WO2008121126A1 - Molécules non-peptidiques pour la détection et le traitement des tumeurs - Google Patents

Molécules non-peptidiques pour la détection et le traitement des tumeurs Download PDF

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WO2008121126A1
WO2008121126A1 PCT/US2007/021002 US2007021002W WO2008121126A1 WO 2008121126 A1 WO2008121126 A1 WO 2008121126A1 US 2007021002 W US2007021002 W US 2007021002W WO 2008121126 A1 WO2008121126 A1 WO 2008121126A1
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onco
tool
subject
composition
tumors
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PCT/US2007/021002
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James E. Summerton
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Summerton James E
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Priority claimed from PCT/US2007/008215 external-priority patent/WO2007117398A2/fr
Application filed by Summerton James E filed Critical Summerton James E
Priority to AU2007350326A priority Critical patent/AU2007350326A1/en
Priority to MX2009010621A priority patent/MX2009010621A/es
Priority to CA002682567A priority patent/CA2682567A1/fr
Publication of WO2008121126A1 publication Critical patent/WO2008121126A1/fr

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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
    • A61K31/185—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

  • TITLE NON-PEPTIDIC MOLECULES FOR DETECTING
  • This invention relates to compositions which when introduced into a subject are selectively sequestered in acidic areas of tumors within the subject for the purpose of detecting and treating tumors.
  • the key advance afforded by the present onco-tool invention is to provide a dramatic many-fold increase in specificity for detecting and treating malignant tumors containing acidic areas. This promises much earlier detection of malignant tumors, as well as a means for completely destroying even late-stage tumors without causing undue harm to the patient - thereby avoiding the devastating damage to patients and avoiding the post-treatment relapses that plague current cancer treatments.
  • Tumor cells at near-normal pH in close proximity to capillaries are fast dividing, while tumor cells in acidic areas more distant from capillaries divide more 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 chemotherapeutics.
  • Conventional cancer therapies were selected in large part for their ability to kill rapidly-dividing cells while sparing the slow-dividing and non-dividing cells typical of most normal tissues. Thus, such cancer therapies are fairly effective in killing the rapidly-dividing tumor cells at near-normal pH close to capillaries. But those same therapies are typically less effective against the non-dividing quiescent cells in acidic areas of a tumor.
  • cancer treatments predominantly kill the well-oxygenated fast-dividing tumor cells while sparing the more treatment-resistant quiescent cells in hypoxic/acidic areas of that tumor.
  • This killing of the fast-dividing cells causes the tumor to go into remission while those killed cells are being disposed of by the body's normal cleanup processes.
  • all too often the surviving treatment- resistant quiescent cells in acidic areas of the tumor slowly regain access to adequate oxygen, nutrients, and waste disposal - eventually allowing them to revert to rapid cell division, with attendant tissue invasion, metastatic spread, and often enhanced resistance to subsequent cancer therapies.
  • a common result of this rejuvenation of the previously-quiescent tumor cells is the dreaded post- treatment relapse that is responsible for most deaths from cancer.
  • Onco-tools of the present invention are designed to exploit the acidity present in most tumors for the purpose of detecting and treating those tumors.
  • Related composition for detecting tumors having acidic areas are designed to exploit the acidity present in most tumors for the purpose of detecting and treating those tumors.
  • 11 C-DMO Carbon 11-labeled ⁇ . ⁇ -Dimethyl ⁇ -oxazolidinedione
  • Figure 2 is a compound which has been used for a number of years for the purpose of detecting acidic areas of tumors (Ginos et al., Journal of Nuclear Medicine, Vol. 23, pages 255 - 258 (1982); Rottenburg et al., Annals of Neurology, Vol. 17 pages 70 - 79 (1985)).
  • This is a low molecular weight substance (MW 129) containing a single weak-acid moiety (pKa of 6.3) and it carries a radioisotope which serves to report the presence of the substance within a tumor to a detector outside of the subject.
  • DMO detects tumors containing acidic areas by the following mechanism.
  • the DMO molecules exist predominantly (93 %) in their anionic hydrophilic form which is repelled from the anionic surface of cells and is rapidly excreted by the kidneys.
  • the DMO enters an acidic area of a tumor a significant fraction of the DMO molecules convert to their non-ionic slightly-lipid-soluble form which is capable of slowly penetrating into the tumor cells. As a consequence, some of the DMO molecules are thereby selectively sequestered in acidic areas of the tumor.
  • the Carbon-11 radioisotope which is incorporated in the DMO structure decays with a half-life of 20 minutes and emits a positron whose photons of annihilation can be readily detected by a PET (positron emission tomography) scan, thereby indicating the presence and position of the tumor.
  • PET positron emission tomography
  • DMO and the onco-tools of the present invention which are formulated for diagnostic use both work by similar mechanisms to achieve the same objective - that being they are selectively sequestered in acidic areas of tumors wherein they serve to report the presence and position of the tumors.
  • the mechanisms of action for DMO and onco-tools are similar, the end results differ considerably because DMO affords only modest specificity for tumors, while onco-tools of the present invention can afford unprecedented tumor specificity due to their unique molecular design.
  • onco-tools of the present invention are uniquely designed to provide a tumor specificity factor which can be many-fold greater than the specificity factor of 6 provided by DMO, and this much greater tumor specificity of onco-tools should allow routine and reliable detection of even very-early-stage tumors just 1 to 2 millimeters in diameter (the size where tumors begin to form hypoxic/acidic areas).
  • tumors of such a small size are generally undetectable by current tumor diagnostics.
  • tumors of such a small size must grow roughly a hundred to a thousand fold in volume (to about 1 centimeter in diameter) before they become large enough to be easily detected by current tumor diagnostics.
  • Chlorambucil shown in Figure 2 is a cytotoxic agent which has been used for a number of years for treating tumors.
  • Chlorambucil is a low molecular weight substance (MW 304) containing a double nitrogen mustard moiety which is particularly toxic to replicating cells due to cross-linking of duplex DNA.
