EP1998760A2 - Compositions outils oncologiques et procédés d'utilisation pour détecter et traiter des tumeurs - Google Patents

Compositions outils oncologiques et procédés d'utilisation pour détecter et traiter des tumeurs

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Publication number
EP1998760A2
EP1998760A2 EP07754699A EP07754699A EP1998760A2 EP 1998760 A2 EP1998760 A2 EP 1998760A2 EP 07754699 A EP07754699 A EP 07754699A EP 07754699 A EP07754699 A EP 07754699A EP 1998760 A2 EP1998760 A2 EP 1998760A2
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EP
European Patent Office
Prior art keywords
onco
tool
subject
composition
radioisotope
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP07754699A
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German (de)
English (en)
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James E. Summerton
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Individual
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Individual
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Priority claimed from US11/395,487 external-priority patent/US20070231256A1/en
Priority claimed from US11/449,495 external-priority patent/US8084610B2/en
Priority claimed from US11/449,508 external-priority patent/US20080124274A1/en
Application filed by Individual filed Critical Individual
Publication of EP1998760A2 publication Critical patent/EP1998760A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • 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 invention relates to compositions effective to be selectively sequestered in acidic areas of tumors within a subject for the purpose of detecting and treating tumors.
  • Acidity an exploitable near-universal characteristic of tumors:
  • tumor cells at near-normal pH in close proximity to capillaries have high metabolic rates and fast cell division, those tumor cells in more acidic areas at a greater distance from capillaries have lower metabolic rates and divide slowly or not at all.
  • these slow-dividing and non-dividing tumor cells referred to as quiescent, are substantially more resistant to cell-damaging agents, such as radiation and toxic chemicals.
  • hypoxic/acidic properties of tumors have been known for over 75 years and it has long been speculated that such properties might be exploitable for therapy. However, until recently the most successful efforts to exploit these properties have focused on the hypoxia. Specifically, substances have been developed which exhibit minimal cytotoxicity in normoxic cells, while exhibiting considerable cytotoxicity in hypoxic cells. One such agent has progressed to the clinical trials stage.
  • Another approach relating to acidity in tumors is based on the fact that the low pH in tumors ionizes weak-base cytotoxic agents and thereby renders them membrane-impermeable, which in turn results in preferential reduction of entry of a number of such weak-base agents into acidic areas of tumors relative to entry of such agents into cells in areas of more normal pH.
  • efforts were instead focused on raising the pH in the tumors as a means to partially de-ionize and thereby enhance the entry of such weak-base cytotoxic agents into cells of the tumor. Such efforts have met with some success.
  • Still another approach that relates more closely to the present invention relied on the fact that the low pH in the interstitial space of tumors will cause partial de-ionization of the weak-acid cytotoxic agent, Chlorambucil (shown in Figure 1).
  • Chlorambucil shown in Figure 1.
  • this well known weak-acid cytotoxic drug in acidic areas of tumors, one would expect that it should show enhanced entry and hence greater cytotoxicity in cells in acidic areas of tumors.
  • This expectation has been tested by Kozin et al., wherein they used established methods to make tumors In tumor-bearing mice more acidic (Cancer Research Vol. 61 , pages 4740 - 4743 (2001)).
  • the object of the present invention is to provide non-peptide compositions and methods for detecting and treating tumors containing acidic areas.
  • Onco-tools of the present invention are a novel class of molecules designed for detecting and treating tumors.
  • Each onco-tool contains two or more pH-switch components which readily convert between an anionic hydrophiiicform at a higher pH and a non-ionic lipophilic form at a tower pH.
  • Each onco-tool also contains a cargo component which is effective to bind a selected radioisotope which is suitable for carrying out the diagnostic or therapeutic role of the onco- tool.
  • Onco-tools consist of relatively small non-peptide synthetic molecules with masses typically in the range of about 300 to 1500 daltons - not counting the mass of the radioisotope.
  • onco-tools exist in their water-soluble negatively-charged form which is designed to be repelled from the negatively- charged surfaces of cells and to be readily excreted by the kidneys.
  • onco-tools enter an acidic region of a tumor, a portion of the onco-tool molecules switch to their non-ionic lipid-soluble form which is designed to rapidly enter any nearby cell.
  • a dose of onco-tool is injected into a person, if that person has a tumor larger than approximately 1 millimeter in diameter, a portion of the injected dose is designed to rapidly enter and remain within cells in acidic regions of the tumor, with the remainder of the dose being excreted by the kidneys.
  • onco-tools are designed to have the key properties of: a) being repelled from cells in normal tissues; b) being sequestered within acidic areas of tumors; and, c) any onco-tool not sequestered in an acidic area of a tumor is designed to be rapidly cleared from the body via the kidneys.
  • Radioisotope Prior to use of an onco-tool a selected radioisotope is linked to that onco-tool. That attached radioisotope carries out the onco-tool's diagnostic or therapeutic role.
  • the diagnostic role is to report the onco-tool's presence within a tumor to a detector outside the body.
  • the therapeutic role is to destroy the tumor.
  • a diagnostic onco-tool is designed to be broadly used in routine annual physical exams to provide very early detection of essentially all tumors larger than microscopic size. Accordingly, a single diagnostic onco-tool may serve as a superior and comprehensive replacement for a wide range of current tumor diagnostic procedures, such as breast mammograms, pap smears, prostate exams, and colonoscopies. Furthermore, it should be appreciated that many tumor types currently cannot be routinely detected until they reach a quite late stage of development wherein they manifest themselves by pain, malfunction of an organ, or some other generic symptom or set of symptoms, which then require exhaustive and very expensive diagnostic procedures in order to identify the root cause of the problem. By the time such a tumor is detected it is often too advanced for any hope of successful treatment (at least with currently approved therapies). In this regard, diagnostic onco-tools offer the promise of an entirely new capability for routine early-stage detection of such difficult-to-diagnose tumors.
  • Therapeutic onco-tools are designed to exploit the acidity characteristic of tumors to provide safe and effective destruction of tumors.
  • One formulation carries a radioisotope which emits short-path-length high linear energy transfer radiation, such as a very-high-energy alpha particle, that is effective to kill the close-by radiation-resistant quiescent cells in acidic areas of the tumor (see Figure 2).
  • the other formulation carries a radioisotope that emits a medium-path- length medium-energy beta particle effective for killing the more-distant radiation- sensitive fast-dividing tumor cells near capillaries.
  • these two formulations are designed to effectively kill the entire tumor, with little or no damage to other cells in the body.
  • therapeutic onco- tools are designed to be effective against all cell types in most or ail tumors, and so offer the promise of far less toxic and far more effective treatment of tumors, with few or no relapses.
  • Figure 1 shows the weak-acid cytotoxic chemotherapeutic, chlorambucil, in both its anionic and its non-ionic forms.
  • Figure 2 illustrates the distribution of acidity in tumors.
  • FIG. 3 illustrates the pH-mediated and solubility-mediated transitions between forms.
  • Figure 4 shows calculated titration curves as a function of lipophilicity of the n ⁇ n-ionic acid form.
  • Figure 5a illustrates expected waters of hydration, showing water molecules H-bonded to a conventional carboxylic acid in its anionic and non-ionic forms.
  • Figure 5b illustrates expected waters of hydration, showing water molecules H-bonded to a carboxylic acid-containing structure in its anionic form and in its internally H-bonded non-ionic form.
  • Figure 6a illustrates an acid-specific H-bond in a structure which forms an internal acid-specific H-bond only in its non-ionic form.
  • Figure 6b illustrates a structure which forms non-acid-specific H-bonds in both its anionic form and in its non-ionic form.
  • Figure 7a shows representative ring structures suitable for advanced pH- switches, including a 4-membered ring structure, a 5-membered ring structure, and a 6-membered ring structure.
  • Figure 7b shows a structure which is unacceptable for use as an advanced pH-switch because of it has an acyclic structure.
  • Figure 8a illustrates the insulation of carboxyl moieties from inductive effects due to an increasing number of carbons separating the carboxyl from an electron-withdrawing group.
  • Figure 8b illustrates a structure having inadequate insulation of its carboxyl from inductive effects.
  • Figure 9a illustrates a structure whose H-bond site is open to solvent.
  • Figure 9b illustrates a structure whose H-bond site which is partially shielded from solvent.
  • Figure 10 shows components of low-barrier H-bonds.
  • Figure 10a shows an H-bond donor, comprising a carboxylic acid, which is suitable for forming low-barrier H-bonds with H-bond acceptor moieties having a pKa value, in the stand alone form, in the range of approximately 3,0 to 6.5.
  • Figure 10b shows representative H-bond acceptor moieties suitable for forming low-barrier H-bonds with a carboxylic acid donor.
  • Figure 10c illustrates representative advanced pH-switches designed to form internal acid-specific low-barrier H-bonds.
  • Figure 11 illustrates the statistical basis for increased specificity of multi- pH-switch onco-tools.
  • Figure 11a illustrates non-sequestered anionic and tumor-sequestered non-ionic forms of an onco-tool with two pH-switches.
