AU2007350326A1 - Non-peptidic molecules for detecting and treating tumors - Google Patents

Description

WO 2008/121126 PCT/US2007/021002 TITLE: NON-PEPTIDIC MOLECULES FOR DETECTING AND TREATING TUMORS 5. INVENTOR: James E. Summerton, Ph.D. RELATED PATENT APPLICATIONS & INCORPORATION BY REFERENCE: 10 This application is a PCT application filed pursuant 35 USC 363 and claims priority based on PCT Patent Application PCT/US2007/008215, filed on 30 March 2007. 25. The following related utility patent applications are incorporated herein by 15 reference and made a part of this application: US Patent Application No. 11/395,487, filed March 30, 2006, and US Patent Application Nos. 11/449,495 and 11/449,508, both filed on June 7, 2006, by the above named inventor, James E. Summerton, Ph.D. If any conflict arises between the disclosure of the invention in this PCT application and that in the related utility patent applications, 20 the disclosure in this PCT application shall govem. Moreover, any and all U.S. patents, U.S. patent applications, and other documents, hard copy or electronic, cited or referred to in this application are incorporated herein by reference and made a part of this application. 25 TECHNICAL FIELD: This invention relates to compositions which when introduced into a subject are selectively sequestered in acidic areas of tumors within the subject for 30 the purpose of detecting and treating tumors.

WO 2008/121126 PCT/US2007/021002 BACKGROUND OF THE INVENTION: Central challenge for tumor diagnostics and therapies A strong case can be made that the central limitation of current tumor 5 diagnostics and therapies is their inadequate specificity for tumors. For diagnostics this limited specificity results in an inability to detect malignant tumors at a sufficiently early stage that current cancer therapies could provide a good chance of a cure. For therapies this inadequate specificity often precludes use of a sufficiently high treatment dose to completely destroy the entire tumor 10 including particularly quiescent cells in hypoxic/acidic areas. As a result, post treatment relapses commonly occur and ultimately kill the patient. Onco-tools meet this specificity challenge The key advance afforded by the present onco-tool invention is to provide 15 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 20 plague current cancer treatments. Acidic areas in tumors In 1930 the famous physiologist Otto Warburg reported that one of the most universal properties of malignant tumors is their acidity. Subsequent 25 research has shown that this acidity arises because for a tumor to grow larger than about 1 to 2 millimeters in diameter it must induce new blood vessels. Such tumor-induced blood vessels are poorly spaced and abnormal. As a result, areas of tumors more than a few tens of microns from tumor capillaries become hypoxic. Cells in such hypoxic areas either die or switch to glycolytic metabolism 30 - resulting in their excreting lactic acid. Because of the poor circulation in a tumor, that excreted lactic acid builds up in the interstitial space surrounding cells 2 WO 2008/121126 PCT/US2007/021002 in the hypoxic areas of the tumor, as illustrated in Figure 1. This results in a pH as low as 6.0 in areas most distant from capillaries, up to about pH 7.0 close to capillaries. For comparison, the pH in the interstitial space in normal tissues is generally tightly regulated between about pH 7.3 and 7.5. 5 Role of acidic areas in post-treatment relapses 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. Compared to the fast-dividing tumor cells, these slow 10 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 15 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. Consequently, 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 20 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. However, during this cleanup process 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 25 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. 30 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. 3 WO 2008/121126 PCT/US2007/021002 Related composition for detecting tumors having acidic areas 11 C-DMO (Carbon 11-labeled 5,5-Dimethyl-2,4-oxazolidinedione), shown in Figure 2, is a compound which has been used for a number of years for the 5 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 10 within a tumor to a detector outside of the subject. DMO detects tumors containing acidic areas by the following mechanism. At a pH of 7.4 characteristic of the extra-cellular space in normal tissues 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. However, when 15 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-1 1 radioisotope which is incorporated in the DMO structure decays with a 20 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. It should be noted that DMO and the onco-tools of the present invention which are formulated for diagnostic use both work by similar mechanisms to 25 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. However, while 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 30 unprecedented tumor specificity due to their unique molecular design. 