  • Chlorambucil also contains a weak-acid moiety (reported pKa of 5.8) which affords preferential entry into acidic areas of tumors (Kozin et al., Cancer
  • Chlorambucil preferentially enters cells in acidic areas of tumors due to the following mechanism.
  • pH 7.4 characteristic of the extra-cellular space in normal tissues the Chlorambucil molecules exist predominantly (97.5 %) in their anionic hydrophilic form which is repelled from the anionic surface of cells and is excreted by the kidneys.
  • Chlorambucil enters an acidic area of a tumor a significant fraction of the molecules convert to their non-ionic npid-soluble form which is capable of rapidly penetrating into the tumor cells. As a consequence, a portion of the Chlorambucil molecules are thereby selectively sequestered in cells in acidic areas of the tumor.
  • the double nitrogen mustard cytotoxic moiety which is incorporated in the Chlorambucil structure then acts to damage those tumor cells containing the Chlorambucil.
  • Chlorambucil and the onco-tools of the present invention which are formulated for therapeutic use both selectively enter tumor cells in acidic areas by a similar mechanism, and then both Chlorambucil and the onco-tools act to damage the cells they have entered.
  • onco-tools can also effectively destroy even those cells of the tumor which the onco-tools have not entered. While there are similarities in the mechanism by which Chlorambucil and onco-tools selectively enter cells in acidic areas of tumors, the end results differ considerably. This is in substantial part because Chlorambucil affords only modest specificity for tumors, while onco-tools of the present invention can achieve unprecedented specificity due to their unique molecular design.
  • Chlorambucil for acidic areas of tumors, at a pH of 6.4 generally achievable in acidic areas of tumors 20.1 % of the Chlorambucil molecules are in their non-ionic cell-penetrating form, while at a pH of 7.4 in normal tissues 2.5% of the Chlorambucil molecules are in their non-ionic cell- penetrating form. Therefore, for a given concentration of Chlorambucil the rate of entry into cells in such acidic areas of a tumor will only be about 8 fold faster than the rate of entry of Chlorambucil into cells in normal tissues - giving a tumor specificity factor of only 8.
  • onco-tools of the present invention are uniquely designed to provide a specificity factor which can be many-fold greater than that provided by Chlorambucil, and this much greater specificity should allow delivery of a sufficient dose of therapeutic onco-tool to completely destroy the entire tumor and thereby prevent post-treatment relapses - without causing undue damage to the patient.
  • this much greater specificity should allow delivery of a sufficient dose of therapeutic onco-tool to completely destroy the entire tumor and thereby prevent post-treatment relapses - without causing undue damage to the patient.
  • current tumor therapies because of their inadequate specificities for tumors, it is generally difficult and often impossible to deliver a sufficiently high dose to completely destroy the entire tumor without also severely damaging or killing the patient.
  • compositions for treating tumors containing acidic areas for treating tumors containing acidic areas
  • Onco-tools are a novel class of molecules designed to achieve these objectives.
  • Onco-tools consist of relatively small non-peptide synthetic molecules with molecular weights typically in the range of about 300 to 1200 daltons - not counting the mass of the radioisotope.
  • An onco-tool contains two or more pH-switch components, each of which includes a weak-acid moiety that readily converts between an anionic hydrophilic form at a higher pH and a non-ionic membrane-penetrating form at a lower pH.
  • Each onco-tool also contains a cargo component which is effective to bind a radioisotope, or which contains a radioisotope, where said radioisotope is suitable for carrying out the diagnostic or therapeutic role of the onco-tool.
  • Onco-tools are sequestered in acidic areas of tumors by virtue of the following processes. At pH 7.4, which is characteristic of the extra-cellular space in normal tissues, the onco-tool molecules exist predominantly in their anionic hydrophilic form which is repelled from the anionic surface of cells and is excreted by the kidneys.
  • the onco-tools perfuse through an acidic area of a tumor the low pH causes a significant fraction of the molecules to convert to their non-ionic membrane-penetrating form which then enters the tumor cells.
  • the cytosol of the tumor cells which typically have an intracellular pH of about 7.4 to 7.6
  • the onco-tools are re-ionized, thereby inhibiting their exit from the tumor cells.
  • onco-tools are selectively sequestered in acidic areas of tumors.
  • 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 cleared from the body via the kidneys.
  • Radioisotope Prior to use of the onco-tool a selected radioisotope will be linked to that onco-tool. That attached radioisotope serves to carry 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.
  • Onco-tools are designed to provide an unprecedented level of specificity for tumors by virtue of their having unique multi-acid structures, engineered pKa values, and adjusted pH-dependent lipophilicities. Together these novel properties are designed to provide a many-fold increase in selective for tumors compared to conventional cancer diagnostics and therapies.
  • New material disclosed in this patent application includes: a) a detailed description of the molecular design strategy which underlies onco-tools 1 unprecedented specificity for acidic areas of tumors; b) new pH-switch structures and their pKa values; c) new onco-tool structures containing fused pH-switches and mixed pH-switches; d) new cargo structures which afford greater latitude in the synthetic steps used for preparing onco-tools; e) additional considerations for selecting radioisotopes for both diagnostic and therapeutic applications; and, f) a method for increasing the percent of an injected onco-tool dose which can be sequestered in acidic areas of tumors.
  • Figure 1 illustrates the distribution of acidity in tumors.
  • Figure 2 shows related compositions which are currently used for detecting and treating tumors containing acidic areas.
  • Figure 3 shows a representative conventional pH-switch structure.
  • FIG. 4 shows representative advanced pH-switch structures.
  • Figure 5 shows representative acceptor moieties for low-barrier H-bonds.
  • Figure 6 shows representative pH-switches and their measured pKa values.
  • Figure 7 shows representative merged pH-switches.
  • Figure 8 shows related pH-switches varying in lipophilicity.
  • Figure 9 shows representative cargo components.
  • Figure 10 shows representative onco-tools with 2 pH-switch components.
