilizing interactions with plasma proteins, and also participates in regulating the permeability of liposomal membranes to small molecules.
- Spingomyelin-based liposomes have also demonstrated superior drug retention and activity when compared to
- phosphatidylcholine-based formulations likely resulting in part from intermolecular hydrogen bonding with neighboring cholesterol molecules and the reduced hydrolysis of sphingomyelin when compared to phospholipids.
- the liposome membrane composition of the present invention can be made by any suitable method known to or later discovered by one skilled in the art.
- lipid components can be used to make the liposomes of the present invention.
- Lipid components usually include, but are not limited to (1) uncharged lipid components, e.g., cholesterol, ceramide, diacylglycerol, acyl(poly ethers) or alkylpoly(ethers); (2) neutral phospholipids, e.g., diacylphosphatidylcholines, sphingomyelins, and
- diacylphosphatidylethanolamines (3) anionic lipids, e.g., diacylphosphatidylserine, diacylphosphatidylglycerol, diacylphosphatidate, cardiolipin, diacylphosphatidylinositol, diacylglycerolhemi succinate, diaclyglycerolhemigluratate, cholesteryl hemi succinate, cholesterylhemiglutarate, and the like; (4) polymer-conjugated lipids, e.g., N-[methoxy- (poly(ethylene glycol)diacylphosphatidylethanolamine, poly(ethylene glycol)-diacylglycerol, poly(ethylene glycol)-ceramide; and (5) cationic lipids, e.g., l,2,-diacyl-3- trimethylammonium-propane (DOTAP), dimethyl dioctadecylammonium bromide (DD
- lipid components can be selected to fulfill, modify or impart one or more desired functions.
- phospholipid can be used as principal vesicle-forming lipid.
- Inclusion of cholesterol is useful for maintaining membrane rigidity and decreasing drug leakage.
- Polymer-conjugated lipids can be used in the liposomal formulation to increase the lifetime of circulation via reducing liposome clearance by liver and spleen, or to improve the stability of liposomes against aggregation during storage, in the absence of circulation extending effect..
- the liposome formulation can include a Bcl-2/Bcl-XL inhibitor compound (e.g., a compound of formula (II) or formula (I)) encapsulated in a unilamellar vesicle formed from one or more liposome-forming lipids (e.g., hydrogenated soy phosphatidylcholine (HSPC)), cholesterol and a hydrophilic polymer-conjugated lipid.
- the hydrophilic polymer conjugated to the lipid is poly-ethylene glycol (PEG) (e.g., methoxy-poly(ethylene glycol)-l,2-distearyl-sn-glyceryl (PEG2000-DSG).
- Hydrophilic polymers e.g., PEG with molecular weights from about 200 to about 30,000 can be employed.
- the molecular weight of PEG moiety of a PEG-lipid is 2000.
- the PEG-lipid can be neutral, such as PEG2000-DSG, or ionically charged, such as N-methoxy-poly(ethylene
- the liposome-forming lipid preferably comprises one or more phospholipids, with the ratio of the phospholipid(s) and the cholesterol selected to provide a desired amount of liposome membrane rigidity while maintaining a sufficiently reduced amount of leakage of the compound of formula (I) from the liposome.
- Ri can be a moiety of the formula -(A)-N(R a )(R ) where A is a linear or branched alkyl moiety, and R a and R b are
- Ra ' or Rb ' is hydrogen and the other is C1-C4 alkyl, or R a and R together form a heterocyclic ring.
- Bcl-2 protein inhibitors include the following compounds: N-(4-(4-((4 ' - chloro(l, 1 ' -biphenyl)-2-yl)methyl)piperazin-l-yl)benzoyl)-4-(((lR)-3-(dimethylamino)-l- ((phenylsulfanyl)methyl)propyl)amino)-3-nitrobenzenesulfonamide) (referred to herein as "Comparator B”):
- Comparator A has shown dose-limiting thrombocytopenia (platelets counts) in clinical trials. This class of compound suffers from toxicity (thrombocytopenia) due to inhibition of BC1-XL (Bajwa et al. (2012), Expert Opin. Ther. Patents 22: 37-55) and the emergence of resistance.
- BC1-XL specific inhibitor with the following structure (referred to herein as "Comparator D"):
- Mcl-1 upregulation has been implicated in the resistance of cancer cells to therapeutics targeting Bcl-2 and BC1-XL.
- Mcl-1 inhibitors for example, a compound of the following structure (referred to herein as "Comparator E"):
- AZD4320 is a dual Bcl-2/ Bcl-X L inhibitor (for IV dosing), that has shown transient platelet counts reduction observed in mouse models, returning to baseline at 72 hrs.
- Liposomes typically have the size in a micron or submicron range and are well recognized for their capacity to carry pharmaceutical substances, including anticancer drugs, such as irinotecan, and to change their pharmaceutical properties in various beneficial ways.
- Methods of preparing and characterizing pharmaceutical liposome compositions are known in the field (see, e.g., Lasic D. Liposomes: From physics to applications, Elsevier, Amsterdam 1993; G. Greroriadis (ed.), Liposome Technology, 3 rd edition, vol. 1-3, CRC Press, Boca Raton, 2006; Hong et al., US Pat. 8, 147,867, incorporated by reference herein in their entirety for all purposes).
- Bcl-2 inhibitor compound liposomes can be prepared by a process that includes the steps of (a) preparing a liposome containing a gradient-generating salt such as ammonium or a substituted ammonium salt (e.g., ammonium sulfate), and (b) subsequently contacting the ammonium-sulfate containing liposome with a Bcl-2 inhibitor compound (e.g., a compound of formula (II) or formula (I)) under conditions effective for the irinotecan to enter the liposome and to permit a corresponding amount of ammonia to leave the liposome (thereby exhausting or reducing the concentration gradient of ammonium sulfate across the resulting liposome).
- a gradient-generating salt such as ammonium or a substituted ammonium salt (e.g., ammonium sulfate)
- a Bcl-2 inhibitor compound e.g., a compound of formula (II) or formula (I)
- the Bcl-2 inhibitor-loaded liposomes of the present invention can be prepared without the use of a transmembrane ion gradient, i.e., by direct entrapment, such as, by forming the liposome in the presence of concentrated aqueous solution of the inhibitor, whereby the drug solution is sequestered in the inner aqueous space of the liposome; by forming the liposome from the mixture of lipids with added drug, whereby the drug is entrapped in the liposome lipid membrane, or by incubation of the liposome with the drug, whereby a portion of the drug is distributed from the micellar phase into the liposome membrane.