4 WO 2008/121126 PCT/US2007/021002 In regard to the specificity of DMO for acidic areas of tumors, at a pH of 6.4 generally achievable in acidic areas of tumors 44.3% of the DMO molecules are in their non-ionic cell-penetrating form, while at a pH of 7.4 in normal tissues 7.4 % of the DMO molecules are in their non-ionic cell-penetrating form. 5 Therefore, for a given concentration of DMO the rate of entry into cells in such acidic areas of a tumor will only be about 6 fold faster than the rate of entry into cells in normal tissues - giving a tumor specificity factor of only 6. In contrast to the case for DMO, onco-tools of the present invention are uniquely designed to provide a tumor specificity factor which can be many-fold 1o 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). In this context it should be appreciated that tumors of such a small size are generally undetectable by current tumor 15 diagnostics. Moreover, 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. Related composition for treating tumors having acidic areas 20 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 25 affords preferential entry into acidic areas of tumors (Kozin et al., Cancer Research Vol. 61, pages 4740 - 4743 (2001)). Chlorambucil preferentially enters cells in acidic areas of tumors due to the following mechanism. At 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 30 repelled from the anionic surface of cells and is excreted by the kidneys. However, when the Chlorambucil enters an acidic area of a tumor a significant 5 WO 2008/121126 PCT/US2007/021002 fraction of the molecules convert to their non-ionic lipid-soluble Torm 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 5 incorporated in the Chlorambucil structure then acts to damage those tumor cells containing the Chlorambucil. It should be noted that both 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 10 and the onco-tools act to damage the cells they have entered. However, it should be noted that with proper selection of the radioisotope cargo, 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 15 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. In regard to specificity of Chlorambucil for acidic areas of tumors, at a pH 20 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 25 the rate of entry of Chlorambucil into cells in normal tissues - giving a tumor specificity factor of only 8. In contrast to the case for Chlorambucil, 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 30 specificity should allow delivery of a sufficient dose of therapeutic onco-tool to completely destroy the entire tumor and thereby prevent post-treatment relapses 6 WO 2008/121126 PCT/US2007/021002 - without causing undue damage to the patient. In this regard it should be appreciated that with 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 5 damaging or killing the patient. Other related compositions for treating tumors containing acidic areas In contrast to the above-described low-specificity agents for detecting (11 C-DMO) and treating (Chlorambucil) tumors containing acidic areas, a number 10 of years ago Dr. James Summerton, the inventor of onco-tools, began the development of novel acid-targeted peptide compositions explicitly designed to exploit the pH differential between normal tissues and acidic areas of tumors, with the objective of providing safer and more effective treatments for a broad range of tumors. Those large peptide compositions, typically with masses in the 15 range of 2,000 to 4,000 daltons, are referred to as "embedder and transporter peptides". Their structures and methods of use for treating tumors containing acidic areas are disclosed in pending US Patent Application No. 11/069,849 and in US Patent No. 7,132,393 issued 7 November 2006. Dr. Summerton is the inventor of both. While such embedder and transporter peptides have been 20 found to be selectively sequestered in acidic areas of tumors in mice, nonetheless, experimental results suggest that such acid-targeted peptides achieve less specificity and efficacy than desired. Those embedder and transporter peptides also appear to be subject to undue re-uptake in the kidneys which is undesirable in diagnostic applications, and unacceptable in therapeutic 25 applications. In view of the limitations of the acid-targeted embedder and transporter peptides, Summerton subsequently embarked on a quest to devise an entirely new class of acid-targeted molecules with the objective of achieving considerably improved specificity, efficacy, and safety relative to the earlier embedder and 30 transporter peptides. The resulting small non-peptide compositions, called "onco tools", have been disclosed by the present inventor, Dr. James E. Summerton, 7 WO 2008/121126 PCT/US2007/021002 only in pending US Patent Application No. 11/395,487 filed on March 30, 2006, and US Patent Application Nos. 11/449,495 and 11/449,508 both filed on June 7, 2006, and in pending PCT Patent Application No. PCT/US2007/008215, filed on 30 March 2007. 