  • Figure 11b illustrates non-sequestered anionic and tumor-sequestered non-ionic forms of an onco-tool with three pH-switches.
  • Figure 12 shows calculated efficacy and specificity factors for multi-pH- switch onco-tools.
  • Figure 12a shows calculated efficacy and specificity factors as a function of pKa value for structures containing one pH-switch.
  • Figure 12b shows calculated efficacy and specificity factors as a function of pKa value for onco-tools containing two pH-switches.
  • Figure 12c shows calculated efficacy and specificity factors as a function of pKa value for onco-tools containing three pH-switches.
  • Figure 12d shows calculated efficacy and specificity factors as a function of pKa value for onco-tools containing four pH-switches.
  • Figure 13 shows a composition containing two advanced pH-switches.
  • Figure 13a shows the structure of this composition.
  • Figure 13b shows a plot of the n-Octanol/buffer partitioning of this 2-pH- switch composition as a function of pH.
  • Figure 14 illustrates representative cargo components in both their precursor and final forms.
  • Figure 15 illustrates two synthetic routes for representative cargo components, including a simple procedure for converting selected precursor forms to their final forms.
  • Figure 16 illustrates representative 2-pH-switch onco-tools.
  • Figure 16a illustrates two representative 2-pH-switch onco-tools containing simple pH-switches.
  • Figure 16b illustrates five representative 2-pH-switch onco-tools containing advanced pH-switches.
  • Figure 16c illustrates ten representative 2-pH-switch onco-tools containing advanced pH-switches designed to form low-barrier H-bonds. Structures 1 and 2 of Figure 16c show two onco-tools wherein the H-bond acceptor moiety of the pH-switches are N-oxide moieties, and wherein each N-oxide moiety serves as the H-bond acceptor for two carboxylic acid H-bond donor moieties.
  • Figure 17 illustrates representative 3-pH-switch onco-tools.
  • Figure 17a illustrates two representative 3-pH-switch onco-tools containing advanced pH-switches.
  • Figure 17b illustrates two representative 3-pH-switch onco-tools containing advanced pH-switches designed to form low-barrier H-bonds.
  • Figure 18 illustrates representative 4-pH-swHch onco-tools.
  • Figure 18a illustrates a 4-pH-switch onco-tool where the H-bond acceptor moieties of the pH-switches are alkoxy amine moieties.
  • Figure 18b illustrates a 4-pH-sw ' rtch onco-tool where the H-bond acceptor moieties of the pH-switches are N-oxide moieties, and wherein each N-oxide moiety serves as the H-bond acceptor for two carboxylic acid H-bond donor moieties.
  • Figure 19 illustrates synthesis of onco-tools containing simple pH- switches, wherein lipophilicity can be adjusted by varying the R group.
  • Figure 20 illustrates a synthetic scheme for advanced pH-switches, wherein lipophilicity can be adjusted by varying the R group.
  • Figure 21 illustrates useful amine-ester and ketone-ester intermediates for onco-tool synthesis.
  • Figure 22 illustrates representative syntheses of pH-switches designed to form an internal acid-specific low-barrier H-bond.
  • Figure 22a shows the synthesis of a pH-switch wherein the H-bond acceptor is a cyanomethyl amine moiety.
  • Figure 22b shows the synthesis of a pH-switch wherein the H-bond acceptor is an N-oxide moiety.
  • Figure 22c shows the synthesis of a pH-switch wherein the H-bond acceptor is a trifluoroethyl amine moiety.
  • Figure 23 shows titration results for a variety of pH-switch structures.
  • Figure 23a shows a conventional titration curve, where the titration was carried out in Methanol/Water, 1:1 by vol., for a simple carboxylic acid (Butyric acid) and an advanced pH-switch (an acid-amide derivative of Camphoric acid), plus that same data plotted in the more informative first derivative form.
  • Figure 23b shows a conventional titration curve, where the titration was carried out in water and each specie was present at 33 milliMolar concentration, for two-pH-switch onco-tools (but with stable Iodine instead of radioactive Iodine) varying in their R group, and a related three-pH-switch structure.
  • Figure 23c shows a titration curve, plotted as the first derivative, for an advanced pH-switch designed to form a low-barrier H-bond. This titration was carried out in water and the pH-switch, comprising an N-oxide/acid structure derived from Camphoric acid, was present at a 5 milliMolar concentration.
  • Figure 24 shows experimentally-determined pKa values for three pH- switch structures, including: the amide/acid advanced pH-swrtch derived from Camphoric acid, shown in Figure 23a; an N-oxide/acid advanced pH-switch designed to form a low-barrier H-bond, derived from lsonipocotic acid; and, the N-oxide/acid advanced pH-switch designed to form a low-barrier H-bond, derived from Camphoric acid, shown in Figure 23c.
  • Figure 25 illustrates synthetic schemes for representative onco-tools containing advanced pH-switches.
  • Figure 25a shows an onco-tool wherein its two pH-swrtches are joined by a di-acylhydrazide structure.
  • Figure 25b shows an onco-tool wherein its two pH-switches are joined by a di-amide structure.
  • Figure 26 illustrates synthetic schemes for representative onco-tools containing advanced pH-switches designed to form a low-barrier H-bond.
  • Figure 26a shows a synthetic scheme for a 2-pH-switch onco-tool wherein a single N-oxide moiety serves as the H-bond receptor moiety for two carboxylic acid H-bond donor moieties.
  • Figure 26b shows a synthetic scheme for a 2-pH-switch onco-tool wherein both of the nitrogens of a hydrazine moiety serve as the H-bond receptor moieties for two carboxylic acid H-bond donor moieties.
  • Figure 26c shows a synthetic scheme for a 2-pH-switch onco-tool wherein cyanomethyl amine moieties serve as the H-bond acceptor moieties for the carboxylic acid H-bond donor moieties.
  • Figure 26d shows a synthetic scheme for a 4-pH-switch onco-tool wherein each N-oxide moiety serves as the H-bond receptor moiety for two carboxylic acid H-bond donor moieties.
  • pH-switch component a structural component of an onco-tool which is capable of undergoing a pH-mediated transition between an anionic hydrophilic form at a higher pH and a non-ionic lipophilic form at a lower pH.
  • An advanced pH-switch has the following properties: a) contains an aliphatic ring structure selected from the group consisting of: 4-membered rings, 5-membered rings, and 6-membered rings; b) contains a carboxylic acid moiety directly linked to the aliphatic ring structure; c) the carboxylic acid moiety is separated from any linked electron- withdrawing group by at least two carbons; d) contains an H-bond acceptor moiety selected from the group consisting of: i) part of the aliphatic ring structure; ii) directly linked to the aliphatic ring structure; and iii) linked through one atom to the aliphatic ring structure; e) the H-bond acceptor moiety has a structure which in its non-ionic form does not serve as an H-bond donor moiety; and f) the carboxylic acid moiety and said H-bond acceptor
  • Cargo component a structural component of an onco-tool which serves to bind a radioisotope that is effective to report the presence of the onco-tool, or is effective to kill cells.
  • the cargo component can exist in either a precursor form ready to bind a radioisotope or a final form which contains a radioisotope.
  • Cargo component in precursor form a cargo component which has a structure that is capable of readily binding to a selected radioisotope, but has not yet bound a radioisotope.
  • An onco-tool with this precursor form of the cargo component is suitable for long-term storage.
  • Cargo component in final form a cargo component that contains at least one bound radioisotope.
  • An onco-tool with this final form of the cargo component is suitable for delivery into a subject for the purpose of detecting and/or killing tumors containing acidic areas.
  • Onco-tool - a non-peptide composition that includes at least two pH-switch components and at least one cargo component.
  • Diagnostic onco-tool - an onco-tool which contains a radioisotope that is effective to report the presence of the onco-tool within a tumor to a detector outside the subject undergoing the diagnostic procedure.
  • Therapeutic onco-tool - an onco-tool which contains a radioisotope that is effective to kill cells.
  • Pual-radioisotope onco-tool therapy strategy - a treatment strategy where at least two different onoo-tools are used to treat tumors, where one onco-tool contains a radioisotope which emits high linear transfer energy radiation, and another onco-tool contains a beta-emitting radioisotope.
  • Interstitial space the area of a tissue or of a tumor which is outside of the vascular bed and outside of the cells.
  • Acidic area of tumor - an area of a tumor where the interstitial space has a pH of less than 7.0.
  • Physiological conditions an aqueous solution with a temperature in the range of 20 deg. C to 40 deg. C and having a sodium chloride concentration between about 0.13 M and 0.17 M.
  • Anionic hvdrophilic form a form which carries at least one negative charge and has an octanol/water partitioning coefficient of less than 1.
  • Non-ionic lipophilic form a form which does not carry an ionic charge and has an octanol/water partitioning coefficient of greater than 1.
  • Specificity factor the ratio: (percent of onco-tool molecules in non-ionic form at pH 6.4) divided by (percent of onco-tool molecules in non-ionic form at pH 7.4).
  • Radioisotope whose emission, such as a gamma ray or positron, generates a signal which is readily detectable outside that subject.