  • Figure 11 shows representative onco-tools with 3 pH-switch components.
  • Figure 12 shows representative onco-tools with 4 pH-switch components.
  • Figure 13 shows representative onco-tools with merged pH-switches.
  • Figure 14 shows representative onco-tools with mixed pH-switch components.
  • FIG. 15 illustrates representative syntheses of several pH-switch components.
  • Figure 16 illustrates representative syntheses of several cargo components.
  • Figure 17 illustrates the assembly of several representative onco-tools.
  • FIG. 18 illustrates the addition of representative radioisotope cargos.
  • Figure 19 shows two representative onco-tools in their final form.
  • pH-switch component - a structural component of an onco-tool which contains an acid moiety and which is capable of undergoing a pH-mediated transition between an anionic hydrophilic form at a higher pH and a non-ionic cell- penetrating form at a lower pH.
  • An advanced pH-switch has the following properties: a) an aliphatic ring structure selected from the group consisting of a 4- membered ring, a 5-membered ring, and a 6-membered ring; b) an acid H-bond donor moiety directly linked to the aliphatic ring structure; c) an H-bond acceptor moiety selected from the group consisting of part of the aliphatic ring structure, directly linked to the aliphatic ring structure, and linked through one atom to the aliphatic ring structure; said H-bond acceptor moiety in its non-ionic form has a structure which cannot serve as an H-bond donor moiety; and, d) said acid H-bond donor moiety and said H-bond acceptor moiety are positioned and oriented such that they are compatible with formation of an internal acid-specific H-bond.
  • Merged pH-switches component an advanced pH-switch type wherein two acid H-bond donor moieties are positioned and oriented so as to allow both H-bond donor moieties to simultaneously H-bond to a single H-bond acceptor moiety.
  • 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.
  • Onco-tool with mixed pH-switches - an onco-tool which contains a mixture of two or more different pH-switch types.
  • 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.
  • Dual-radioisotope onco-tool therapy strategy - a treatment strategy where at least two different onco-tools are used to treat tumors, where one onco-tool contains a radioisotope which emits an alpha particle, and another onco-tool contains a radioisotope which emits a beta particle.
  • 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 7.0 or lower.
  • Anionic form - a form which carries at least one negative charge.
  • Non-ionic form - a form which does not carry an ionic charge.
  • Efficacy factor the percent of the onco-tool molecules which are in their non- ionic form at pH 6.4.
  • Tumor 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).
  • the pH-Switch Components a) Conventional pH-switches b) Advanced pH-switches i) acid-specific H-bond ii) minimal conformational freedom iii) acid moiety insulated from inductive effects iv) partial shielding of H-bonding site v) low-barrier H-bond c) Merged pH-switches d) Adjust lipophilicity of pH-switches 3.
  • 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 e) Onco-tools with merged pH-switches f) Onco-tools with mixed pH-switches
  • Each onco-tool includes 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 cell-penetrating form at lower pH.
  • Each onco-tool also includes a cargo component which is effective to bind a selected radioisotope, or which has bound a radioisotope, where said radioisotope is suitable for carrying out the diagnostic or therapeutic role of the onco-tool.
  • 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 cell-penetrating form within the available very limited pH difference between normal tissues and acidic areas of tumors. While this pH difference between normal and tumor is typically only about 0.3 to 0.7 pH unit for most tumors, it should be appreciated that simple and safe interventions (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 interventions the pH difference between normal and acidic areas of tumors can typically be increased to 1.0 pH unit or greater - ie., pH 6.4 or less in tumors and pH 7.4 in normal tissues.
  • tumor specificity factor this ratio of the percent in the non-ionic form at pH 6.4 divided by the percent in the non-ionic form at pH 7.4 the "tumor specificity factor" because it serves as a measure of how specifically the molecules can be sequestered in acidic areas of tumors.
  • monoclonal antibodies targeted against tumor cells typically show about a 4 to 8 fold preference for binding tumor cells relative to binding non-tumor cells. This suggests that conventional acid-containing agents, such as 11 C-DMO and Chlorambucil, are only about on a par with or slightly better than monoclonal antibodies in regard to specificity for tumors.
  • the first entails adjusting the number of non-polar groups in the onco-tool structure in order to optimize its cell-penetrating ability.
  • adding non-polar groups serves to increase the lipophilicity of the non-ionic form of the onco-tool, which in turn serves to substantially increase the rate at which the non-ionic form can penetrate into cell membranes.
  • steps to increase lipophilicity can only be carried so far before the anionic form of the onco-tool becomes so lipophilic that rather than being repelled from cells due to its negative charge, it instead can bind to cells due to its excessive lipophilicity.
  • the resultant hydrophobic-type binding to cells at pH 7.4 would result in a loss of tumor specificity.
  • the second molecular design strategy which was a considerable challenge to implement and which appears to be unique to onco-tools, is to adjust the pKa of at least one of the onco-tool's acid moieties to within a fairly narrow range calculated to be suitable for achieving both high tumor specificity and adequate efficacy.
  • the novel means used to adjust the pKa of the acid moieties also provides an additional valuable contribution to both the efficacy and the specificity of the onco-tools by virtue of substantially amplifying the lipophilicity differential between the anionic and the non-ionic forms of the onco-tool.
  • the unique means devised for adjusting the pKa of the acid moieties in onco- tools entails designing special compact structures called "advanced pH-switches" which are capable of forming an internal acid-specific low-entropy hydrogen bond.
  • advanced pH-switches special compact structures
  • the acid moieties incorporated in such advanced pH-switch structures have pKa values significantly greater than the pKa values of similar acid moieties which are not appropriately positioned for forming an internal acid-specific H-bond.
  • acid moieties in these advanced pH-switches have pKa values in the range of 5.5 to 6.6, which, as illustrated in the table below, fall within the range of pKa values suitable for achieving both very high specificity and adequate efficacy in multi-acid onco-tools.