- a transmembrane ion gradient i.e., by direct entrapment, such as, by forming the liposome in the presence of concentrated aqueous solution of the inhibitor, whereby the drug solution is sequestered in the inner aqueous space of the liposome; by forming the liposome from the mixture of lipid
- Liposomes can be prepared in multiple steps comprising the formation of an ammonium sulfate containing liposome, followed by loading of a Bcl-2 inhibitor compound into the liposome as the ammonium sulfate leaves the liposome.
- the first step can include forming the ammonium sulfate containing liposome by hydrating and dispersing the liposome lipids in the solution of ammonium sulfate.
- the lipid dispersion can be formed into liposomes having the average size of 75-125 nm (such as 80-120 nm, or in some embodiments, 90-115 nm), by extrusion through track-etched polycarbonate membranes with the defined pose size, e.g., 100 nm.
- the ammonium sulfate solution can have a concentration of about 0.25M, and a pH (e.g., about 5.2-5.3) that is selected to prevent unacceptable degradation of the liposome phospholipid during the dispersion and extrusion steps (e.g., a pH selected to minimize the degradation of the liposome phospholipid during these steps). Then, the non-entrapped ammonium sulfate can be removed from the liposome dispersion, e.g., by dialysis, gel chromatography, ion exchange or ultrafiltration prior to irinotecan encapsulation.
- a pH e.g., about 5.2-5.3
- the resulting Bcl-2 inhibitor compound liposomes can contain a sulfate salt of the Bcl-2 inhibitor compound(s) of formula (I) or formula (II).
- These Bcl-2 inhibitor liposomes can be stabilized by loading enough of the Bcl-2 inhibitor compound into the liposomes to reduce the amount of ammonium in the resulting liposome composition to a level that results in less than a given maximum level of lyso-PC formation after 180 days at 4 °C, or less than a given maximum level of lyso-PC accumulation rate in the liposome composition during storage in a refrigerator at about 4 °C, or, more commonly, at 5 ⁇ 3 °C, measured, e.g., in mg/mL/month, or % PC conversion into a lyso-PC over a unit time, such as, mol% lyso-PC/ month.
- the ammonium exchanged from the liposomes into the external medium during the loading process, along with any unentrapped Bcl-2 inhibitor compound, is typically removed from the liposomes by any suitable known process(es) (e.g., by gel chromatography, dialysis, diafiltration, ion exchange or ultrafiltration).
- the liposome external medium can be exchanged for an injectable isotonic fluid (e.g. isotonic solution of sodium chloride), buffered at a desired pH.
- the liposomes encapsulating the Bcl-2 inhibitor compounds disclosed herein preferably interrupt protein-protein interaction (PPI) of pro-apoptosis proteins (Bid, Bim, Bad, Bak, Bax, Noxa, etc. ) with anti-apoptosis proteins (Bcl-2 family: Bcl-2, BC1-XL, Mcl-1, etc.) leading to cell apoptosis (intrinsic apoptosis pathway), bind at a large hydrophobic, flexible contact surface (protein/protein), and/or have higher molecular weights about 1000.
- the liposomes can be used as mono or combination therapies (with chemo agents).
- preferred liposomes are PEGylated liposomal formulations with reduced major distribution into platelets (to reduce the possibility of thrombocytopenia) compared to the free (non-encapsulated) form of the encapsulated Bcl-2 inhibitor compound.
- Novel Bcl-2 inhibitor compounds were identified using the assay in Example 1, including compounds of formula (I) listed in Table 3. The cellular activity of certain Bcl-2 inhibitor compounds were measured against cell lines
- Comparator F was used as an excellent tool compound for inducing thrombocytopenia. Platelet count in in vivo could be used as an indication of target engagement.
- novel BC1-2/BC1-XL inhibitor compounds are disclosed, including compounds of formula (I) and forumula (II) as described herein.
- the examples of the compounds of formula (I) and (II) are provided herein permit one to identify numerous additional BC1-2/BC1-XL inhibitor compounds within the scope of formula (I) or formula (II) in addition to those described in Example 8 and elsewhere herein, including: (a) additional compounds of formula (I) where R is C or a heteroatom (such as N) and Ri is a moiety comprising an amine (e.g., an alkyl substituted amine having a pK a of at least about 7.0, preferably about 8.0-10.5 and most preferably about 9.0-10.0) selected to provide an IC5 0 value of less than 100 nM against each of Bcl-2 and BC1-XL (as measured by the assay of Example 1 below); and (b) additional compounds of formula (II) where R ' is C or a heteroatom
- liposomes encapsulating Bcl-2 Bcl-X L inhibitor compounds are provided, where the liposome-encapsulated BC1-2/BC1-XL inhibitor compounds have an activity against each of Bcl-2 and BC1-XL measured by an IC5 0 value of up to about ⁇ l - ⁇ using the assay of Example 1.
- the liposomes encapsulate the novel BC1-2/BC1-XL inhibitor compounds disclosed herein (e.g., BC1-2/BC1-XL inhibitor compounds of formula (I) and formula (II)).
- the liposomes encapsulate a BC1-2 BC1-XL inhibitor Comparator compound disclosed herein.
- the BC1-2/BC1-XL inhibitor liposomes can provide an extended release of the encapsulated Bcl-2/Bcl-X L inhibitor compound, as described in Example 5.
- the BC1-2 BC1-XL inhibitor liposomes disclosed herein can be prepared to reduce incidence of thrombocytopenia in the mouse models disclosed herein to levels that are acceptable to advance the Bcl-2/Bcl-X L inhibitor compounds as clinical candidates for human testing for the treatment of cancer.
- the administration of BC1-2/BC1-XL inhibitor encapsulated in liposomes as disclosed herein to a mouse model can result in PLT reduction over 3 days post administration that is less than the resulting PLT reduction of the non- encapsulated BC1-2/BC1-XL inhibitor.
- the BC1-2/BC1-XL inhibitor encapsulated in a liposome does not result in the reduction of PLT levels below about 400 PLT/microliter (preferably not below about 500 PLT/microliter and most preferably not below about 600 PLT/microliter) for at least about 21 days after inj ection in a mouse in any one of the test described in Examples 6 and 7 below.
- Compounds having inhibitory activity at Bcl-2, Bcl-X L and/or Mcl-l can be indentified using the assay of Example 1.
- the activity of compounds at targets Bcl-2, BC1-XL and Mcl-l was determined using the assay of example l .
- the activity of compounds at Bcl-2, Bcl-X L and Mcl-l were determined using the following TR- FRET based assay.
- Synthetic peptides specific for Bcl-2, BC1-XL and MCL-1 can be purchased or otherwise obtained by custom peptide synthesis. Synthetic genes can be obtained for GST- tagged Bcl-2, BC1-XL and Mcl-l fragments. Proteins can be purified and used for assay development.