  • the pT value resembles the pKa value for a substance, the pT value differs in that it is influenced both by the pKa of the substance and the lipophilicity of the substance.
  • the pT value is useful for describing carboxyl-containing substances whose non-ionic form is strongly lipophilic and so can drop out of aqueous solution during titrations, or can partition into a non-polar phase during partitioning assays. Further, the pT value is measured in the presence of approximately a physiological concentration of saline so it better reflects properties of the test substance in an in vivo environment.
  • Radioisotope which emits high linear energy transfer radiation a radioisotope which emits an alpha particle or which emits an Augar and/or Coster-Kronig electron.
  • pH-Switch Components a) Adjust lipophilicity of pH-switch b) Incorporate a novel acid-specific internal H-bond in pH-switch c) Incorporate multiple pH-switch components in onco-tool 2.
  • Cargo Component a) Structural requirements b) Precursor and final forms c) Selection of radioisotope cargo i) for detecting tumors ii) for treating tumors
  • Onco-Tool Structures a) Structural requirements b) Onco-tools with 2 pH-switches c) Onco-tools with 3 pH-switches d) Onco-tools with 4 pH-switches
  • Onco-tools comprising the present invention are a novel class of molecules designed for detection and treatment of tumors.
  • Each onco-tool contains two or more pH-switch components which readily undergo a pH- mediated transition between an anionic hydrophilic form at higher pH and a non- ionic lipophilic form at lower pH.
  • Each onco-tool also contains a cargo component which is effective to bind a selected radioisotope that is suitable for carrying out the diagnostic or therapeutic role of the onco-tool.
  • an onco-tool is to achieve adequate specificity for acidic areas of tumors it must have a structure such that in aqueous solution at pH 7.4 nearly all of the onco-tool molecules exist in a negatively-charged form (anionic) which lacks affinity for the negatively-charged surfaces of cell membranes.
  • a o significant portion (greater than 1%) of the onco-tool molecules should switch to a non-ionic lipophilic form that readily enters cell membranes.
  • a principal challenge in designing onco-tools is to devise a structure wherein a sufficient portion of the onco-tool molecules undergo the transition between the anionic hydrophilic form and the non-ionic lipophilic form within the available very limited pH difference 5 between normal tissues and acidic areas of tumors. While this pH difference is commonly only about 0.5 to 0.7 pH unit, it should be appreciated that simple steps (described in Section C later herein) can be easily implemented to substantially increase this pH difference between normal tissues and acidic areas of tumors. With such steps the pH difference between normal and acidic areas of 0 tumors can typically be increased to 1.0 pH unit or greater.
  • pH-switch structures have been devised wherein each contains a weak acid moiety (generally a carboxylic acid) that carries a negative charge at pH 7.4. Then, because this essential weak-acid moiety still s exhibits substantial hydrophilic character in its non-ionic free-acid form, in order to achieve adequate efficacy two molecular design strategies have been utilized in order to provide sufficient lipophilicity for rapid membrane entry when the onco- tool enters an acidic area in a tumor .
  • a weak acid moiety generally a carboxylic acid
  • inco ⁇ orating excessive lipophilic moieties as the sole means for increasing the lipophilicity of the non-ionic form can be counter productive. This is because when a very high lipophilicity is generated solely by inco ⁇ oration of an excessive number of lipophilic moieties then even the anionic form of the pH-switch can begin to have an affinity for cell surfaces - resulting in sequestering of the onco-tool in normal tissues as well as in tumors. This can be a particular problem when the lipophilic moieties form one large surface and the hydrophilic moieties are well separated from this lipophilic surface. When the onco-tool is also sequestered in normal tissues it will cause high background signal in the diagnostic application, and significant toxicity in the therapeutic application.
  • E ⁇ Incorporate a novel acid-specific internal H-bond in pH-switch to amplify the solubility differential between the hydrophilic and lipophilic forms.
  • both the hydrophilicity of the anionic form and the lipophilicity of the non-ionic form of the pH-swifches could be substantially increased by devising a structure effective to form a pH-dependent internal acid-specific hydrogen bond.
  • the inventor further postulated that such an internal H- bond would raise the pH at which the pH-switch transitioned between its anionic form and its non-ionic form, thereby making more of the onco-tool available in the non-ionic form for entry into cells in acidic areas of tumors.
  • That predicted H-bond-mediated increase in lipophilicity of the non-ionic form and concomitant increase in hydrophilicity of the anionic form is based on the expectation that in an aqueous environment such an internal H-bond will displace several (probably two) bound waters from the free-acid form.
  • Figure 5a shows the expected waters of hydration directly H-bonded to a standard carboxyl in its anionic form and in its non-ionic free-acid form.
  • Figure 5b shows corresponding expected waters of hydration directly H-bonded to the anionic form and to the non-ionic free-acid form of a structure effective to form the desired internal acid-specific H-bond.
  • both the standard carboxyl and the carboxyl effective to form an internal H-bond lose their counter-ion in going from the anionic form to the non-ionic free-acid form.
  • the standard carboxyl is expected to have the same number (probably 4) of waters directly H-bonded to both the anionic form and to the non-ionic free-acid form
  • the inventor believes that for a carboxyl able to form an internal H-bond there will be a net loss of several (probably two) directly-H-bonded waters when going from the anionic form (probably 5 H-bonded waters) to the non-ionic free-acid form (probably 3 H-bonded wafers) - and this loss of several H-bonded waters will substantially amplify the hydrophilicity/lipophilicfty differential between the two forms.
  • the inventor predicted that if such an internal acid-specific H-bond could be formed in an aqueous environment, then the resulting loss of several waters of hydration would significantly increase an onco-tooPs ability to rapidly enter cells in acidic areas of tumors, while also significantly increasing the onco-tool's repulsion from cells in normal tissues.
  • incorporation of such an internal acid-specific H-bond was predicted to increase both the specificity and the efficacy of an onco-tool by differentially altering the number of water molecules bound to each of the two forms of its component pH-switches.
  • the inventor also predicted (and has now demonstrated) that formation of an internal acid-specific H-bond in the pH-switch component should favor the transition of the carboxyl moiety of a pH-switch component to its free-acid form, resulting in a significant increase in the pKa value for that carboxylic acid moiety.
  • an increase in the pKa of the carboxyl moiety is desirable in the context of a strategy for dramatically increasing the onco-tool's specificity for tumors by incorporating two or more pH-switches in that onco-tool.
  • a pH-Switch which has a structure designed to form an internal acid-specific H-bond is called an "advanced pH-switch”.
  • the structure must contain a carboxyl moiety which is positioned in suitable proximity to an H-bond acceptor moiety for formation of an H-bond.
  • carboxyl moiety When that carboxyl moiety is in its free-acid form it must serve as the H-bond donor, and the proximal H-bond acceptor moiety must be such that in its non- ionic form it can only serve as an H-bond acceptor, and cannot serve as an H- bond donor.
  • the inventor refers to an H-bond formed by such a structure as an "internal acid-specific H-bond".
  • Figure 6a shows a representative pH-switch structure which can form only an internal acid-specific H-bond.
  • Figure 6b illustrates a similar, but unacceptable structure which can form both an internal H-bond when the carboxyl is in its anionic form and an internal H-bond when the carboxyl is in its free-acid form.
  • the inventor calls this dual H-bonding capability "internal non-acid-specific H-bonding," and his experimental results indicate that such non-acid-specific H-bondi ⁇ g is unacceptable because it fails to provide the desired increase in the lipophilicity of the acid form, and it fails to raise the pH at which the structure switches from its anionic hydrophilic form to its non-ionic lipophilic form.
  • Example 2 describes experimental results wherein in a 1 to 1 mixture of water and methanol the structure in Figure 6a forms an internal acid-specific H-bond under mildly-acidic conditions - evidenced by a significant increase in the pH of the transition between anionic and free-acid forms (that is, an increase in the pKa value for the carboxyl moiety).
  • this same example also shows that the very similar structure in Figure 6b, designed to form non-acid-specific H-bonds, fails to increase the pH of the transition between anionic and non-ionic forms under these conditions.
  • H-bond acceptor moiety and the carboxyl serving as the H-bond donor moiety should be held in close proximity to each other by a structure which has minimal conformational freedom. This limited conformational freedom can be achieved by using a suitable ring structure.
  • Molecular modeling and experimental work suggests that 4-membered, 5-membered, and 6-membered aliphatic rings are preferred for this purpose.
  • Figure 7a shows representative 4- membered, 5-membered and 6-membered aliphatic ring structures which allow only limited conformational freedom between the H-bond donor (OH of the carboxylic acid) and suitably-positioned H-bond acceptor moieties.
  • Figure 7b shows a somewhat similar structure which is unacceptable because its acyclic structure allows excessive conformational freedom between the H-bond donor and H-bond acceptor moieties.
  • Example 3 describes experimental results showing that in a 1:1 mix of methanol and water a structure with minimal conformational freedom between H-bonding moieties can afford a significant increase in the pKa value of the carboxyl moiety, while a similar structure with substantially greater rotational freedom between H-bonding moieties fails to provide a corresponding increase in the pKa value of the carboxyl moiety - and so is presumably failing to form a stable internal acid-specific H-bond under these conditions.