  • those same internal H-bonds which serve to increase the pKa of the acid moieties in advanced pH-switches also serve to substantially amplify the lipophilicity differential between the anionic and non-ionic forms of the onco-tools. This is because formation of that internal H-bond causes the displacement of waters of hydration (probably two water molecules displaced) when the internal H-bond forms at a pH achievable in tumors (pH 6.4).
  • the table below shows calculated tumor specificity and efficacy factors for molecules having 1 , 2, 3, and 4 acid moieties of varying pKa values. Note that the pKa values of 5.5, 6.0, and 6.6 are actual values for acid moieties in advanced pH- switches (structures b, d, and e of Figure 6) which have been synthesized in the course of this onco-tools development program.
  • Tumor specificity factor 4 20 90 403
  • Efficacy factor 61% 38% 23% 14% Values in the above table suggest that it should be possible to develop multi- acid onco-tools which have tumor specificities that are far greater than the specificities afforded by current cancer diagnostics and therapeutics (whose specificities are generally below 10), while still providing an acceptable efficacy factor.
  • an efficacy factor as low as about 0.1% may be adequate, but a higher efficacy factor may be more desirable for therapeutic applications where it is necessary to load the tumor cells in acidic areas with a considerable dose of radioisotope sufficient to achieve (via a crossfire effect) complete killing of nearby fast-dividing tumor cells in less acidic areas closer to capillaries.
  • the acid moieties must be sufficiently far apart that ionization of one acid moiety does not significantly suppress ionization of any neighboring acid moieties in that molecule. Based on a survey of known di-acids, it is estimated that this lower limit on separation of acid moieties is about 5 to 7 Angstroms. 2) The acid moieties must be sufficiently close together that ionization of any one acid moiety is sufficient to cause the entire molecule to be repelled from the negatively-charged surfaces of cells. It is estimated that this upper limit on separation of acid moieties is in the range of about 15 to 20 Angstroms.
  • the binomial expansion gives: a 2 + 2ab + b 2 , and the a 2 term constitutes the portion of the molecules which are in the non-ionic cell-penetrating form, while the 2ab and b 2 terms together constitute the portion of the molecules which contain at least one acid moiety in the anionic form.
  • the efficacy factor for an onco-tool containing two acid moieties is:
  • Efficacy factor [ (a 2 ) / ( a 2 + 2ab + b 2 ) ] x 100 where a and b are calculated using the Henderson-Hasselbalch equation and a pH value of 6.4.
  • Tumor specificity factor (Efficacy factor at pH 6.4) / (Efficacy factor at pH 7.4)
  • the binomial expansion gives: a 3 + 3a 2 b + 3ab 2 + b 3 , and the a 3 term constitutes the portion of the molecules which are in the non-ionic form, while the 3a 2 b, 3ab 2 , and b 3 terms together constitute the portion of the molecules which contain at least one acid moiety in the anionic form.
  • the efficacy factor for the 3-acid onco- tool is:
  • Efficacy factor [ (a 3 ) / ( a 3 + 3a 2 b + 3ab 2 + b 3 ) ] x 100 Using the above, one can calculate for molecules containing differing numbers of acid moieties the percent of the molecules of a given specie which will be in the non-ionic form at pH 6.4 (as in tumors) and at pH 7.4 (as in normal tissues). Since only the non-ionic form can penetrate into and through cell membranes, one can use these values both to provide the efficacy factor (ie., percent in non-ionic form at pH 6.4), and to calculate the specificity factor (ie., percent in non-ionic form at pH 6.4 / percent in non-ionic form at pH 7.4)
  • an onco-tool If 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 (anionic) form which repels from the negatively-charged surfaces of cell membranes. Conversely, at the pH present in acidic areas of tumors a significant portion (probably about 0.1% or more) of the onco-tool molecules should switch to a non-ionic form that readily enters cells. These special pH-mediated properties of the onco-tools are predominantly imparted by the pH-switch components. Following are descriptions of the various classes of pH-switch structures used in onco-tools.
  • a conventional pH-switch contains a simple acid moiety, typically with a pKa value in the range of about 4.7 to 5.1.
  • Figure 3 shows a representative conventional pH-switch structure.
  • the methyl groups on the carbon alpha to the carboxylic acid afford a modest increase in the pKa of the adjacent carboxylic acid - to about 5.0.
  • Those methyls alpha to the carboxylic acid also serve to increase lipophilicity and thereby enhance the cell-penetrating ability of the non- ionic form of the molecule in which they are incorporated.
  • novel means devised for achieving this pKa adjustment also serves to increase both the specificity and the efficacy of onco-tools by substantially amplifying the lipophilicity differential between the anionic and the non-ionic forms of the onco-tool - by virtue of loss of two waters of hydration upon formation of an internal acid-specific H-bond.
  • a pH-switch which has a structure designed to form an internal acid-specific H-bond is called an "advanced pH-switch".
  • a number of such advanced pH-switches are shown in Figure 4. Results from molecular modeling and from extensive experimental work suggest that the following two properties are essential in order for an advanced pH-switch to form an acceptably stable internal acid-specific H-bond in aqueous solution.
  • the structure must contain an acid moiety which is positioned in suitable proximity to an H-bond acceptor moiety for formation of an H-bond.
  • an acid moiety When that acid 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".
  • a structure which can form both an internal H-bond when the acid moiety is in its anionic form and an internal H-bond when the acid moiety is in its free-acid form (referred to as a non-acid-specific H-bond) is unacceptable because it has been found to fail to provide the desired increase in the lipophilicity of the acid form, and it has been found to fail to raise the pH at which the structure switches from its anionic hydrophilic form to its non-ionic lipophilic form.
  • the H-bond acceptor moiety and the acid moiety 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 suggest that 4-membered, 5-membered, and 6-membered aliphatic rings are preferred for this purpose.
  • At least one additional property selected from the following three properties, is desirable to achieve formation of an internal H-bond in aqueous solution.
  • the acid moiety 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 acid moiety due to inductive effects from electron-withdrawing groups.