- Mcl-l (aa 171-327) E. coli expressed, RBC custom production
- HTRF reagent MAb Anti GST-Tb cryptate 61 GSTTLA (CisBio)
- Bcl-2, Bcl-X L and MCL-1 protein fragments were designed with a C-terminal GST tag. Summaries for protein production and purification are shown below. In case detailed protocol is required for any of the steps, it can be provided separately.
- BL21(DE3) cells were induced with 0.5 mM IPTG and grown in TB at 18 °C
- BL21(DE3) cells were induced with 0.5 mM IPTG and grown in TB at 18 °C
- BL21(DE3) cells were induced with 0.5 mM IPTG and grown in 2XTY at
- Buffer 1 100 mM Potassium Phosphate pH 7.5; 50 mM NaCl; 1 mM EDTA; 0.01% NP40 (see, Zhang, H., et al. Anal Biochem, 2002. 307(1): p. 70-5 and Leverson, J.D., et al., Cell Death Dis, 2015. 6: p. el590).
- Buffer 2 20 mM Tris, pH 7.5; 50 mM NaCl; 0.01% NP40 (see Du, Y., et al. Assay Drug Dev. Technol, 201 1. 9(4): p. 382-93.)
- FIGs. 18A, 18B, 18C, and 18D are graphs showing the fluorescent peptide titrations at multiple protein concentrations.
- FIGs. 19A, 19B, 19C and 19D are graphs showing protein titrations at two f-peptide concentrations.
- FIGs. 20A, 20B, 20C, and 20D are graphs showing extended range protein titrations at single f-peptide concentration (see table below for protein/peptide pair concentrations).
- HTRF technology enables multiple plate readings without negative effect on signal.
- plates were scanned every 30 min for 2.5 hours.
- Signal stability as a function of time is shown below. At least 30 min incubation is required prior to reading in order to achieve maximum signal in assay.
- Bcl-2/fbak shows highest decrease in signal with prolonged incubation, while other pairs are less affected. A 60 minute incubation would be acceptable for all protein/peptide pairs.
- FIGs. 21A, 21B, 21C and 21D are graphs showing the signal stability over time.
- Protein and peptide concentrations were as shown in Table 2. Compounds were serially diluted in 100% DMSO and 15 nL of each compound concentration was delivered into protein solution using ECHO (Labcyte). Following 15 min pre-incubation, peptide was added to assay wells and mixture was further incubated for 15 min prior to addition of anti-GST-Tb mAb. Plates were read every 30 min for 2.5 h. Data from -60 min incubation was used for EC50 determinations. EC50 plots are shown below (FIGs. 22A, 22B, 22C, 22D, 22E and 22F). Mcl-l/fbak pair showed similar behavior to Mcl-l/fNoxa. EC50 plots for Mcl-l/fNoxa are shown. Data is summarized below (Table 2B).
- Table 2D Selected protein concentrations for displacement IC5 0 determinations.
- Example 2 Cellular activity of compounds of Formula (I) and Formula (II) against selected cell lines
- FIG 1C is a table (Table 3A) of the IC5 0 values measured for compounds on cell viability in multiple myeloma cells incubated with drug for 24 h, followed by washing and additional 72 h incubation.
- the IC 50 values for compounds of formula (I) are plotted in the graph of FIG. 1A.
- the IC5 0 values for compounds of formula (II) are plotted in the graph of FIG. IB.
- C concentration of drug
- y normalized CTG value
- a top asymptote (represents maximum cell kill)
- b bottom asymptote (constrained 0.8-1.2)
- IC50 slope: logistic curve slope.
- concentration range is optimal according to these rules: (1) if the lowest concentration kills more than 70% of the cells the concentration range is deemed too potent (2) if the highest concentration kills less than 30% of the cells, the concentration range is deemed low or the cell line is too resistant.
- Figures 2A-2D are graphs showing the dose response of docetaxel (DTX) in MDA- MB23 1 breast carcinoma cells when combined with Comparator C, Compound 6, Compound 2, or Comparator E at three different concentrations.
- DTX docetaxel
- Example 4-L Aqueous stock solution of Compound 6 dimesylate.
- Free base Compound 6 (68.7 mg, 0.0743 mmol) in a powder form was thoroughly mixed with 0.1485 ml (0.1485 mmol) of 1 N aqueous methanesulfonic acid. To the resulting orange cake, 2 ml of distilled water was added, stirred with intermittent 1 -2 min heating on a 65 °C water bath and subjected to 2 cycles of bath-sonication (5-10 sec.) until the clear solution having about 32 mg/ml of Compound 6 was obtained.
- the solution was adjusted to 20 mg/ml Compound 6 (as free base) by adding 1.29 ml of distilled water, and passed through a 0.45-micron polyvinylidenefluoride (PVDF) syringe filter.
- the solution had pH 2.80.
- Distearoyl phosphatidylcholine (DSPC; Lipoid GMBH, Germany; 1002.4 mg), cholesterol "high purity” (Choi; Dishman, China; 327.3 mg), and PEG(2000)- distearoylglycerol (PEGDSG; Sunbright GS-020, NOF, Japan; 334 mg) were combined in a closed glass vial with 2.5 ml of 100% ethanol (Sigma, USA, molecular biology grade) and heated on a 70 °C water bath until the lipids were completely dissolved.
- the target molar ratio of lipid components was DSPC:Chol :PEGDSG 3 :2:0.3.
- the ethanolic solution of the lipids was quickly added to 22.8 g of 0.25 M ammonium sulfate, pH 5.27, preheated to 67.8 °C, with intensive stirring, and the stirring continued on a 70 °C water bath to form a suspension of multilamellar liposomes (MLV) as about 50 mM phospholipid.
- MLV multilamellar liposomes
- the MLV suspension was extruded through the stack of 4xl00-nm and 1x200 nm track- etched polycarbonate filters (Whatman Nuclepore, USA) three times using Lipex thermobarrel extruder (Northern Lipids, Canada) with circulating water at 70 °C.
- the extruded liposomes were allowed to reach the room temperature and passed through 0.2 - micron PES sterile filter.
- Particle size was determined by DLS using Malvern Zeta-sizer Nano; average liposome size (Xz, by the cumulants method) was 100.2 nm, polydispersity index (Pdl) 0.036.
- the drug-liposome mixture was heated with stirring on a 65 °C water bath for 22 min. and chilled on ice. Ionic strength (IS) was adjusted to 0.1 M NaCl by adding 0.9 ml of 3 M NaCl stock solution.
- the IS-adjusted loading mixture (pH 4.86) was titrated with 1 M HEPES-Na buffer, pH 7.0, total 0.25 ml, to the final pH 5.62, and passed through a sterile 0.2-micron PES syringe filter.