5 SUMMARY OF THE INVENTION: The objectives of the present invention are to provide compositions and 10 methods for detecting and 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 15 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. 20 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. However, when the onco-tools perfuse through an 25 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. Once within 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. By this mechanism onco-tools are 30 selectively sequestered in acidic areas of tumors. As a consequence of these processes, when a dose of onco-tool is 8 WO 2008/121126 PCT/US2007/021002 injected into a subject, it that subject has a malignant tumor larger than about 1 to 2 millimeters in diameter (the size where tumors begin to form hypoxic/acidic areas), a portion of the injected dose will become sequestered within cells in acidic regions of the tumor, with the remainder of the dose being excreted by the 5 kidneys. Stated differently, 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. Prior to use of the onco-tool a selected radioisotope will be linked to that onco-tool. That 10 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 15 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' 20 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 25 method for increasing the percent of an injected onco-tool dose which can be sequestered in acidic areas of tumors. 30 9 WO 2008/121126 PCT/US2007/021002 BRIEF DESCRIPTION OF THE FIGURES: Figure 1 illustrates the distribution of acidity in tumors. Figure 2 shows related compositions which are currently used for detecting and 5 treating tumors containing acidic areas. Figure 3 shows a representative conventional pH-switch structure. Figure 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. 10 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. 15 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. Figure 15 illustrates representative syntheses of several pH-switch components. Figure 16 illustrates representative syntheses of several cargo components. 20 Figure 17 illustrates the assembly of several representative onco-tools. Figure 18 illustrates the addition of representative radioisotope cargos. Figure 19 shows two representative onco-tools in their final form. 25 DEFINITIONS OF TERMS: The terms used herein have the following specific meanings, unless otherwise noted. 30 The words "comprising," "having," "containing," and "including," and other forms thereof, are intended to be equivalent in meaning and be open ended in that an 10 WO 2008/121126 PCT/US2007/021002 item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. The words "consisting," "consisting of," and other forms thereof, are intended to 5 be equivalent in meaning and be closed ended in that an item or items following any one of these words is meant to be an exhaustive listing of such item or items and limited to only the listed item or items. PH-switch component - a structural component of an onco-tool which contains an 10 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. Advanced PH-switch component - a pH-switch component designed to form an 15 internal acid-specific hydrogen bond. 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 20 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 25 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. 30 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 11 WO 2008/121126 PCT/US2007/021002 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 5 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 10 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 forr n of the cargo component is 15 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.. 20 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 25 subject undergoing the diagnostic procedure. Therapeutic onco-tool - an onco-tool which contains a radioisotope that is effective to kill cells. 30 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 12 WO 2008/121126 PCT/US2007/021002 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 5 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. 10 Significant portion - about 0.1% or greater. Anionic form - a form which carries at least one negative charge. Non-ionic form -- a form which does not carry an ionic charge. 15 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 20 form at pH 6.4) divided by (percent of onco-tool molecules in non-ionic form at pH 7.4). Effective to report the presence - a radioisotope whose emission, such as a gamma ray or positron, generates a signal which is readily detectable outside 25 that subject. 