  • a principal challenge in forming a lone H-bond in an aqueous environment is to preferentially form that H-bond in the presence of the vast concentration of competing H-bond donors and H-bond acceptors comprising the surrounding water.
  • the inventor postulated that the desired intramolecular H-bond might be more favored if the H-bonding site were partially shielded from the bulk water by parts of the pH-switch structure.
  • 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 were predicted to appreciably favor the desired intramolecular H-bond in pH- switches.
  • Figure 5 shows a variety of H-bonding moieties which are appropriate for forming low-barrier H-bonds in pH-switches. Structures b, c, d, and e in Figure 6 show several representative pH-switch structures which titration results indicate form H-bonds of the low-barrier type.
  • a) contains an aliphatic ring structure selected from the group consisting of: 4- membered rings, 5-membered rings, and 6-membered rings; b) contains an acid moiety which is directly linked to the aliphatic ring structure; c) said acid moiety in its non-ionic form serves as the H-bond donor moiety; 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) said acid H-bond donor moiety and said H-bond acceptor moiety are positioned in close proximity
  • the cargo component is a structural component of an onco-tool which serves to bind 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 cells 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. 9 illustrates several representative cargo components in both their precursor forms and in their radioisotope-containing final forms ready for diagnostic or therapeutic use.
  • FIG. 9 shows several selected cargo components, in both their precursor forms and their final forms, that satisfy the particular requirements for use in onco-tools.
  • Figure 16 shows synthetic schemes for preparing two such cargo components in their precursor forms. One scheme entails adding a vinyl tri-alkyl tin moiety. Similar synthetic schemes have been described by: Thibonnet, et al., Tetrahedron Letters, Vol.
  • Scheme a of Figure 18 also illustrates a simple procedure for converting the vinyl tin precursor form to the final radioisotope-containing form, as has been described by Zalutsky, page 96 of Chapter 4 titled: Radiohalogens for Radioimmunotherapy, in the book: Radioimmunotherapy of Cancer, Ed. by Abrams and Fritzberg, Pub.
  • 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
  • the selected radioisotope which is to be bound to an onco-tool has a short half-life (a few minutes to a few hours) it is often desirable to make, ship, and store the onco-tool in its precursor form, and then to add the radioisotope cargo shortly before delivering the onco-tool in its final form into the subject to be diagnosed or treated.
  • radioisotope which has a moderately long half-life (about 12 hours or longer) then that radioisotope can be incorporated into the onco-tool in a production setting, the final product purified and quantitated by experienced production personnel, and the resultant onco-tool in its final ready-to- use form then rapidly shipped to the end user - thereby facilitating immediate use by non-specialists as soon as the onco-tool arrives at the medical facility where it is to be used.
  • radioisotopes In onco-tools used for detecting tumors one has considerable latitude in selecting the radioisotope which is to generate the signal suitable for detection outside the subject.
  • radioisotopes with favorable properties for use in diagnostic onco-tools are listed below. These particular radioisotopes generate gamma rays (detectable by a 2-D scan with a gamma camera or a 3-D SPECT scan) and/or positrons (whose photons of annihilation are detected by a PET scan) and they have half-lives sufficiently long that they can be bound to the onco-tool and then that onco-tool in its final ready-to-use form rapidly shipped to the end user.
  • a tumor cell in a hypoxic/acidic area typically can be as much as about 140 microns from the better-oxygenated fast- dividing tumor cells at near-normal pH near capillaries - though in rare cases this distance can probably be larger by two or three fold (probably up to about 400 microns). Because of the mechanism by which onco-tools are sequestered in tumors, onco-tools will likely be at their highest concentration in the most acidic areas of the tumor furthest from capillaries, with a decreasing gradient of onco-tool concentration with increasing pH closer to capillaries.
  • the key challenge in using onco-tools for killing the entire tumor is to assure that the emissions from the radioisotopes sequestered in acidic areas are effective to destroy both the relatively-radiation-resistant quiescent cells in acidic areas as well as the more-radiation-sensitive fast-dividing tumor cells up to about 400 hundred microns from the highest concentrations of sequestered onco-tools.
  • a phenomenon referred to as a crossfire effect is exploited.
  • Radioisotopes of choice for this purpose emit beta particles with particle energies of about 600 thousand electron volts or greater, giving mean path lengths in biological tissues of about 400 microns or greater. It should be noted that such beta-emitting radioisotopes with relatively long path lengths can also be used in combination with radioisotopes with shorter path length emissions.
  • the table below shows a variety of beta-emitting radioisotopes which are suitable for use in therapeutic onco-tools.
  • Achieving a radiation dose sufficient to kill the fast-dividing tumor cells in less acidic areas requires that sufficient onco-tool be sequestered in acidic areas to assure complete killing of the nearby better-perfused more-radiation-sensitive fast- dividing tumor cells closer to capillaries. Determining what dose is required to achieve such killing via a crossfire effect will need to be determined empirically.
  • the onco-tool's very high specificity for tumors should allow one to deliver a dose far more than adequate for complete killing of the entire tumor via a crossfire effect - without incurring undue damage to the patient's normal cells, including fast-dividing cells such as in the bone marrow and lining the intestinal track - such damage commonly being the dose-limiting factor in current tumor treatments.
  • quiescent cells in acidic areas of tumors are generally not undergoing DNA replication they can be significantly more resistant to killing by radiation compared to fast-dividing tumor cells in better oxygenated areas of the tumors. While such quiescent cells can probably be completely killed just by an adequate dose of onco-tool containing a beta-emitting radioisotope, an alternative is to use a combination of both an onco-tool containing a beta-emitting radioisotope for killing the more radiation-sensitive fast-dividing tumor cells near capillaries and a second onco-tool containing an alpha-emitting radiohalogen, Astatine-211, for decisively killing the more-radiation-resistant quiescent cells in acidic areas.
  • Alpha particles emitted by Astatine-211 while having quite short path lengths (only about 50 to 80 microns, which is about 3 to 4 cell diameters) are nonetheless quite effective at killing cells because they deposit a ferocious 2 million electron volts of ionizing energy while traversing a single cell diameter and this is often sufficient to devastate even a non-dividing (quiescent) cell.