  • ⁇ i ⁇ Carboxyl insulated from inductive effects of electron- withdrawin ⁇ groups
  • the carboxylic acid which is to serve as the H-bond donor moiety should be separated from any linked electron-withdrawing group by at least two, and preferably three or more carbons. This avoids any excessive reduction in the pKa value of that carboxyl due to inductive effects from electron-withdrawing groups.
  • the molecular structures and their corresponding pKa values shown in Figure 8a illustrate how an increasing number of aliphatic carbons can progressively insulate a carbo ⁇ yl from the pKa-reducing effects of a phenyl group.
  • Figure 8b shows a structure wherein inductive effects from the proximal amide moiety apparently causes an undue reduction in the pKa of the carboxylic acid moiety.
  • Example 4 describes experimental results suggesting that even with minimal conformational freedom between H-bond donor and acceptor moieties, if there is inadequate insulation of the carboxyl from inductive effects then the pH of the transition between anionic and free-acid forms will be too low to be useful in a pH-switch.
  • At least one additional property selected from the following two properties, is desirable to achieve formation of an internal H-bond in aqueous solution.
  • low-barrier H-bond is used herein to mean a non-covalent bond formed between an H-bond donor moiety and an H-bond acceptor moiety, where the pKa values of the two isolated moieties are within about 2 pH units of each other. It should be noted that this definition includes what can also be construed as an internal salt wherein the hydrogen is closer to the acceptor moiety than to the donor moiety. Such low-barrier H-bonds are commonly found to be exceptionally strong and so can appreciably favor the desired intramolecular H- bond in pH-switches.
  • Figures 10a and 10b show a variety of H-bonding moieties which are appropriate for forming low-barrier H-bonds in pH-switches.
  • Figure 10c illustrates several representative pH-switch structures designed to form H-bonds of the low-barrier type.
  • Example 6 describes the syntheses and testing of two representative pH-switches (structures i and iii in Figure 10b), along with experimental evidence for formation of the desired low-barrier H-bonds in aqueous solution.
  • a) contains an aliphatic ring structure selected from the group consisting of: 4-membered rings, 5-membered rings, and 6-membered rings; b) contains a carboxylic acid moiety directly linked to the aliphatic ring structure; c) the carboxylic acid moiety is separated from any linked electron- withdrawing group by at least two carbons; d) contains an H-bond acceptor moiety selected from the group consisting of: i) part of the aliphatic ring structure; ii) directly linked to the aliphatic ring structure; and iii) linked through one atom to the aliphatic ring structure; e) the H-bond acceptor moiety has a structure which in its non-ionic form does not serve as an H-bond donor moiety; and f) the carboxylic acid moiety and the H-bond acceptor moiety are
  • the pKa of an acid is the pH of an aqueous solution of that acid wherein half of the acid molecules are anionic and half are non-ionic. If one knows the pKa of an acid, and the pH of its aqueous solution, then the Henderson-
  • the inventor has devised a novel design strategy to dramatically increase the specificity of onco-tools over the apparent theoretical maximum of 10 which might appear to be imposed by the small pH differential between normal and tumor tissues.
  • the strategy is to incorporate two or more pH-switches into a single onco-tool, where the carboxyl of each pH-switch is sufficiently distant (probably greater than about 5 angstroms apart) from the carboxyl of its neighboring pH-switch that ionization of one carboxyl does not significantly affect the ionization of its neighboring carboxyl.
  • all of the pH-switches of the multi-pH-switch onco-tool should be in sufficiently close proximity within the onco-tool molecule that all of the component pH-switches must be in their non-ionic form before that onco-tool can closely approach and then breach the negatively-charged outer surface of a cell membrane in order to enter its lipophilic interior, as is required for that onco- tool to be sequestered in acidic areas of tumors.
  • Bt is estimated that alt carboxyls of a multi-pH-switch onco-tool should be no more than approximately 15 to 20 angstroms from each other in order to adequately satisfy this latter requirement.
  • Figure 11a illustrates in an abstract form the various anionic and non-ionic forms of an onco-tool containing two pH-switch components
  • Figure 11b illustrates likewise the various anionic and non-ionic forms of an onco-tool containing three pH-switch components. It should be appreciated that it is the concentration of the non-ionic form which is of particular interest because, when properly designed, it should be only this non-ionic form of the onco-tool which is capable of efficiently contacting and entering cell membranes - with all the other forms containing at least one anionic moiety being repelled from the anionic cell surfaces by electrostatic forces.
  • the efficacy factor is the percent of the onco-tool molecules which are in their non-ionic form at pH 6.4. This factor provides a measure relating to how efficiently the onco-tool will enter cells in acidic areas of tumors.
  • the specificity factor is the ratio: (percent in non-ionic form at pH 6.4) divided by (percent in non-ionic form at pH 7.4).
  • This factor provides a measure relating to the relative rate of entry of the onco-tool into cells in acidic areas of tumors (at pH 6.4) relative to the rate of entry of the onco-tool into cells in normal tissues (at pH 7.4).
  • pH 6.4 acidic areas of tumors
  • pH 7.4 normal tissues
  • Figure 12 shows plots of calculated efficacy and specificity factors for a) compositions with 1 pH-switch, b) onco-tools with 2 pH-switches, c) onco-tools with 3 pH-switches, and d) onco-tools with 4 pH-switches - as a function of the pKa values of their component pH-switches.
  • These calculated efficacy and specificity factors demonstrate that compositions containing a single carboxyl moiety, such as chlorambucil, inherently have rather poor specificities for selective entry into cells in acidic areas of tumors (specificity factor is always less than 10).
  • the specificity factor can be over 50 for onco-tools containing 2 pH-switches, and over 200 for onco-tools containing 3 pH-switches, and over 900 for onco-tools containing 4 pH-switches.
  • incorporation of an internal acid-specific H-bond in a pH-switch provides a suitable means for adjusting the pKa value of the pH-switch to a range appropriate for obtaining such dramatically improved specificities in these multi- pH-switch onco-tools.
  • the cargo component is a structural component of an onco-tool which serves to incorporate a radioisotope whose emission is effective to report the presence of the onco-tool, or is effective to kill cells.
  • the cargo component should satisfy the following three design requirements. i) The cargo component in its precursor form should be effective to readily and efficiently incorporate, with minimal manipulations, its selected radioisotope. ii) The radioisotope that is bound to the cargo component in its final form should remain so bound during the course of the diagnostic procedure or through the course of the therapeutic process wherein emissions from the radioisotopes are killing cells of the tumor.
  • the cargo component in its final form which includes a bound radioisotope, should be sufficiently small and of such a composition that it does not have an undue impact on the pH-dependent hydrophilicity/lipophilicity properties of the onco-tool. Stated differently, if the final form of the cargo component contributes excessive hydrophilicity it can suppress entry of the non- ionic form of the onco-tool into ceils in acidic areas of tumors - thereby reducing efficacy. Conversely, if the final form of the cargo component contributes excessive lipophilicity it can cause undue sequestering in normal tissues - thereby reducing specificity.
  • FIG. 14 illustrates a number of prospective cargo components in both selected precursor forms and in their final radioisotope-confaining forms ready for diagnostic or therapeutic use.
  • Figure 15 also illustrates a simple procedure for converting the precursor forms to their final radioisotope-containing forms, previously described by Zalutsky, page 96 of Chapter 4 titled: Radiohaiogens for Radioimmunotherapy, in the book: Radioimmunotherapy of Cancer, Ed. by Abrams and Fritzberg, Pub. by Marcel Dekker, Inc. (2000).
  • Procedures for preparing other suitable cargo components and incorporating the radioisotope to give the final form are described in: Zalutsky et al., Proc. Nat. Acad. Sci. USA, Vol. 86, Pages 7149 - 7153 (1989); and, Vaidyanathan & Zalutsky, Nature Protocols, Vol. 1, Pages 1655 -1661 (2006).
  • the bound radioisotope of the onco-tool which determines the application of that onco-tool. If the bound radioisotope emits a signal which is readily detectable outside the body then that onco-tool can serve to detect tumors containing acidic areas. Conversely, if the radioisotope has an emission which is effective to kill cells then that onco-tool can serve for the treatment of tumors containing acidic areas.
  • the onco-tool contains a radioisotope, such as lodine-131, which both emits a signal which is readily detectable outside the body (eg., gamma ray) and has an emission which is effective to kill cells (eg., beta particle) then that onco-tool can serve both for diagnosis and treatment of tumors containing acidic areas.
  • a radioisotope such as lodine-131
  • Successful treatment of a tumor faces two challenges.
  • One challenge is to completely kill all the treatment-sensitive fast-dividing tumor cells at near normal pH close to tumor capillaries.
  • the other more forbidding challenge is to completely kill all of the treatment-resistant quiescent tumor cells in acidic areas of the tumor.
  • Therapeutic onco-tools were initially devised solely as a means for killing treatment-resistant quiescent cells in acidic areas of tumors.