30 13 WO 2008/121126 PCT/US2007/021002 OUTLINE OF THE SPECIFICATION: A. Molecular Design of Onco-Tools 1. Design Strategy for Unprecedented Specificity 5 2. The pH-Switch Components a) Conventional pH-switches b) Advanced pH-switches i) acid-specific H-bond ii) minimal conformational freedom 10 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 15 3. Cargo Component a) Structural requirements b) Precursor and final forms c) Selection of radioisotope cargo i) for detecting tumors 20 ii) for treating tumors 4. Onco-Tool Structures a) Structural requirements b) Onco-tools with 2 pH-switches c) Onco-tools with 3 pH-switches 25 d) Onco-tools with 4 pH-switches e) Onco-tools with merged pH-switches f) Onco-tools with mixed pH-switches B. Synthesis and Testing of Components and Onco-Tools 30 1. Preparation of representative pH-switches a) Conventional pH-switches 14 WO 2008/121126 PCT/US2007/021002 b) Advanced pH-switches c) Merged pH-switches 2. Preparation of representative cargo components 3. Preparation of representative onco-tools and conversion to final form 5 a) Synthesis of onco-tools with cargo component in precursor form b) Conversion of onco-tool to final form by adding radioisotope 4. Testing pH-switches a) Titration b) Partitioning 10 5. Testing onco-tools a) Assess sequestering in cultured cells at pH 6.4 and pH 7.4 b) Assess disposition in normal mice c) Assess disposition in tumor-bearing mice d) Pre-clinical and clinical testing 15 C. Methods of Using Onco-Tools 1. Diagnostic method 2. Therapeutic method 20 a) Single-radioisotope method b) Dual-radioisotope method 3. Treat to increase pH in urine to protect kidneys from damage 4. Treat to decrease pH in tumors for increased efficacy and specificity 5. Treat to decrease rate of excretion by kidneys for increased efficacy 25 6. Monitor bladder for improved safety 7. Flush bladder for improved detection and safety 8. Use multiple onco-tools varying in pKa to increase efficacy 9. Comprehensive method for detecting and treating tumors 10. Strategy for dealing with micro-metastases 30 15 WO 2008/121126 PCT/US2007/021002 MODES FOR CARRYING OUT THE INVENTION: A. Molecular Design of Onco-Tools 5 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 10 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 15 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 20 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. 1. Design Strategy for Unprecedented Specificity 25 Inherent in the structure of onco-tools are acid moieties which undergo a switch between an anionic cell-repelling form at a higher pH and a non-ionic cell penetrating form at a lower pH. For such structures one can use the Henderson Hasselbalch equation [ pH = pKa - log (acid/anion) ] to calculate the fraction of the molecules which will be in their non-ionic cell-penetrating form at any selected 30 pH. For practical purposes the two pH values of interest are pH 6.4 which is generally achievable in the interstitial space in acidic areas of tumors, and pH 7.4 16 WO 2008/121126 PCT/US2007/021002 which is typical for the interstitial space in normal tissues. To illustrate, for a molecule containing a typical carboxylic acid moiety having a pKa of 4.8 one can calculate that at pH 6.4 (in acidic area of tumor) 2.5% of the molecules will be in their non-ionic cell-penetrating form, while at pH 7.4 (in normal tissue) only 0.25% 5 of the molecules will be in their non-ionic cell-penetrating form. Thus, the percent of the molecules in their cell-penetrating form will be 10 times as high in acidic areas of a tumor as in normal tissues - ie., 2.5% / 0.25% = 10. I call 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 10 how specifically the molecules can be sequestered in acidic areas of tumors. To put this tumor specificity factor of 10 in perspective, 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 15 only about on a par with or slightly better than monoclonal antibodies in regard to specificity for tumors. Strategy for dramatically increasing specificity Relatively simple calculations utilizing the Henderson-Hasselbalch 20 equation, combined with a binomial expansion, indicate that if certain structural design constraints are met then the specificity of a molecule for acidic areas of a tumor should be dramatically increased by incorporating multiple acid moieties in the molecule. To illustrate, below are calculated tumor specificity values for molecules containing 1, 2, 3, and 4 typical acid moieties, each with a pKa of 4.8. 25 Number of acid moieties in molecule Tumor specificity factor 1 10 2 96 3 935 30 4 9,147 17 WO 2008/121126 PCT/US2007/021002 From this table it can be seen that as the number of acid moieties goes up linearly the specificity factor goes up exponentially. The daunting cost of increasing specificity The foregoing table shows that a multi-acid design strategy can afford dramatically greater specificities than are achieved by any current tumor diagnostic or therapeutic. However, it turns out that with this design strategy the dramatic 5 increases in specificity are counter balanced by corresponding dramatic decreases in efficacy. Thus, in designing acid-targeted molecules for detecting and treating tumors an additional crucial factor must also be taken into account, that being what I call the "efficacy factor". This is the percent of the molecules which are in their non ionic cell-penetrating form at the pH 6.4 achievable in hypoxic/acidic areas of tumors. 