  • a beta particle deposits only about 0.02 million electron volts of ionizing energy while traversing a single cell diameter. 4. Onco-Tool Structures
  • each onco-tool must contain two or more pH-switch components. This "two or more" requirement is essential in order to achieve a specificity which is substantially greater (preferably over 30) than is typically provided by current cancer therapies (specificities typically in the range of about 4 to 8). Further, 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 0.1% 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.
  • the following sections describe onco-tools with 2, 3, and 4 pH-switches, as well as onco-tools which contain merged pH-switches and which contain mixed pH- switches. With respect to such onco-tool structures, it should be appreciated that any selected combination of components will generally require optimization of lipophilicity in order to achieve adequate efficacy and a desirable balance between efficacy and specificity. Procedures for such optimizations are described and illustrated later herein in Section B and in Figures and Examples relating to that section.
  • Onco-tools with 2 pH-switches These onco-tools have the simplest structures and are generally the easiest to synthesize. They typically have specificity factors potentially ranging from about 20 to about 90.
  • Figure 10 illustrates a variety of representative two-pH-switch onco- tools which satisfy the key structural requirements for onco-tools. Structure a in Figure 10 shows an onco-tool which contains two conventional pH-switches. Structures b, c, d, and e in Figure 10 show onco-tools, each of which contains two advanced pH-switches. c) Onco-tools with 3 pH-switches
  • Onco-tools containing 3 pH-switches are more complex than the 2-pH-switch onco-tools described above, and are generally more challenging to synthesize.
  • the 3-pH-switch onco-tools have the merit of affording appreciably higher specificity factors, potentially ranging from about 100 to about 800.
  • Figure 11 shows two representative three-pH-switch onco-tools which satisfy the key structural requirements for onco-tools.
  • Onco-tools containing mixed pH-switches are shown in Figure 14.
  • the particular merit of using mixed pH-switches in an onco-tool is it provides an easy and versatile method for adjusting the efficacy and specificity of the onco-tool. It can also provide a simple means for incorporating the cargo component.
  • the first step in developing an onco-tool is to prepare and assess the properties of prospective pH-switch types, as well as variations within each type which serve to impart a wide range of lipophilicities to the structures.
  • pH-switches Conventional pH-switches can typically be made from known compounds in a few steps. For instance, 2,2-Dimethylglutaric acid is converted to its dimethylester and aqueous alkali is then used to convert the di-ester to the mono-acid/mono-ester. The Curtis Rearrangement is then used to generate the amino/ester structure, which is suitable for reacting with one or more other pH-switches plus a cargo componento to give a complete onco-tool, such as structure c of Figure 14. Many other conventional pH-switches can also be easily made using well known reactions.
  • Figure 15 shows a few of the many possible synthetic routes which can bes used to make advanced pH-switches of the types shown in Figure 4.
  • Yongfu Li at GENE TOOLS, LLC has5 developed a simple and convenient method for converting camphanic acid to the useful intermediate: 1-Carboxy-1,2,2-trimethyl-3-keto cyclopentane using lead tetraacetate, followed by alkaline hydrolysis (personal communication).
  • FIG. 15 also shows a synthetic scheme for making representative merged pH-switches (structure a in Figure 7, where R is a hydrogen). 2. Preparation of representative cargo components
  • Figure 9 shows several cargo components suitable for use in onco-tools.
  • Figure 16 illustrates several of the many possible synthetic routes which can be used for preparing the precursor forms of several variations of preferred cargo components.
  • Figure 17 illustrates the synthesis of four representative onco-tools with their cargo component in the precursor form, including one onco-tool that contains merged pH-switches, and another onco-tool that contains mixed pH-switches.
  • Figure 18 illustrates two reactions effective for converting the cargo component of an onco-tool from its precursor form to its final radioisotope-containing form.
  • Figure 19 shows two representative onco-tools in their final radioisotope- containing form.
  • Simple titration assays of pH-switches provide both pKa values and useful information about the pH-dependent solubility properties of pH-switch structures.
  • deareated water be used in order to avoid interference by dissolved carbonic acid (pKa 6.37).
  • pKa 6.37 dissolved carbonic acid
  • 0.2 milliMole of the pH switch in its sodium salt form is suspended in 20 ml of deareated water and the pH is adjusted to between 9 and 10 using NaOH or HCI.
  • the titration is then carried out by adding 2 microLiter aliquots of 5 N HCI while stirring rapidly. After each addition the pH is allowed to stabilize and is then recorded.
  • the titration results are plotted as delta pH on the Y axis and pH on the X axis (referred to as a first-derivative plot).
  • the pH value at the minimum in such a plot corresponds to the pKa value for that pH-switch. It is recommended that a corresponding titration also be carried out on a conventional carboxylic acid (propionic acid, pKa 4.87) in order to validate one's procedures and equipment.
  • the pH-switch is designed to form a low-barrier type H-bond, and so contains a weakly-basic H-bond acceptor moiety such as shown in Figure 5, one will typically see two minimums in the first derivative plot - where the minimum at the higher pH generally corresponds to the pKa value for the acid moiety and the minimum at the lower pH generally corresponds to the pKa value for the H-bond acceptor moiety.
  • the first- derivative plot will appear fairly symmetrical about the minimum corresponding to the pKa value.
  • the above first-derivative plot can appear seriously skewed and during the titration as the pH corresponding to the pKa value is approached one typically observes light scattering (if oiling out) or precipitate formation on each addition of HCI.
  • pH-switch components which are to be used in an onco-tool it is useful to have a reasonable measure of the lipophilicity of their non-ionic cell- penetrating form. This is obtained by assessing their partitioning between water and n-octanol.
  • a reasonable partitioning value for a pH-switch can be obtained by adding 0.2 milliMole of the pH-switch in its non-ionic form to a 50 ml centrifuge tube and adding 20 ml of deareated water and 20 ml of n-octanol. The tube is capped and shaken vigorously and then centrifuged.