  • onco-tools are designed to be sequestered only in acidic areas of tumors, and so it was presumed that such onco-tools would have to be used in conjunction with more conventional cancer therapies, such as radiation or chemotherapy, where the conventional therapies would serve for killing the treatment-sensitive fast-dividing tumor cells in more neutral areas of tumors closer to capillaries - because such areas would be largely devoid of onco-tools.
  • more conventional cancer therapies such as radiation or chemotherapy
  • the conventional therapies would serve for killing the treatment-sensitive fast-dividing tumor cells in more neutral areas of tumors closer to capillaries - because such areas would be largely devoid of onco-tools.
  • More recently the inventor has devised a dual-radioisotope strategy wherein onco-tools alone can be used to destroy the entire tumor, thereby obviating the need for co-treatment with more toxic and less specific conventional cancer therapies.
  • This dual-radioisotope strategy entails using at least two onco- tool formulations effective to kill cells.
  • the onco-tool formulation used to kill the treatment-resistant quiescent tumor cells should contain a radioisotope which emits high linear energy transfer radiation in order to kill all of the radiation-resistant quiescent tumor cells in or near which radioisotope-carrying therapeutic onco tool is positioned.
  • Radioisotope which emits an alpha particle that releases a vast amount of energy over a very short distance (a few cell diameters), such that the released energy is highly effective to kill even cells which are highly resistant to most radiation.
  • the best radioisotope for this purpose appears to be the radiohalogen, Astatine 211.
  • This radioisotope is generated from natural Bismuth 209 in a medium-energy cyclotron equipped with an alpha particle beam.
  • Astatine-211 has a half-life of 7.2 hours and emits two alpha particles with energies of 5.87 and 7.45 million electron volts, which have been shown to devastate cells within the approximately 50 to 80 micron path lengths of the emitted alpha particles (a few cell diameters). Just a few such alpha emissions can kill even the most radiation- resistant quiescent tumor cell.
  • Radioisotopes which emit high linear energy transfer radiation are those which emit Augar and/or Coster-Kronig electrons. Such radioisotopes, if positioned within the cell to be killed, can cause devastating damage to the cell's DNA and so are extremely cytotoxic when localized in close proximity to the cell nucleus. Radioisotopes of this type which are particularly suitable for therapeutic onco-tools include: Bromine-77 (half-life of 57 hours) and lodine-123 (half-life of 13 hours).
  • the onco-tool formulation used to kill the treatment-sensitive fast-dividing tumor cells closer to capillaries should contain one or more radioisotopes which emit particles that release sufficient energy along their path to kill treatment- sensitive fast-dividing tumor cells. More importantly, those emitted particles must have a sufficiently long path length that they are effective against cells up to several hundred microns from where the onco-tool is sequestered - allowing them to kill the treatment-sensitive fast-dividing tumor cells in more neutral areas of the tumor, which can be up to several hundred microns from acidic areas where onco-tools are sequestered. Beta-emitting radioisotopes best satisfy this requirement.
  • radioisotope Half-life While there are a number of beta-emitting radioisotopes which can be used in this dual-isotope therapy strategy, the following radiohalogens have favorable properties for this application, including mean path lengths of greater than 600 microns. Radioisotope Half-life
  • Onco-Tool Structures a) Structural requirements Each, onco-tool must contain two or more pH-switch components. This
  • each onco-tool must have a structure such that at pH 7.4 it exists almost completely in an anionic hydrophilic form, but at pH 6.4 a significant portion (preferably 1% or more) shifts to a non-ionic lipophilic form effective to be sequestered in acidic areas of tumors.
  • Each onco-tool must also contain a cargo component which is effective to bind a radioisotope, or which contains a radioisotope suitable for reporting the presence of the onco-tool and/or suitable for killing cells.
  • Figure 16 illustrates a variety of representative two- pH-switch onco-tools which satisfy the key structural requirements for onco- tools.
  • Figure 16a illustrates onco-tools containing two simple pH-switches.
  • Figure 16b illustrates onco-tools containing two advanced pH-switches.
  • Figure 16c illustrates onco-tools containing two advanced pH-switches designed to form low-barrier H-bonds.
  • FIG. 17 illustrates a variety of representative three-pH-switch onco-tools which satisfy the key structural requirements for onco-tools.
  • Figure 17a illustrates onco-tools containing three advanced pH-switches and
  • Figure 17b illustrates onco-tools containing three advanced pH-switches designed to form low-barrier H-bonds.
  • Onco-tools containing 4 pH-switches are even more complex than the 3- pH-switch onco-tools described above, and are generally still more challenging to synthesize.
  • the 4-pH-switch onco-tools have the potential for even higher specificity factors, potentially ranging from about 400 to about 2500.
  • Figure 18 illustrates two representative onco-tools which satisfy the key structural requirements for onco-tools and contain four advanced pH-switches designed to form a low-barrier H-bond.
  • Figure 19 illustrates a representative synthetic route for onco-tool structures containing simple pH-swrtches, with lipophilicity adjusted by varying the R group.
  • Figure 20 illustrates a synthetic route for structures containing an advanced pH-switch type, with lipophilicity adjusted by varying the R1 group.
  • FIGS. 21 and 22 illustrate synthetic routes for structures containing a number of advanced pH-switch types designed to form a low-barrier H-bond.
  • Figure 21 shows useful amine-ester and ketone-ester intermediates for preparing pH-switches and for preparing onco-tools. Synthetic procedures for making key core components of such intermediates have been reported in the following sources: 1) Goldman, Jacobsen, and Torssell, Synthesis in the Camphor Series. Alkylation of Quinones with Cycloalkyl Radicals. Attempted
  • Figure 22 then illustrates representative synthetic routes for converting several such intermediates to various pH-switches designed to form an internal acid-specific low-barrier H-bond.
  • Simple titration assays provide useful information about the pH-dependent solubility properties of pH-switch structures, as well as their pKa values.
  • Example 7 describes titration procedures which have been used for studying pH- switch components.
  • Figure 23 shows representative results from a variety of past titrations.
  • Figure 23a shows a conventional titration curve, where the titration was carried out in Methanol/Water, 1:1 by vol., for a simple carboxylic acid (Butyric acid) and an advanced pH-switch (an acid-amide derivative of Camphoric acid), In the companion plot the same titration results are plotted in the more informative first derivative form.
  • Figure 23b shows conventional titration curves, where the titration was carried out in water and each specie was present at 33 milliMolar concentration. Titration results are shown for three related tw ⁇ -pH-switch onco-tools (but with stable Iodine instead of radioactive Iodine) which vary only in their R group.
  • Figure 23c shows a titration curve, plotted as the first derivative, for an advanced pH-switch designed to form a low-barrier H-bond. This titration was carried out in water and the pH-switch, comprising an N-oxide/acid structure derived from Camphoric acid, was present at a 5 milliMolar concentration.
  • Figure 24 shows experimentally-determined pKa values for three pH- switch structures, including: an amide/acid advanced pH-switch derived from Camphoric acid; an N-oxide/acid advanced pH-switch designed to form a low- barrier H-bond, derived from lsonipocotic acid; and, an N-oxide/acid advanced pH-switch designed to form a low-barrier H-bond, derived from Camphoric acid (also shown as structure i of Figure 10c, and the structure shown in Figure 23c).
  • Figure 19 illustrates a synthetic route for preparing a representative onco- tool containing simple pH-switches.
  • Figure 25 illustrates synthetic schemes for representative onco-tools containing advanced pH-switches.
  • Figure 25a shows an onco-tool wherein its two pH-switches are joined by a di-acylhydrazide structure.
  • Figure 25b shows an onco-tool wherein its two pH-switches are joined by a di-amide structure.
  • Figure 26 illustrates synthetic schemes for representative onco-tools containing advanced pH-switches designed to form a low-barrier H-bond.
  • Figure 26a shows a synthetic scheme for a 2-pH-switch onco-tool wherein a single N-o ⁇ ide moiety serves as the H-bond acceptor moiety for two carboxylic acid H-bond donor moieties.
  • Figure 26b shows a synthetic scheme for a 2-pH- switch onco-tool wherein both of the nitrogens of a hydrazine moiety serve as the H-bond acceptor moieties for two carboxylic acid H-bond donor moieties.
  • Figure 26c shows a synthetic scheme for a 2-pH-switch onco-tool wherein cyanomethyl amine moieties serve as the H-bond acceptor moieties for the carboxylic acid H- bond donor moieties.
  • Figure 26d shows a synthetic scheme for a 4-pH-switch onco-tool wherein each M-oxide moiety serves as the H-bond acceptor moiety for two carboxylic acid H-bond donor moieties.
  • the resulting 2-pH- switch product comprises 2 isomers, one where both of the pH-switch components are cis with respect to the amine and ester, and the other product where one of the pH-switch components is cis and one of the pH-switch components is trans with respect to the amine and ester moieties.
  • the trans pH-switch component cannot form an internal acid-specific low-barrier H-bond.
  • its pKa will typically be approximately 5.0 in an aqueous solution.