- the filtered, pH- and ionic strength-adjusted loading mixture was purified from an non- encapsulated drug by column chromatography on a 1 Ox volume of Sepharose CL-4B (GE Healthcare, USA), using 5 mM HEPES-Na, 144 mM NaCl, pH 6.5 buffer (HBS-6.5) for elution.
- the void volume fractions containing drug-loaded liposomes were combined and manually concentrated about 3-fold by diafiltration through a hollow fiber cartridge (C02- E500-10-N, Spectrum, USA).
- Liposome size Xz 108.7 nm, Pdl 0.028, the drug concentration (determined as in Example 4-L2) 3.34 mg/ml; drug/phospholipid ratio 139.5 ⁇ 2.6 g/mol (quantitative loading).
- Example 4-L Treatment of Compound 6 liposomes with serum albumin.
- the concentrated Compound 6 liposomes were treated with serum albumin as follows. Bovine serum albumin (BSA; A7906, Aldrich, USA) was dissolved in 0.85% aqueous NaCl to give 5% (w/w) solution, and the solution was passed through 0.2-micron syringe sterile filter. One volume part of the BSA solution was added to four volume parts of the Compound 6 liposomes to achieve the final 10 mg/ml of BSA, and the mixture was stirred at 37 °C for 50 min.
- BSA bovine serum albumin
- the liposomes were purified from BSA using Sepharose CL- 4B column chromatography, concentrated on a hollow fiber cartridge essentially as described above for the removal of non-encapsulated drug, and aseptically passed through 0.2-micron PES sterile filter.
- the diafiltrate from the last the concentrating step was spectrophotometrically assayed for the free drug (at 308 nm) and residual free albumin (at 280 nm). Found free Compound 6 0.0065 mg/ml (0.17%), residual albumin 0.056 mg/ml. Treatment with albumin caused only a very minor loss of the liposome-associated drug (2.0%), which is within the error range of the
- Example 4-L3 For preliminary in vivo studies, the protocol of Example 4-L3 was performed on the scale of 2 mg of the drug (1 ml of the liposome loading mixture) at the
- Post-load mixture (pH 4.82) was adjusted to pH 5.61 before the Sepharose CL-4B size exclusion chromatography purification step.
- 1 -ml portions of IS- and pH adjusted loading mixture were applied onto PD-10 columns (GE Healthcare, USA) filled with 10 ml of Sepharose CL-4B.
- the liposome fraction was collected between 3.0 and 4.5 ml, while the free drug fraction, being micellar in nature (volume-average micelle diameter about 8-15 nm), appears at the elution volume of 5.5 ml.
- Example 4-L6 Formulation of Compound 1 into PEGylated liposomes using ammonium sulfate gradient.
- the extruded liposomes were chromatographed on a Sephadex G-25 (GE Healthcare, USA) column in distilled water as eluent. The void volume fraction containing liposomes was collected, and the phospholipid concentration was determined to be 27.1 mM.
- MES morpholinoethanesulfonic acid
- NaCl morpholinoethanesulfonic acid
- pH 5.2 to the final 10 mM MES
- 0.05 ml of 20 mg/ml Compound 1 bis-mesylate solution prepared according to Example 4-Ll
- the drug-liposome mixture was heated with stirring on a 65 °C water bath for 20 min. and chilled on ice.
- Ionic strength (IS) was adjusted to 0.1 M NaCl by adding 0.0175 ml of 3 M NaCl stock solution.
- the IS-adjusted loading mixture (pH 4.30) was purified from an non- encapsulated drug by column chromatography on a 1 Ox volume of Sepharose CL-4B (GE Healthcare, USA), using 5 mM HEPES-Na, 144 mM NaCl, pH 6.5 buffer (HBS-6.5) for elution.
- Example 4-L7 Formulation of Compound 5 into PEGylated liposomes using ammonium sulfate gradient.
- the protocol of Example 4-L6 was followed to encapsulate Compound 5, except that the amount of 1 M morpholinoethanesulfonic acid (MES)-NaOH buffer pH 5.2 was increased to 0.01 ml (to the final 20 mM MES).
- the IS-adjusted loading mixture (pH 3.90) was purified from an non-encapsulated drug by column chromatography on a lOx volume of Sepharose CL-4B (GE Healthcare, USA), using 5 mM HEPES-Na, 144 mM NaCl, pH 6.5 buffer (HBS-6.5) for elution.
- Example 4-L8 Formulation of Compound 4 into PEGylated liposomes using ammonium sulfate gradient.
- Example 4-L6 The protocol of Example 4-L6 was followed to encapsulate Compound 4, except that the amount of 1 M morpholinoethanesulfonic acid (MES)-NaOH buffer pH 5.2 was increased to 0.01 ml (to the final 20 mM MES).
- the IS-adjusted loading mixture (pH 4.70) was purified from an non-encapsulated drug by column chromatography on a lOx volume of Sepharose CL-4B (GE Healthcare, USA), using 5 mM HEPES-Na, 144 mM aCl, pH 6.5 buffer (HBS-6.5) for elution.
- Example 4-L10 Formulation of Compound 8 into PEGylated liposomes using ammonium sulfate gradient.
- Liposomes composed of DSPC , Cholesterol, PEGDSG, and a fluorescent lipid label DiIC18(3)-DS (Molecular probes, USA) in the molar ratio of 3 :2:0.3 :0.0015, respectively, containing entrapped 0.25 M ammonium sulfate were prepared essentially as described in Example 4-L3, except that the calculated amount of DiIC18(3)-DS was added to the ethanolic solution of other lipids as 25 mg/ml stock solution in dimethylacetamide. After removal of unentrapped ammonium sulfate on a Sephadex G-25 column, the liposome PhL concentration was 23.6 mM.
- Ionic strength (IS) was adjusted to 0.1 M aCl by adding 0.035 ml of 3 M NaCl.
- the IS - adjusted loading mixture (pH 4.86) was purified from an non-encapsulated drug by column chromatography on a 1 Ox volume of Sepharose CL-4B (GE Healthcare, USA), using HBS- 6.5 buffer for elution.
- Example 4-L1 1. Formulation of Compound 2 into PEGylated liposomes using ammonium sulfate gradient.
- Liposomes composed of DSPC , Cholesterol, PEGDSG, and a fluorescent lipid label DiIC18(3)-DS (Molecular probes, USA) in the molar ratio of 3 :2:0.3 :0.0015, respectively, containing entrapped 0.25 M ammonium sulfate were prepared as in Example 4-L10. After removal of unentrapped ammonium sulfate on a Sephadex G-25 column, the liposome PhL concentration was 25.3 mM.