  • the top octanol phase is drawn off and the lower aqueous phase titrated with NaOH to pH 9.
  • the amount of NaOH required to reach pH 9 then provides a measure of the proportion of the original onco-tool which has partitioned into the aqueous phase.
  • the non-ionic form of the pH-switch will partition predominantly into the n-octanol phase.
  • the non-ionic form of the pH-switch will partition in substantial part into the aqueous phase.
  • the non-ionic cell-penetrating form of a pH- switch can be moderately hydrophilic (ie., partition predominantly into water) and still function reasonably effectively in an onco-tool. This is evidenced in the case of 11C- DMO which has been used for years in the nuclear medicine field for detecting acidic areas in tumors.
  • 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 15 minutes to a given onco-tool containing a radioisotope (typically lodine-131). In one culture well the onco-tool is in medium buffered at pH 6.4.
  • the onco-tool is in medium buffered at pH 7.4. After incubating 15 minutes at 37 deg. C, the onco-tool- containing medium is removed and the cells are washed thoroughly with 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.
  • 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.
  • results from the initial cell testing may be so far from acceptable that instead of minor adjustments in structure, instead one should select different pH-switch components and/or a different cargo component.
  • 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.3. 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.4. 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, combined with methods of using onco-tools described in Section C below, can be readily adapted for preclinical and clinical testing of onco-tools of the present invention.
  • 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 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). Increased sensitivity can typically be obtained by waiting additional time (eg., one to a few 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 can serve to greatly lower background signal from normal tissues and allow detection of even quite small tumors. In this regard, over the course of an hour to a few hours much 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.
  • additional time e., one to a few hours
  • the rate of excretion of non-sequestered onco-tool can be increased by increasing the subject's fluid intake, particularly if that fluid contains a diuretic.
  • the rate of excretion of onco-tool can be decreased by pre-treatment with Probenecid, as described later herein in Section C.5.
  • 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.
  • modern imaging equipment such as 2-D gamma ray scanners, 3-D SPECT 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 is preferably one which emits a beta particle which has a mean path length in biological tissues greater than about 200 microns, and preferably a mean path length of about 400 microns or greater.
  • Such beta-emitting radioisotopes are effective both to kill cells containing the radioisotope (ie., cells in acidic areas of the tumor), and, via a crossfire effect, to kill cells up to a few hundred microns from cells containing the radioisotope (ie., cells in less acidic areas of the tumor close to capillaries).
  • radioisotopes which have high linear energy transfer emissions are highly effective for killing the more- radiation-resistant quiescent cells in acidic areas of tumors, but the short path length of such emissions (typically about 50 to 80 microns, or about 3 to 4 cell diameters) makes onco-tools containing such radioisotopes rather ineffective for killing the fast- dividing cells in less acidic areas of tumors near capillaries - because such areas will be relatively devoid of sequestered onco-tool.
  • an onco-tool containing a beta-emitting radioisotope can be somewhat less effective against the more-radiation-resistant quiescent cells of the tumor, but an onco-tool containing an appropriate beta-emitting radioisotope has the particular merit of being effective for killing the more-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 fast- dividing tumor cells up to several hundred microns from the sequestered onco-tool.
  • 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 the alpha particle-emitter Astatine- 211) to thoroughly destroy the proximal more-radiation-resistant quiescent cells in acidic areas of the tumor, and the other onco-tool contains a beta-emitting radioisotope to kill the more distant more-radiation-sensitive fast-dividing cells in higher-pH regions near tumor capillaries, which can be largely devoid of sequestered onco-tool.
  • a radioisotope having a high linear energy transfer emission preferably the alpha particle-emitter Astatine- 211
  • the other onco-tool contains a beta-emitting radioisotope to kill the more distant more-radiation-sensitive fast-dividing cells in higher-pH regions near tumor capillaries, which can be largely devoid of sequestered onco-tool.
  • kidneys One of the functions of the kidneys is to maintain the pH in the body at very close to 7.4. To carry out this function the kidneys can excrete urine ranging from moderately basic to fairly acidic. In this process 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. If the urine is acidic at this point then an onco-tool in this acidic environment is expected to bind and enter and remain within the cells lining the proximal tubules of the kidneys - by the same basic mechanism by which onco-tools enter tumor cells from an acidic extra-cellular environment.
  • the excreted urine is sufficiently acidic (below about pH 7.0) a portion of the onco-tool will switch to its non-ionic cell-penetrating form. If that switch to a cell-penetrating form occurs in a region where the urine has good access to cell membranes, such as is the case for cells lining the proximal tubules of the kidneys, then the onco-tool is expected to enter such cells. In the case of a diagnostic onco-tool this entry of onco-tool into cells lining the proximal tubules can lead to excessive signal emanating from the kidneys, which could obscure a tumor in or near the kidneys.
  • One safe and effective substance for rendering the urine basic is the carbonic anhydrase inhibitor drug, Acetazolamide.
  • Acetazolamide One safe and effective substance for rendering the urine basic is the carbonic anhydrase inhibitor drug, Acetazolamide.
  • the pH in acidic areas of tumors can be further reduced by as much as 0.7 pH unit by use of a combination of glucose and meta- iodobenzylguanidine (Kuin et al., (1994) Cancer Research 54. 3785 - 3792).
  • the pH in acidic areas of tumors can be further reduced by vasodilator drugs which are routinely used to treat persons with hypertension (Adachi and Tannock (1999) Oncology Research IJ., 179 - 185).
  • vasodilator drugs are routinely used to treat persons with hypertension (Adachi and Tannock (1999) Oncology Research IJ., 179 - 185). Such drugs are probably effective because the abnormal vasculature of tumors generally lacks vasoconstrictor nerve fibers.
  • Such treatments 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 lower pKa values - which can result in a significant increase in the onco-tool's specificity.
  • an onco-tool carries one or more negative charges and is very hydrophilic, and so it can be excreted very rapidly by the kidneys.