  • the cis pH-switch component generally can form the desired internal acid-specific low-barrier H-bond, which typically raises the pKa of the contained carbo ⁇ yl moiety to about 6.0 or higher.
  • the desired cis/cis form should generally be readily separable from the undesired cis/trans form by partitioning between an organic phase, such as Tetrahydrofuran, t- Butylmethylether, or Dichloromethane, and an aqueous phase buffered at round 6.0.
  • an organic phase such as Tetrahydrofuran, t- Butylmethylether, or Dichloromethane
  • aqueous phase buffered at round 6.0 aqueous phase buffered at round 6.0.
  • the desired cis/cis form should generally preferentially partition into the organic phase, while the undesired cis ⁇ rans form should preferentially partition into the aqueous buffer phase.
  • the same basic separation strategy can also be used to separate the desired all-cis form of the 4-pH-switch onco-tool of Figure 26d from the multiple forms containing at least one trans pH-switch.
  • FIG. 12 The efficacy and specificity plots in Figure 12 provide guidance on desirable pKa values for the pH-switch components as a function of how many pH-switch components the selected onco-tool is to contain.
  • Figure 4 provides guidance as to the impact of changing the partition coefficient of the non-ionic form of the onco-tool - where the partition coefficient is typically adjusted by changing the lipophilicity of the R groups of the pH-swrtch components.
  • synthesis of various prospective pH-switch types and their R-group variants, such as described in Section B.1. above, and subsequent titration studies of those pH-switches, as described in Section B.2. above, provide valuable information for selecting suitable pH-switch components for a prospective onco-tool.
  • Figure 14 illustrates a number of cargo components suitable for incorporation into onco-tools.
  • the optimization process is begun by preparing a set of prospective onco- tool structures incorporating the selected components, wherein onco-tools of the set exhibit a wide range of lipophilicities due to their containing different R1 and R2 groups.
  • Each onco-tool of the set should also contain a suitable radioisotope, such as lodine-131 , which affords easy detection and quantitation in biological systems.
  • a preferred biological system for such initial testing comprises mammalian cells cultured in serum-containing medium buffered at pH 7.4 to emulate normal tissues, and buffered at pH 6.4 to emulate acidic areas of tumors. Briefly, two different wells of cultured cells are exposed for one hour to a given onco-tool. In one culture well the onco-tool is in pH 6.4 medium. Jn the other culture well the onco-tool is in pH 7.4 medium. After incubating 1 hour at 37 deg.
  • the onco-tool-containing medium is removed and the cells are washed thoroughly wrth medium of the same pH, and then radioisotope retained by the cells is counted to provide a measure of the relative quantity of onco-tool which has been sequestered under each of the two pH conditions.
  • the preferred onco-tool structures are those which are maximally sequestered by the cells at pH 6.4, but minimally sequestered by the cells at pH 7.4.
  • the first application of the above two optimization steps should generally serve to identify a reasonably narrow range of onco-tool lipophilicities which give acceptable efficacy (related to amount sequestered at pH 6.4) and specificity (related to the ratio: amount sequestered at pH 6.4 / amount sequestered at pH 7.4) values.
  • Such initial testing may instead serve to indicate that the selected onco-tool components are inadequate and different pH-swrtch components and/or a different cargo component should be used in one's prospective onco-tool structures.
  • the above cell culture assays are relatively fast, simple, provide quantitative results, and are amenable to initial testing of a substantial number of prospective onco-tool structures.
  • this initial cell culture screening system does not perfectly emulate the true complexity of a living subject. Accordingly, it is desirable to next take a reasonable number of the most promising prospective onco-tool structures, identified in the above iterative optimization process, and test them in living mammals.
  • As a first step in such animal testing it is useful to test these promising onco-tool structures in normal mice (pre-treated to assure that their urine is slightly basic; see Section C.7. below), as described in Example 9. Such tests in normal mice allow one to discard any onco-tools which are found to exhibit an excessive affinity for normal tissues.
  • Onco-tools will work best when two ancillary methods are used.
  • One such method entails pre-treating the mice to prevent re-uptake of onco-tool into the cells lining the proximal tubules of the kidneys. This re-uptake is blocked by rendering the urine slightly basic, as described in Section C.7. below.
  • the other method entails pre-treating the mice to further increase the acidity (reduce the pH) in hypoxic/acidic areas of their tumors.
  • Three pre-treatments for this purpose are described later herein in Section C.5. At least one, and preferably a combination of two or three such pre-treatments should be employed in order to adjust the tumor micro-environment so as to be best suited for effective and specific onco-tool activity.
  • Procedures for testing radioisotope-containing substances in live animals, including humans, are well known in the nuclear medicine field, and particularly in the sub-field of radio-immunotherapy. Such known methods can be readily adapted for testing onco-tools by incorporating the methods of using onco-tools described in Section C. below.
  • onco-tools exploit the acidity which is a near-universal characteristic of tumors
  • onco-tools should be effective to detect most or all types of tumors with sizes ranging from near-microscopic to very large.
  • the following method of using onco-tools for detecting tumors is suitable for many research applications, as well as for both veterinary medicine and human medicine.
  • the diagnostic method generally includes, but is not limited to, the following steps: Step 1.
  • Step 2 Provide a diagnostic onco-tool in its final form - either by contacting the precursor form of the onco-tool with a suitable radioisotope which is effective to report its presence within a tumor to a detector outside the living subject, or by obtaining directly from a supplier the final form of a diagnostic onco-tool already containing such a radioisotope.
  • Step 2 Deliver that diagnostic onco-tool into the subject - typically by intravenous injection.
  • Step 3 Wait a suitable period of time for onco-tool to be sequestered in acidic areas of any tumors which may be present (eg., from about 10 minutes to about 50 minutes).
  • a suitable period of time for onco-tool to be sequestered in acidic areas of any tumors which may be present eg., from about 10 minutes to about 50 minutes.
  • additional time hours for excretion through the kidneys of most of that portion of the onco-tool dose which has not been sequestered in acidic areas of tumors. Waiting this additional time serves to greatly lower background signal from normal tissues and allow detection of even quite small tumors.
  • most of the injected dose of onco-tool should be excreted by the kidneys, with significant retention of onco-tool only occurring if one or more tumors are present.
  • Step 4 The final step in the diagnostic method is to scan the subject with equipment suitable for detecting the emission from the radioisotope component of the onco-tool in order to assess if significant onco-tool has been sequestered in one or more tumors.
  • equipment suitable for detecting the emission from the radioisotope component of the onco-tool in order to assess if significant onco-tool has been sequestered in one or more tumors.
  • modem imaging equipment such as gamma ray scanners and PET scanners, tumors should show up as an obvious radioisotopic hot spot at the site of each tumor.
  • onco-tools exploit the acidity which is a near-universal characteristic of tumors
  • onco-tools should be effective to treat most or all types of tumors with sizes ranging from near-microscopic to very large.
  • the following methods of using onco-tools for treating tumors are suitable for many research applications, as well as for both veterinary medicine and human medicine.
  • the therapeutic methods generally include, but are not limited to the following.
  • the therapeutic method generally includes, but is not limited to, the following two steps:
  • Step 1 Provide a therapeutic onco-tool in its final form containing a radioisotope effective to kill cells. This can be done either by contacting the precursor form of the onco-tool with a suitable radioisotope, or by obtaining directly from a supplier the final form of the therapeutic onco-tool already containing such a radioisotope.
  • Step 2 Deliver that therapeutic onco-tool into the subject - typically by intravenous injection.
  • the. radioisotope effective to kill cells can be either one whose emission has a high linear energy transfer, such as an alpha particle, or one which emits a beta particle.
  • the particular merit of using a radioisotope having a high linear energy transfer emission, such as the alpha- emitting radioisotope Astatine-211, is that it is appreciably more effective than beta-emitting radioisotopes for killing the quiescent cells in acidic areas of tumors.
  • their emissions generally have a quite short path length (eg., 80 microns or less).
  • At-211 it may be necessary to use such onco-tools in combination with conventional cancer therapies in order to also assure the destruction of the fast- dividing tumor cells in areas of more normal pH near capillaries.
  • the preferred alpha emitter, At-211 has a short half- life (7 hours) and so shipping this radioisotope, or an onco-tool containing this radioisotope, a substantial distance from the site where it is generated can be a problem.
  • At-211 must be generated in a high-end cyclotron with an alpha beam capability, and at present there are only relatively few sites where such equipment is available.
  • An alternative single-radioisotope method entails providing a therapeutic onco-tool containing a beta-emitting radioisotope. While beta emissions are significantly less effective than alpha emissions for killing the quiescent cells in acidic areas of tumors, this lesser effectiveness of beta emissions can be compensated for by using a much greater dose. While this much greater dose will compromise specificity somewhat, nonetheless, it has the potential to give acceptable therapeutic results because of the exceptionally high level of specificity for tumors achievable by onco-tools.