- Hydrogenated soy phosphatidylcholine (HSPC, Lipoid, Germany), cholesterol, PEGDSG, and DiIC18(3) -DS at the molar ratio of HSPC:Chol:PEGDSG: DiIC18(3)-DS of 3 :2:0.15 :0.003 were combined with 100% ethanol (1.43 ml for each 1 g of the total lipid) and heated in a closed vial on a 70 deg.C water bath until complete dissolution.
- DiIC 18(3)-DS was added as a 25 mg/ml stock solution in dimethylacetamide.
- the lipid solution was added to the stirred TEA-SOS solution preheated to more than 65 deg. C on a 68 deg.
- the liposomes were purified from unentrapped TEA-SOS by gel- chromatography on Sepharose CL-4B in water; the elimination of non-encapsulated TEA- SOS was verified by conductivity measurements in the liposome-containing void volume fraction (1 micro-S/cm or less). After purification, TEA-SOS liposomes had phospholipid concentration of 26.4 mM.
- a volume of 0.316 ml of the purified TEA-SOS liposomes was mixed with 0.1 1 ml of 50% dextrose USP (to final 50 mg/ml dextrose), 0.324 ml of distilled water (for volume adjustment), 0.25 ml of 20 mg/ml Compound 1 (Example 4-Ll) to final 5 mg/ml of the drug and drug/phospholipid ratio 600 g/mol, and 0.007 ml of 1 M MES-NaOH buffer pH 5.2 to the final pH of 3.78.
- the mixture was incubated with stirring on a 65 deg.C water bath for 30 min. and chilled on ice.
- One-half ml of the post-incubation loading mixture was adjusted to 0.14 M NaCl by adding 0.0245 ml 3 M NaCl and chromatographed on Sepharose CL-4B, eluent HBS-6.5, to remove non-encapsulated drug.
- the drug-loaded liposomes obtained in the void volume fractions were passed through 0.45 micro-m and 0.2 micro-m polyethersulfone syringe filters.
- Example 4-L13 Formulation of Compound 8 into PEGylated liposomes using 0.25 M triethylammonium sulfate gradients at various drug-lipid ratios.
- Solution of triethylammonium sulfate was prepared by neutralizing calculated amount of 2 N sulfuric acid (volumetric standard) with neat triethylamine to pH 6.0-6.5, and diluting the neutralized solution with distilled water to the final
- triethylammonium sulfate concentration of 0.25 M Liposomes with entrapped 0.25 M triethylammonium sulfate were prepared essentially as described in Example 4-L3, except that DSPC:Chol:PEGDSG molar ratio was 3 :2:0.18, 0.25 M 0.25 M triethylammonium sulfate solution was used instead on 0.25 M ammonium sulfate, and extrusion step included 4 passages through the polycarbonate filter stack. The extruded liposomes were purified from non-encapsulated triethylammonium sulfate by chromatography on
- triethylammonium sulfate liposomes were combined with Compound 8 dimesylate solution (Example 4-Ll) at the final drug concentration of 2 mg/ml in the presence of 88.4 mg/ml dextrose and 10 mM MES-NaOH buffer (pH 5.2 at 1 M MES) to obtain
- Example 4-L14 Formulation of Compound 8 into PEGylated liposomes using 0.35 M triethylammonium sulfate gradients at various drug-lipid ratios.
- Solution of triethylammonium sulfate was prepared by neutralizing calculated amount of 2 N sulfuric acid (volumetric standard) with neat triethylamine to pH 6.0-6.5, and diluting the neutralized solution with distilled water to the final
- Example 4-L15 Formulation of Compound 8 into PEGylated liposomes in the absence of ammonium gradient.
- Liposomes of DSPC, Cholesterol and PEGDSG were prepared according to Example 4-L13, except that instead of 0.25 M triethylammonium sulfate, 0.85% soluti on of sodium chloride was used.
- Example 4-L16 Formulation of Compound 8 into PEGylated liposomes containing ionic PEG-lipid derivative and 0.25 M ammonium sulfate
- PEG(2000)-oxycarbonyl)-distearoylphosphatidylethanolamine (PEGDSPE) at the molar ratio of 3 :2:0.3, containing 0.25 M ammonium sulfate, were purified from unentrapped ammonium sulfate on a Sephadex G-25 column (eluent water), and incubated 24 min. with stirring on a 68 deg. C water bath in the presence of 2 mg/ml Compound 8 in 88.4 mg/ml dextrose, 10 mM MES-Na (pH 5.2 at 1 M MES)and drug/phospholipid ratio of 100 g/mol, at pH 4.40.
- PEGDSPE PEG(2000)-oxycarbonyl)-distearoylphosphatidylethanolamine
- the resulting liposomes were chilled on ice, adjusted to 0.1 M NaCl by addition of 3M NaCl stock solution, and purified from non-encapsulated drug by chromatography on a Sepharose CL-4B column, eluent HBS-6.5. The purification step was repeated once again, and the void volume fraction containing drug-loaded liposomes was analyzed for the drug and phospholipid content. Drug/phospholipid ratio in the loaded, purified liposomes was 68.5 g/mol, or 68.5% of its pre-purifi cation value (encapsulation efficiency 68.5%).
- Example 4-L17 Formulation of compound Comparator B into PEGylated liposomes using ammonium sulfate gradient.
- FIG. 23 shows data obtained from a study comparing the effects of the free and liposomal Compound 6 (formulation 1) at 10 mg/mg, showing that liposomal drug has less thrombocytopenic effect.
- Commercially obtained compound Comparator B (Selleck Chemicals) was mixed with two equivalents of 1 M methanesulfonic acid, distilled water was added to achieve 10 mg/ml of Comparator B, and the suspension was agitated, with intermittent warming up on a 65 °C water bath and brief sonication in an ultrasound bath until most of the drug was dissolved. The solution was passed through 0.2-micron
- PEG(2000)DSG essentially as described in Example 4-L5, except that the aliquots of the components were calculated to achieve, prior to the 65°C incubation step, the final concentration of 1.9-2.0 mg/ml of the drug, 88.2 mg/ml of dextrose, 10 mM MES-NaOH buffer (pH 5.2 at 1 M MES), and the liposome phospholipid at the drug/phospholipid ratios of 125, 200, or 300 g/mol. Post-incubation mixtures were adjusted to 0.1 M NaCl, and the pH was adjusted to pH 5.4-5.5 with 1 M HEPES_Na buffer.
- the liposome were purified from the non-encapsulated drug by Sepharose gel chromatography as in Example 4-L5, and the purified liposomes were passed through 0.45- micron PVDF filter. Only the 125 g/mol formulation was partially passable through 0.2-micron PES filter, while two other were not.
- the liposomes had the following properties (Table 4B):
- Example 4-L18 Formulation of Compound 2 into cholesterol-free PEGylated liposomes with an acidic phospholipid using ammonium sulfate gradient.