  • sequestering of an onco-tool in the poorly perfused acidic areas of a tumor is in serious competition with excretion of the onco-tool by the kidneys, and this competition between sequestering in tumors and excretion by the kidneys can unduly limit the fraction of an injected dose of onco-tool which ends up in tumors.
  • the kidneys are excreting acidic urine
  • 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 one to a few hours).
  • Such monitoring will allow emergency intervention (such as injecting an additional dose of Acetazolamide to further increase the pH of the urine) 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.
  • therapeutic onco-tools with too low of a pKa may be too poorly sequestered in mildly-acidic areas closer to capillaries in the tumor, thereby leading to inadequate treatment of such areas.
  • 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 effectively sequestered in higher-pH regions of the tumor closer to capillaries, and where another onco-tool with a lower pKa is maximally sequestered in lower-pH regions of the tumor which are further from capillaries.
  • 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 (differing only in the contained radioisotope, or in dose administered to the subject) in general if a given onco-tool structure is effective to detect a tumor, then that same onco-tool structure, but possibly with a different radioisotope or at a much higher concentration, 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 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.
  • 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 may alternatively entail providing at least two onco-tools, where one contains a radioisotope which emits an alpha particle and the other contains a radioisotope which emits a beta particle. It may also entail providing a combination of onco-tools where one has a higher pKa and the other has a lower pKa, such that the two together are more effectively sequestered throughout the tumors. Step 6. The one or more provided onco-tools are delivered into the subject - generally by intravenous injection.
  • micro-metastases When a tumor reaches a substantial size (such as on the order of about 1 centimeter or larger) it often begins to metastasize, wherein single tumor cells or small aggregates of tumor cells are released from the parent tumor, and those released cells then colonize distant sites in the body. These colonies of cells, called micro-metastases, can then grow into new progeny tumors.
  • the difficulty this presents for the onco-tool therapy method is that in the period of time between formation of the micro-metastases and the time it takes such micro-metastases to grow to a size sufficient to generate their own acidic areas (about 1 to 2 millimeters in diameter), those sub-millimeter progeny tumors typically cannot be detected or killed by onco-tools.
  • any progeny micro-metastases smaller than about 1 to 2 millimeters in diameter are expected to survive the onco- tools treatment and ultimately lead to a relapse - though such a relapse may not occur for a number of years after the initial onco-tool treatment.
  • a strategy for solving this micro-metastases problem is to wait after the initial onco-tool therapy for a period of time sufficient for any micro-metastases that might have been present at the time of the therapy to grow to a size where they develop acidic areas (typically about 1 to 2 millimeter in diameter).
  • micro-metastases should not be allowed to grow for a much longer period of time sufficient for them to reach the much larger size (probably about 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.
  • 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 the post-treatment onco-tool diagnostic process at appropriate intervals (perhaps every year or two) continuing for a sufficient length of time (perhaps 6 to 10 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. If and when one of the repeat diagnostic procedures does detect one or more tumors, then the patient would be again treated as described earlier herein.
  • each prospective onco-tool structure should be tested in a relatively simple biological system wherein the onco-tool is 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 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 12- well culture plates. Next, the culture medium is removed from four wells of cells and replaced with the onco-tool-containing medium buffered at pH 7.4.
  • culture medium is removed from another four wells of cells and replaced with the onco-tool medium buffered at pH 6.4.
  • the plates are then incubated at 37 deg. C for 15 minutes.
  • the onco-tool-containing medium is removed and replaced with onco-tool-free medium of the same pH, swirled briefly, and removed. This wash procedure is repeated a total of 4 times.
  • 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 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.
  • the 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.
  • 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
  • mice Following a suitable period of time (on the order of 1 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.
  • a combination of two or three such pre-treatments to selectively reduce pH in the tumors should be employed in order to maximally acidify the hypoxic areas of the tumors - thereby maximizing efficacy and specificity of the onco-tools.
  • an additional pre-treatment with Probenecid may also prove beneficial for increasing efficacy - by virtue of slowing excretion of the onco-tool by the kidneys and thereby allowing greater amounts of the onco-tool to be sequestered in acidic areas of the tumors.
  • 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

Selon l'invention, les onco-instruments sont de compositions permettant de détecter et de traiter les tumeurs contenant des régions hypoxiques/acides caractéristiques des tumeurs malignes dont la taille est supérieure à une taille microscopique. Les onco-instruments présentent les propriétés suivantes : ils sont rejetés par les cellules des tissus normaux, ils sont séquestrés dans les cellules des régions acides des tumeurs, et tout onco-instrument non séquestré dans une tumeur est éliminé de l'organisme par les reins. Préalablement à l'utilisation, on fixe un radio-isotope sélectionné sur l'onco-instrument. L'onco-instrument permet alors de signaler la présence de tumeurs (usage à des fins diagnostiques) ou de détruire lesdites tumeurs (usage à des fins thérapeutiques). Les onco-instruments sont conçus pour offrir un niveau sans précédent de spécificité face aux tumeurs en raison de leur structure multi-acide unique, de leur valeur pKa manufacturée et de leur lipophilicité dépendant du pH. Les propriétés nouvelles dont ils sont dotés sont conçues pour offrir une avancée considérable concernant la sélectivité face aux tumeurs en comparaison de la sélectivité permise dans le cadre conventionnel du diagnostic et du traitement du cancer.
PCT/US2007/021002 2007-03-30 2007-09-29 Molécules non-peptidiques pour la détection et le traitement des tumeurs WO2008121126A1 (fr)

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AU2007350326A AU2007350326A1 (en) 2007-03-30 2007-09-29 Non-peptidic molecules for detecting and treating tumors
MX2009010621A MX2009010621A (es) 2007-03-30 2007-09-29 Moleculas no petidicas, para detectar y tratar tumores.
CA002682567A CA2682567A1 (fr) 2007-03-30 2007-09-29 Molecules non-peptidiques pour la detection et le traitement des tumeurs

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