  • the beta emitter, lodine-131 has several useful properties: it is readily available in large quantities at a moderate price; because of its relatively long half-life (8 days) it can be shipped and stored for reasonable periods of time before use; and, while it has only a moderate mean path length (about 900 microns), nonetheless that is sufficient to kill cells throughout the tumor, including the fast-dividing tumor cells in regions closer to neutral pH near capillaries - which will be relatively devoid of sequestered onco-tool.
  • radioisotopes which have high linear energy transfer emissions are optimal for killing the radiation-resistant quiescent cells in acidic areas of tumors, but the short path length of such emissions makes an onco-tool containing such radioisotopes rather ineffective for killing the fast- dividing celts in distal areas of tumors near capillaries where the pH is closer to neutral - because such areas will be relatively devoid of sequestered onco-tool.
  • an onco-tool containing a beta-emitting radioisotope is relatively ineffective against the radiation-resistant quiescent cells of the tumor, but that onco-tool containing the beta-emitting radioisotope can be quite effective for killing the radiation-sensitive fast-dividing cells in areas of tumors where the pH is closer to neutrality - even when that onco-tool is only present in the acidic areas of the tumor. This is because the greater path length of the beta particles allow them to reach and kill those more sensitive tumor cells.
  • a preferred method for treating tumors is to use a combination of two therapeutic onco-tools, where one onco-tool contains a radioisotope having a high linear energy transfer emission (preferably selected from Astatine-211 , Bromine-77, and lodine-123) to kill the proximal radiation- resistant quiescent cells in acidic areas of the tumor, and the other onco-tool contains a beta-emitting radioisotope to kill the distal radiation-sensitive fast- dividing cells in higher-pH regions near tumor capillaries, which are relatively devoid of sequestered onco-tool.
  • a radioisotope having a high linear energy transfer emission preferably selected from Astatine-211 , Bromine-77, and lodine-123
  • the other onco-tool contains a beta-emitting radioisotope to kill the distal radiation-sensitive fast- dividing cells in higher-pH regions near tumor capillaries, which are relatively devoid of sequestered onco-tool.
  • Onco-tools offer the highly desirable properties of being able to both detect and treat most or all types and sizes of tumors, ranging from near-microscopic to very large. Because the diagnostic and the therapeutic onco-tools can be virtually identical - but typically differing only in the contained radioisotope - in general if a given onco-tool structure is effective to detect a tumor, then that same onco-tool structure, but typically with a different radioisotope, should also be effective to treat that same tumor. These special properties of onco-tools facilitate a comprehensive method for detecting tumors in living subjects, followed by treatment of any tumors so detected. This comprehensive method is suitable for both veterinary medicine and human medicine. It includes, but is not limited to, the following steps.
  • Step 1 The first step it to provide a diagnostic onco-tool in its final form containing a radioisotope which is effective to report its presence within a tumor to a detector outside the living subject.
  • Step 2. The next step is to deliver that diagnostic onco-tool into the subject - typically by intravenous injection.
  • Step 3 The subsequent step is to wait a suitable period of time for onco- tool to be sequestered in acidic areas of any tumors which may be present. This step may also include waiting additional time for excretion through the kidneys of most of that portion of the onco-tool dose which has not been sequestered in acidic areas of tumors. During this period of time the subject may also be given fluid, particularly fluid that contains a diuretic, to increase the excretion of that portion of the onco-tool dose which has not been sequestered in acidic areas of tumors.
  • Step 4 The last step in the detection process is to scan the subject with equipment suitable for detecting the emission of the radioisotope of the onco-tool in order to assess if significant onco-tool has been sequestered in one or more tumors.
  • equipment suitable for detecting the emission of the radioisotope of the onco-tool in order to assess if significant onco-tool has been sequestered in one or more tumors.
  • modem imaging equipment such as gamma ray scanners and PET scanners, tumors should show up as an obvious radioisotopic hot spot at the site of each tumor.
  • one or more therapeutic onco-tools are provided. While this can be a single onco-tool containing a beta-emitting radioisotope, it is generally preferred to provide two or more onco-tools, where one contains a radioisotope which emits high linear energy transfer radiation, and another contains a beta-emitting radioisotope.
  • Step 6 The one or more provided onco-tools are delivered into the subject - generally by intravenous injection.
  • any progeny micro-metastases smaller than about 1 millimeter in diameter are expected to survive the onco-tools treatment and ultimately lead to a relapse - though such a relapse may not occur for several years after the initial onco-tool treatment.
  • a strategy for solving this micro-metastases problem is to wait a period of time after the initial onco-tool therapy (steps 5 and 6 in Section 3 above) sufficient for any micro-metastases, that might be present and survived the initial treatment, to grow to a size where they develop acidic areas (typically about 1 millimeter in diameter).
  • micro-metastases should not be allowed to grow to the much larger size (probably on the order of 1 centimeter in diameter) where they too begin to metastasize.
  • micro-metastases that might have escaped the first onco-tool treatment are in this proper size range (large enough to contain acidic areas, but not so large as to have begun metastasizing), one again carries out the onco-tool detection and treatment process (repeat of steps 1 through 6 in Section 3 above).
  • a complication in the above strategy is that tumors exhibit a wide range of growth rates, and so it is difficult to predict how long it will take for any micro- metastases which might have escaped the first treatment to reach a size where they contain acidic regions. Therefore, the prudent course is to repeat diagnostic steps 1 through 4 above at appropriate intervals (perhaps once a year) continuing for a sufficient length of time (perhaps 4 to 6 years) to virtually assure that if any micro-metastases did escape destruction in the initial onco-tool therapy, then such micro-metastases would have grown to a size sufficient to generate acidic regions and so be detected in one of the subsequent repeat diagnostic procedures (repeats of steps 1 through 4 above).
  • the pH in acidic areas of tumors can be further reduced by vasodilator drugs which are routinely used to treat persons with hypertension
  • Such treatments but preferably a combination of two or more such treatments to further reduce the pH in acidic areas of tumors should increase the sequestering of onco-tool in the now-more-acidic areas of the tumor, as well as lead to an increase in the areas of the tumor which are sufficiently acidic to sequester onco-tool. Both of these effects serve to increase the efficacy and the specificity of the onco-tool.
  • Making the tumor more acidic also allows one to use an onco-tool with pH- switches having a lower pKa - which can result in a significant increase in the onco-tooFs specificity.
  • One strategy for dealing with a wide pH range within a tumor is to use a combination of two or more onco-tools, where one onco-tool with a higher pKa is maximally effective in higher-pH regions of the tumor closer to capillaries, and where another onco-tool with a lower pKa is maximally effective in lower-pH regions of the tumor which are further from capillaries.
  • kidneys Treat To Increase pH In Urine To Protect Kidneys From Damage
  • One of the functions of the kidneys is to maintain the pH in the body at very close to 7.4.
  • the kidneys can excrete urine ranging from moderately basic to fairly acidic.
  • a substance is filtered from the blood in the glomerulus of the kidney, after which that substance (dissolved in urine) passes through the proximal tubule where critical components excreted in the glomerulus are reabsorbed by cells lining the proximal tubule.
  • an onco-tool in this acidic environment has the potential of binding and entering the cells lining the proximal tubules of the kidneys, much as the onco-tool enters tumor cells from an acidic extracellular environment.
  • the excreted urine is sufficiently acidic the onco-tool will switch to its non-ionic lipophilic form. If that switch to a lipophilic form occurs in a region where the urine is in direct contact with cell membranes, such as is the case for cells lining the proximal tubules of the kidneys, then the onco-tool can enter such cells.
  • a prudent course in any diagnosis or treatment with onco-tools is about one to two hours prior to delivering the onco-tool into the subject, first treat the subject with this drug, or other substance effective to raise and maintain the pH of the urine at a pH above about 7.4.
  • the kidneys may also be desirable to continually monitor the pH of the urine entering the bladder by using a micro-pH-probe at the end of a catheter.
  • the preferred period of time for such monitoring is from just before injection of the onco-tool until such time as most of the onco-tool not sequestered in acidic areas of tumors has been excreted by the kidneys (typically about 3 to 6 hours).
  • Such monitoring will allow emergency intervention (such as injecting an additional dose of Acetazolamide) in the event the pH of the newly excreted urine begins to drop below about pH 7.4.
  • Irrigating of the bladder during the period of time when most of the onco- tool dose is being excreted by the kidneys serves an additional purpose, that being the solution carried out of the bladder can be passed into a shielded storage vessel where it can be contained until the radioisotope has decayed to a safe level (typically 10 half-lives of the radioisotope).
  • a safe level typically 10 half-lives of the radioisotope.
  • the sodium salts of propionic acid (a hydrophilic carboxylic acid) and octa ⁇ oic acid (a lipophilic carboxylic acid) at a concentration of 33 milliMolar in water were titrated with 5 M HCI.
  • the mid-point of the transition between salt and acid (pKa value) was found to be an expected 4.83 for propionic acid, but a surprisingly-high 5.5 for octanoic acid.
  • the first derivative of the titration curve was symmetrical for the propionic acid, but highly unsymmetrical for the octanoic acid.