- DSPC Distearoylphosphatidylglycerol
- PEGDSPE PEGDSPE
- a fluorescent lipid label DiIC18(3)-DS (Molecular probes, USA) in the molar ratio of 2: 1 :0.2:0.002 were co-dissolved in a chloroform-methanol mixture, and the solution was evaporated to dryness in vacuum at 60°C.
- the lipid residue was dissolved in 100% ethanol, mixed with 0.25 M ammonium sulfate and processed to form liposomes as described in Example 4-L3, except that the number of passes through the polycarbonate membrane stack was five.
- Compound 2 bis-mesylate stock solution prepared as described in Example 4-Ll
- Morpholinoethanesulfonic acid (MES)-NaOH buffer (pH 5.2) was added to the final MES concentration of 10 mM, the mixtures were heated with stirring on a 65 deg. C water bath for 30 min. and chilled on ice.
- the loaded liposomes were purified from unencapsupated drug by chromatography on a 1 Ox volume of Sepharose CL-4B (GE Healthcare, USA) columns eluted with 5% aqueous dextrose.
- Ionic strength (IS) of the liposomes was adjusted to 0.1 M NaCl with 3 M NaCl, and the pH was adjusted to 7.0 by adding 1 M HEPES-NaOH buffer, pH 7.3, to 10 mM HEPES.
- the pH- and IS- adjusted liposomes were passed through 0.2-micro-m polyethersulfone syringe filter.
- the liposomes had the following characteristic:
- the liposomes prepared without cholesterol and with an acidic phospholipid showed very effective loading of Compound 2 via ammonium sulfate gradient method (>90% loading), while keeping the average liposome size small (z-average particle size 97-100 nm) and narrow size distribution (Pdl ⁇ 0.1) up to the drug phospholipid ratio of 225 g/mol, in spite of the fact that an ionic PEG-lipid derivative (PEGDSGE)was used instead of PEGDSG for liposome PEGylation.
- PEGDSGE ionic PEG-lipid derivative
- Example 4-Ll Formulation of Compound 2 into cholesterol-free PEGylated liposomes with an acidic phospholipid using tri ethyl ammonium sucrose octasulfate gradient.
- Solution of TEA-SOS was prepared as described in Example 4-L12, except that the concentration of TEA-SOS was 0.46 N (0.0575 M).
- DSPC, DSPG, PEGDSPE, and DiIC18(3)-DS in the molar ratio of 2: 1 :0.2:0.002 were co-dissolved in a chloroform- methanol mixture, and the solution was evaporated to dryness in vacuum at 60°C.
- the lipid residue was dissolved in 100% ethanol, mixed with 0.46 N TEA-SOS, and processed to form liposomes as described in Example 4-L17.
- Unentrapped TEA-SOS war removed by repeated chromatography on a Sepharose CL-4B column eluted with 5% aqueous dextrose until the conductivity of the liposome eluate was less than 12 micro-S/cm.
- the drug/phospholipid ratios of 150, 200, 250, or 300 g/mol (calculated for the drug free base content).
- the drug-liposome mixtures (pH 5.15.-5.23, no buffer added) were heated with stirring on a 68 deg. C water bath for 30 min. and chilled on ice.
- the loaded liposomes were purified from unencapsupated drug by chromatography on a lOx volume of Sepharose CL-4B (GE Healthcare, USA) columns eluted with 5% aqueous dextrose.
- Ionic strength (IS) of the liposomes was adjusted to 0.1 M NaCl with 3 M NaCl, and the pH was adjusted to 7.0 by adding 1 M HEPES-NaOH buffer, pH 7.3, to 10 mM HEPES.
- the pH- and IS-adjusted liposomes prepared at drug/phospholipid ratios of 150 and 200 g/mol were passed through 0.2-micro-m polyethersulfone syringe filter.
- the liposomes prepared at drug/phospholipid ratios of 250 and 300 g/mol did not pass through 0.2-micro-m polyethersulfone syringe filter and were characterized as is.
- the liposomes had the following characteristics:
- Example 4-L20 Formulation of Compound 2 into cholesterol-free PEGylated liposomes with an acidic phospholipid using tri ethyl ammonium sucrose octasulfate gradient at various concentrations of TEA-SOS.
- Example 4-L12 were prepared as described in Example 4-L12.
- DSPC, DSPG, PEGDSPE, and DiIC18(3)- DS in the molar ratio of 2: 1 :0.2:0.003 were co-dissolved in a chloroform-methanol mixture, the solution was divided into three portions and evaporated to dryness in vacuum at 60°C.
- the lipid residues were dissolved in 100% ethanol, mixed with 0.43 N, 0.65 N, or 1.083 N TEA-SOS, and processed to form liposomes as described in Example 4-L17 (four passes through the polycarbonate membrane stack).
- Unentrapped TEA-SOS was removed by repeated chromatography on a Sepharose CL-4B column eluted with 5.1%, 7.8%, or 14.0% aqueous dextrose, respectively, until the conductivity of the liposome eluate was less than 13 micro-S/cm.
- the eluted liposomes (1 1.7-14.3 mM phospholipid, Xz 87.6-92.4 nm, Pdl 0.049- 0.076) were mixed with 37.4 mg/ml Compound 2 bis-mesylate stock solution (prepared as described in Example 4-L1) to reach the drug phospholipid ratios of 150 or 300 g/mol (calculated for the drug free base content).
- the drug-liposome mixtures were heated with stirring on a 65 deg. C water bath for 30 min. and chilled on ice.
- the loaded liposomes were purified from unencapsupated drug by chromatography on a lOx volume of Sepharose CL-4B (GE Healthcare, USA) columns eluted with 5.1%, 7.8%, or 14.0% aqueous dextrose, respectively.
- the liposomes prepared at drug/phospholipid ratio of 150 g/mol were passed through 0.2-micro-m polyethersulfone syringe filter.
- the liposomes had the following
- Example 4-L21 Formulation of Compound 2 into cholesterol-free PEGylated liposomes with various amounts of acidic phospholipid using triethylammonium sucrose octasulfate gradient.
- DSPC, DSPG, PEGDSPE, and DiIC18(3)-DS were co-dissolved in a chloroform-methanol mixture at the molar ratios of 2.4:0.6:0.2:0.003 ("20mol.% DSPG"), 2.7:0.3 :0.2:0.003 ("10mol.% DSPG”), and 2.85:0.15 :0.2:0.003 ("5 mol.% DSPG").
- a lipid composition at 10 mol .% DSPG containing PEGDSG instead of PEGDSPE in the same molar amount was prepared. The solutions were evaporated to dryness in vacuum at 60°C.
- the lipid residues were dissolved in 100% ethanol, mixed with 0.43 N TEA-SOS, and processed to form liposomes as described in Example 4-L17.