  • Titration in aqueous solution of the acyclic structure of Figure 7b showed an expected pH of transition value of 4.95 (typical of simple carboxylic acids), with no precipitation of the acid form. Sn contrast, titration of the 5-membered ring structure of Figure 7a showed an unexpectedly-high value of 5.6 for the pH of transition between anionic and non-ionic forms. Titration of that ring structure was also accompanied by massive precipitation when the pH dropped below about 5.8, and the shape of the titration curve was highly asymmetric.
  • Example 5 Advantage of partially shielding H-bonding site from aqueous solvent
  • Example 6 Preparation and titration of two pH-switch structures designed to form an internal acid-specific low-barrier H-bond
  • this pKa 6.0 value is due to an internal low-barrier H-bond forming between the carboxyl H-bond donor and the N-oxide H-bond acceptor, and that internal H-bond acts to drive the equilibrium toward the acid form - ie., raise the pKa.
  • the cis(3-amine-1-acid) derivative of camphoric acid was reductively alkylated with an excess of acetaldehyde and the resulting tertiary amine product converted to the N-oxide with meta-Chloroben ⁇ oylperoxide to give structure i in Figure 10b.
  • This product in its sodium carboxylate form, was then titrated in water.
  • the first derivative of its titration plot shows two minimums, one at pH 4.2 (which is approximately that expected for the N-oxide moiety), and another minimum at pH 6.5, which is substantially higher than would be expected for either the N-oxide moiety or the carboxylic acid moiety.
  • the inventor's molecular modeling has shown that only the twisted boat conformation of the isonipocotic derivative can form the internal acid-specific H-bond, it is likely that the strength of that H-bond is attenuated by the energy cost of converting from the chair to the twisted boat conformation.
  • the 5-membered aliphatic ring of the camphoric derivative can readily and at low energy cost shift between differing pucker conformations, and so there is little energy cost to adopting the conformation suitable for forming the internal acid-specific H-bond.
  • essentially the full energy of that H-bond is available for favoring the acid form over the salt form - hence raising the pKa to the exceptionally high value of 6.5.
  • Aqueous titrations Typically new structures that may be suitable as advanced pH-switch components are first assessed for their pT value, that is, the pH value at the midpoint of their transition between anionic and non-ionic forms. This entails preparing the sodium salt form of the structure and dissolving it at a concentration of 5 milliMolar in 50 ml of 0.15 Molar NaCI (deareated to minimize carbonic acid content). A small magnetic stir bar is added and the pH of the solution adjusted to about 9 with a small amount of 1 M NaOH. While stirring, 5 microliter portions of 1 JvI HCI are added and the pH is recorded at 1 minute after addition of each HCI portion.
  • the titration curve will generally be strongly skewed. In such cases it is difficult to differentiate between solubility effects and pKa effects on the mid-point of the transition between anionic and non-ionic forms of the pH-switch. In such cases the solubility effect can be avoided by titrating in a semi-aqueous solvent where both the salt and acid forms of the pH-switch are fully soluble. A 1:1 mix of methanol and water generally serves for this purpose. In this solvent the first derivative titration curves again become symmetric.
  • the titration plot can also show a substantially increased pKa value relative to a very similar structure which cannot form an internal acid-specific H-bond - and this increased pKa value is indicative of an infernal acid-specific H-bond forming under the conditions of the titration.
  • each prospective onco-too! structure should be tested in a relatively simple biological system wherein the onco-tool is s exposed to the principal biological environments and structures it will encounter in a living subject - including particularly mammalian cells exposed to serum- containing medium buffered at pH 7.4 to emulate blood and normal tissues, and buffered at pH 6.4 to emulate acidic areas of tumors.
  • Such a suitable test system entails preparing two preparations of isotonic o culture medium.
  • One should contain 10% serum and be strongly buffered at pH 7.4 with 50 milliMolar HEPES buffer (pKa 7.5).
  • the other should contain 10% serum albumin and be strongly buffered at pH 6.4 with 50 milliMolar BisTRIS buffer (pKa 6.5).
  • lodine-131 containing onco-tool should be added at equal concentration to each culture medium.
  • HeIa cells should first be grown to confluency in
  • the cells are lysed with 1 ml of detergent solution and that lysis solution removed and counted in a scintillation or gamma counter to provide a measure of the 5 relative quantity of onco-tool which has been sequestered under each of the two pH conditions.
  • a preferred onco-tool structure is one which is maximally sequestered by the cells at pH 6.4, but only minimally sequestered by the cells at pH 7.4.
  • the above cell culture test system for onco-tools allows an initial quick and quantitative assessment of the probable efficacy and specificity properties of a substantial number of prospective onco-tools.
  • this initial assessment should next be followed up for the most promising onco-tool structures with tests in living mice. It is recommended that the mice first be pre-treated with a carbonic anhydrase inhibitor, such as Acetazolamide, to assure that their urine remains basic for a number of hours.
  • a suitable quantity of lodine-131- containing onco-tool in phosphate-buffered saline should be injected, preferably intravenous, such as into the tail vein.
  • mice should be periodically monitored for a period of up to about 24 hours (such as by briefly positioning under a suitable gamma counter or gamma camera) to determine the rate of excretion of the labeled onco-tool.
  • the main purpose of this testing in normal mice is to identify any onco- tools which are unduly sequestered in normal tissues - presumably due to excessive lipophilicity, or possibly due to some structural element that has an unexpected affinity for normal tissues or some particular organ, etc.
  • Onco-tools that are rapidly and thoroughly excreted from normal mice, and so pass this preliminary animal test, should next be tested in tumor-bearing mice, as described in the following Example.
  • mice a) with a carbonic anhydrase inhibitor, such as Acetazolamide, to raise the pH in the urine; and, b) with one or a combination of substances effective to selectively reduce the pH in tumors.
  • a carbonic anhydrase inhibitor such as Acetazolamide
  • substances effective to selectively reduce the pH in tumors.
  • substances include: i) glucose (Naeslund & Swenson (1953) Acta Obstet. Gyneocol. Scand.
  • the tumor-bearing mice are injected (preferably intravenous) with the lodine-131- containing onco-tool.
  • a suitable period of time on the order of 5 to 24 hours to allow normal excretion by the kidneys of that portion of the administered dose which is not sequestered in tissues and/or tumors of the mice, the mice are killed and the major organs and any obvious tumors excised. Each organ and tumor and the remaining carcass is then counted in a gamma counter.
  • onco-tools will work best when both types of pre-treatments are used, where one type serves to prevent re-uptake of onco-tool into the cells lining the proximal tubules of the kidneys. This re-uptake is blocked by rendering the urine slightly basic.
  • the other type serves to further increase the acidity (reduce the pH) in hypoxic/acidic areas of tumors.
  • a combination of two or three such pre-treatments to selectively reduce pH in the tumors should be employed in order to adjust the tumor micro-environment so as to be best suited for effective and specific onco-tool activity.
  • compositions and methods of the present invention are, however, susceptible to modifications and alternate constructions from the illustrative embodiments discussed above which are fully equivalent. Consequently, it is not the intention to limit the disclosed compositions and methods to the particular embodiments disclosed. On the contrary, the intention is to cover all modifications and alternate constructions coming within the spirit and scope of the compositions and methods as generally expressed by the following claims, which particularly point out and distinctly claim the subject matter of the disclosed compositions and methods.

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Abstract

L'une des caractéristiques les plus universelles des tumeurs malignes est leur acidité. Les outils oncologiques sont de petites molécules synthétiques non peptides conçues pour exploiter cette acidité pour une détection et destruction précoce des tumeurs. Chaque outil oncologique présente une structure qui est anionique et hydrophile au pH 7.4 et crée une répulsion par rapport aux surfaces à charges négatives des cellules de tissus normaux. Quand un outil oncologique pénètre dans un environnement acide tel qu'une tumeur, une partie des molécules outils oncologiques passent à leur forme lipophile non ionique qui est conçue pour pénétrer les cellules, notamment les cellules des zones acides des tumeurs. Avant d'utiliser un outil oncologique, on lui relie un radioisotope sélectionné. Si ce radioisotope émet un rayonnement détectable à l'extérieur du corps, l'outil oncologique convient à la détection de tumeurs. Si ce radioisotope émet un rayonnement capable de tuer des cellules, l'outil oncologique convient au traitement de tumeurs.
EP07754699A 2006-03-30 2007-03-25 Compositions outils oncologiques et procédés d'utilisation pour détecter et traiter des tumeurs Withdrawn EP1998760A2 (fr)

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US11/395,487 US20070231256A1 (en) 2006-03-30 2006-03-30 Compositions and methods for detecting and treating tumors containing acidic areas
US11/449,495 US8084610B2 (en) 2006-06-07 2006-06-07 Compositions and methods for detecting and treating tumors containing acidic areas
US11/449,508 US20080124274A1 (en) 2006-06-07 2006-06-07 Compositions and methods for detecting and treating tumors containing acidic areas
PCT/US2007/008215 WO2007117398A2 (fr) 2006-03-30 2007-03-30 Compositions outils oncologiques et procédés d'utilisation pour détecter et traiter des tumeurs

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