- Unentrapped TEA-SOS war removed by repeated chromatography on a Sepharose CL-4B column eluted with 5% aqueous dextrose until the conductivity of the liposome eluate was less than 25 micro-S/cm.
- the eluted liposomes (23.5- 25.0 mM phospholipid) were mixed with 40.3 mg/ml Compound 2 bis-mesylate stock solution (prepared as described in Example 4-L1) to reach the drug phospholipid ratio of 150 g/mol (calculated for the drug free base content).
- the drug-liposome mixtures (pH 4.5.-5.4, no buffer added) were heated with stirring on a 65 deg. C water bath for 30 min. and chilled on ice.
- the loaded liposomes were purified from unencapsupated drug by chromatography on a lOx volume of Sepharose CL-4B (GE Healthcare, USA) columns eluted with 5% aqueous dextrose.
- the eluted liposomes were passed through 0.2-micro-m PES syringe filter.
- the liposomes had the following characteristics:
- Example 4-L22 Formulation of Compound 2 into the liposomes containing the drug in a membrane-entrapped form.
- compounds of the invention can be formulated to be contained within the bilayer membrane of the liposomes, that is, in the membrane-entrapped form, by the following general protocol.
- the drug e.g., Compound 2
- chloroform-methanol mixture (1 : 1 by volume) at the concentration of 10-50 mg/ml, and brought into dihydrichloride salt form by addition of the calculated amount of 1.25 M HC1 solution in isopropanol (Sigma-Aldrich, USA).
- the solution is added to the liposome lipids dissolved in chloroform, optionally with addition of methanol, and the drug- lipid solution is evaporated in vacuum at 40-60 deg. C to dryness.
- the dry lipid cake is dissolved in 100% ethanol to form approximately 50% (w/w) solution at 70-75 deg. C, and mixed, at this temperature, with 10 volumes of pre-heated 5 mM HEPES-NaOH buffer, 144 mM NaCl, pH 6.5 (HBS-6.5 buffer).
- the drug-lipid suspension is extruded through polycarbonate membranes as described in Example 4-L3.
- the extruded liposome suspension is passed through a Sepharose CL-4B column, eluted with 10 mM HEPES-NaOH, 140 mM NaCl buffer pH 7.25 (HBS-7.25 buffer) to purify the liposomes from extraliposomal (e.g., micellar) drug.
- the purified liposomes are sterilized by passage through 0.2 micro-m (e.g., PES) filter.
- the following liposomes containing Compound 2 in the membrane-entrapped form were prepared:
- All lipid compositions also included 0.2 mol. parts of PEGDSPE and 0.002 mol. parts of DiIC 18(3)-DS. Choi: cholesterol. DL ratio - drug/phospholipid ratio.
- Example 4-L23 Formulation of Compound 2 into the liposomes containing the drug in a membrane-entrapped form in the presence of various anionic lipids.
- the Sepharose- purified and 0.2 micro-m filtered liposomes had the following characteristics:
- Liposomal formulations of Bcl-2 inhibitor Compound 6 were prepared as described in Example 4-L5.
- Free drug injection solution was prepared according to Example 4-L2.
- the liposomes and the free drug were administered intravenously at a dose of 15 mg drug kg or 10 mg/kg, respectively, to three 6 week-old female CD-I mice (Charles River) (body weight about 25 g).
- Blood samples were collected into lithium heparin tubes by bleeding from saphenous vein at 0.08, 0.25, 1.5, 4, 8 24 and 48h (post injection, p.i.) time points. Plasma was separated from the cell fraction by centrifugation at 10000 rpm for 5 min.
- Drugs were extracted by incubation of plasma samples with 200 ⁇ of 1% acidic acid in methanol (l%Ac/MeOH) at least 2 hours at -80oC. Plasma proteins were spin down by centrifugation at 15000 rpm for 20 min. Then 75 ⁇ of supernatant was transferred to HPLC vials (Thermo Scientific, Cat# C401 1-LVl) and additional 75 ⁇ of 1% Ac/MeOH were added. Drug content was analyzed by HPLC with each sample measured in duplicate. The data were expressed as the drug concentration in plasma plotted against post injection time in hours (Figure 3). PK parameters were calculated for each individual mouse using
- PK Solutions Pharmacokinetics data analysis package "PK Solutions” (Summit Research Services, CO, USA) and average values for each treatment group are presented in Table 5.
- the liposome is formulated to reduce the blood clearance rate of the Bcl-2/Bcl-X L inhibitor compounds from the blood and/or to reduce the release of the Bcl- 2/BCI-XL inhibitor compounds from the liposome in plasma.
- the liposome is contacted with albumin (e.g., BSA) under conditions effective to increase the concentration of the BC1-2/BC1-XL inhibitor compound in mouse plasma over at least 4 hours, and preferably over at least 48 hours, as described in FIG. 3 and Example 5.
- albumin e.g., BSA
- treatment with albumin may help to remove a portion of the inhibitor that remains bound to the outer leaflet of the liposome bilayer membrane and potentially creates either a membrane defect that, upon contact with blood, facilitates drug leakage from the liposome interior, or promotes opsonization of the liposomes resulting in faster liposome clearance.
- Sequestration of Bcl-2 inhibitors by serum albumin is documented (Vogler M, et al., Blood, vol. 117, p. 7145-7154, 2011).
- Example 6 Effect of liposomal encapsulation on the thrombocytopenic activity of Bcl- 2/Bcl-X L inhibitors
- Blood samples 50-100 ⁇ were drawn into micro-collection tubes with EDTA dipotassium salt (SARSTEDT lot# 4072801) by bleeding from saphenous vein at 0 (pre- injection), 6, 24, 48 and 72 hour post injection.
- SARSTEDT lot# 4072801 EDTA dipotassium salt
- the mice received injection of the buffered physiological saline, and the blood was collected and processed similarly. Platelets were counted immediately after blood collection using a Veterinary Hematology System "Hemavet 950" (The Americas Drew Scientific Inc, Oxford, CT).
- albumin treatment also eliminated the fast initial blood clearance phase in the pharmacokinetics of the liposomal Bcl- 2 inhibitor, it is suggested, without being bound by a theory, that treatment with albumin prevents quick release of a portion of the encapsulated drug shortly after the injection, further reducing the exposure of platelets to the free (released) inhibitor.
- Example 7 Comparison of the thrombocytopenic activity of free (nonencapsulated) compounds Compound 2 and Compound 6 in mice.
- Drug solutions were sterilized with 0.2 micron NalgeneTM 13mm Syringe Filters. Drug concentration in the solutions was determined by HPLC. 5-6 week-old female CD-I mice (Charles River) were injected intravenously via the tail vein with a single bolus of free (non-encapsulated) Compound 2 or Compound 6 at 1, 2 or 5 mg/kg. Blood samples (50-100 ⁇ ) were collected into tubes pre-filled with EDTA dipotassium salt (SARSTEDT, lot# 4072801) by bleeding from saphenous vein at 0, 6, 24, 48 and 72 hour time points. Platelets were counted immediately after blood collection using a Veterinary Hematology System "Hemavet 950" (The Americas Drew Scientific Inc, Oxford, CT).
- the measurements at time 0 represent a background blood test performed prior to injection of the test liposome Formulations 1 and 2.
- the measurements at 6 hours were taken 6 hours after injection of test liposome Formulations 1 and 2 on the same day as the background blood test reading taken at time 0. (This is different from the method of obtaining data shown in FIGs. 7A, 7B and 7C, where the background reading indicated as time in each graph was obtained the day before administration of the test compounds, and the next data point in each curve was obtained 6 hours after administration of the test compound on the day after the background reading was obtained.)
- CISO 3 H 13 mL was stirred at 120 °C overnight.
- the reaction mixture was cooled to r.t. and poured into ice/water.
- the resultant was extracted with EA (50 mL x 3).
- the organic layer was washed with water (40 mL x 2) and brine (40 mL), dried over Na 2 SC>4, filtered and concentrated to afford 2.5 g of white solid, which was redissolved in isopropanol (70 mL).
- the solution was cooled to -60 °C.
- Ammonium hydroxide (1 1 mL) was added dropwise. After stirring at -60 °C for 1 hour, HCl (6 M, 8 mL) was added to quench the reaction.
- Compound 9 and Compound 10 can be synthesized by steps similar to the other compounds disclosed herein.
- intermediate 28 (1.2 g, 2.2 mmol) in DCM (30 mL) was added intermediate 18 (1.1 g, 2.7 mmol), DMAP (0.8 g, 6.6 mmol) and EDCI (0.9 g, 4.4 mmol). The mixture was stirred at r.t. overnight. DCM (150 mL) was added and the organic layer was washed with water (30 mL x 3) and brine (30 mL), and then concentrated to dryness. The residue was purified by prep-HPLC to afford 1.1 g of Compound 10 as a white solid.
Abstract
Description
Claims
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EP17738848.5A EP3402485A1 (en) | 2016-01-11 | 2017-01-11 | Inhibiting b-cell lymphoma 2 (bcl-2) and related proteins |
JP2018554661A JP2019501225A (en) | 2016-01-11 | 2017-01-11 | Inhibition of B cell lymphoma 2 (BCL-2) and related proteins |
AU2017206731A AU2017206731A1 (en) | 2016-01-11 | 2017-01-11 | Inhibiting B-cell lymphoma 2 (Bcl-2) and related proteins |
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Cited By (6)
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CN110143941A (en) * | 2019-06-04 | 2019-08-20 | 北京四环制药有限公司 | A kind of synthetic method of Barrow Sa Weimabo ester intermediate |
WO2021007303A1 (en) * | 2019-07-10 | 2021-01-14 | Recurium Ip Holdings, Llc | Nanoparticle formulation of bcl-2 inhibitor |
WO2021119439A1 (en) * | 2019-12-11 | 2021-06-17 | The Regents Of The University Of Michigan | Compositions and methods for systemic delivery of bcl-2 and bcl-xl antagonists |
US11318134B2 (en) | 2018-01-10 | 2022-05-03 | Recurium Ip Holdings, Llc | Benzamide compounds |
WO2023288100A1 (en) * | 2021-07-16 | 2023-01-19 | Celator Pharmaceuticals, Inc. | Liposomal formulations of bcl inhibitors |
WO2024012557A1 (en) * | 2022-07-15 | 2024-01-18 | Berrybio (Hong Kong) Limited | Anti-apoptotic bcl-2 family protein degraders, pharmaceutical compositions, and therapeutic applications |
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2017
- 2017-01-11 AU AU2017206731A patent/AU2017206731A1/en not_active Abandoned
- 2017-01-11 WO PCT/US2017/012992 patent/WO2017123616A1/en active Application Filing
- 2017-01-11 JP JP2018554661A patent/JP2019501225A/en active Pending
- 2017-01-11 MA MA043871A patent/MA43871A/en unknown
- 2017-01-11 EP EP17738848.5A patent/EP3402485A1/en not_active Withdrawn
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US5043164A (en) * | 1989-01-17 | 1991-08-27 | The University Of Tennessee Research Corporation | Blood-stable, cholesterol-free liposomes |
US20150182460A1 (en) * | 2004-05-03 | 2015-07-02 | Merrimack Pharmaceuticals, Inc. | Liposomes useful for drug delivery |
US20070027135A1 (en) * | 2005-05-12 | 2007-02-01 | Milan Bruncko | Apoptosis promoters |
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Cited By (12)
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US11318134B2 (en) | 2018-01-10 | 2022-05-03 | Recurium Ip Holdings, Llc | Benzamide compounds |
US11344546B2 (en) | 2018-01-10 | 2022-05-31 | Recurium IP Holding, LLC | Benzamide compounds |
US11590126B2 (en) | 2018-01-10 | 2023-02-28 | Recurium Ip Holdings, Llc | Benzamide compounds |
US11813259B2 (en) | 2018-01-10 | 2023-11-14 | Recurium Ip Holdings, Llc | Benzamide compounds |
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CN110143941A (en) * | 2019-06-04 | 2019-08-20 | 北京四环制药有限公司 | A kind of synthetic method of Barrow Sa Weimabo ester intermediate |
CN110143941B (en) * | 2019-06-04 | 2021-05-25 | 北京四环制药有限公司 | Synthesis method of intermediate of balusavir mefene |
WO2021007303A1 (en) * | 2019-07-10 | 2021-01-14 | Recurium Ip Holdings, Llc | Nanoparticle formulation of bcl-2 inhibitor |
EP3972601A4 (en) * | 2019-07-10 | 2023-07-12 | Recurium IP Holdings, LLC | Nanoparticle formulation of bcl-2 inhibitor |
WO2021119439A1 (en) * | 2019-12-11 | 2021-06-17 | The Regents Of The University Of Michigan | Compositions and methods for systemic delivery of bcl-2 and bcl-xl antagonists |
WO2023288100A1 (en) * | 2021-07-16 | 2023-01-19 | Celator Pharmaceuticals, Inc. | Liposomal formulations of bcl inhibitors |
WO2024012557A1 (en) * | 2022-07-15 | 2024-01-18 | Berrybio (Hong Kong) Limited | Anti-apoptotic bcl-2 family protein degraders, pharmaceutical compositions, and therapeutic applications |
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