10 It is crucial that this efficacy factor have a reasonably high value because it constitutes the fraction of the molecules at any given time which are in a form able to penetrate into cells in acidic areas of tumors. If the efficacy factor is too small then insufficient molecule will be sequestered in the tumors. The following table illustrates the relationship between tumor specificity factor and efficacy factor for 15 molecules containing 1, 2, 3, and 4 typical acid moieties, each with a pKa of 4.8. Number of acid moieties in molecule Specificity factor Efficacy factor 1 10 2.5% 2 96 0.06% 20 3 935 0.0015% 4 9,147 0.000036% From the above table it is seen that as the number of acid moieties in the molecule is increased linearly the specificity factor increases exponentially, while the 25 efficacy factor decreases exponentially. The result is that with an increasing number of acid moieties the structures become dramatically more specific for acidic areas of 18 WO 2008/121126 PCT/US2007/021002 tumors, but concomitantly their efficacy can become vanishingly low - even for structures containing just two acid moieties. This brings us to the crucial question: can a multi-acid structure be designed and developed wherein very high specificity is achieved while avoiding much of the 5 accompanying efficacy decrease ? Design strategy for both unprecedented specificity and adequate efficacy The principle design challenge for onco-tools was to devise and implement a means for achieving the dramatically increased level of specificity that comes from io incorporating multiple acid moieties, while at the same time avoiding much of the efficacy decrease that can also come from incorporating those same multiple acid moieties. To achieve this end two design strategies are used in combination. The first entails adjusting the number of non-polar groups in the onco-tool structure in order to 15 optimize its cell-penetrating ability. In this regard, 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. However, 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 20 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 (in normal tissues) 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 25 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 30 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. In 19 WO 2008/121126 PCT/US2007/021002 the course of research carried out in support of the present invention a variety of such advanced pH-switches have been synthesized, and it has been demonstrated that 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 5 appropriately positioned for forming an internal acid-specific H-bond. More important, 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. 10 It is also noteworthy that 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 15 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 20 course of this onco-tools development program. Number of acid moieties in molecule 1 2 3 4 pKa of acid moieties = 5.5 25 Tumor specificity factor: 9 81 727 6542 Efficacy factor: 11% 1.3% 0.14% 0.02% pKa of acid moieties = 6.0 Tumor specificity factor: 7 55 411 3059 Efficacy factor: 28% 8% 2.3% 0.7% 30 pKa of acid moieties = 6.6 Tumor specificity factor: 4 20 90 403 Efficacy factor: 61% 38% 23% 14% 20 WO 2008/121126 PCT/US2007/021002 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 5 factor. In this regard, with suitable adjustment of the lipophilicity of the onco-tool 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 io less acidic areas closer to capillaries. Constraints on molecular structure The positioning of the component parts of the onco-tool should satisfy three requirements in order to achieve the specificity advantages resulting from 15 incorporation of multiple acid moieties 1) 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. 20 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. 3) When the onco-tool is in its anionic form the hydrophilic and the lipophilic 25 surfaces of the molecule should be fairly well dispersed - with no large lipophilic patches. This is because lipophilic surface areas of substantial size in the anionic form of the onco-tool can lead to undesirable hydrophobic interactions with cell surfaces and other structures in normal tissues - resulting in reduced specificity for tumors. 30 Calculating efficacy and specificity values One can calculate for a molecule containing 1 or more acid moieties the percent of those molecules which will be in the non-ionic cell-penetrating form at 21 WO 2008/121126 PCT/US2007/021002 both pH 6.4 (achievable in the interstitial space in acidic areas of tumors) and pH 7.4 (characteristic of the interstitial space in normal tissues). To do such calculations simply use the Henderson-Hasselbalch equation to calculate the ratio of the portion of the acid moieties which are in the free-acid form (a) and in the anionic form (b) at 5 a selected pH (ie., 6.4 for acidic areas of tumors, and 7.4 for normal tissues). For molecules containing multiple acid moieties having the same pKa values one then also uses a binomial expansion: (a + b)" where n is the number of acid moieties in the molecule. 10 Thus for a molecule containing 2 acid moieties 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: 15 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. To calculate the tumor specificity factor one calculates the efficacy factor for 20 the molecule at a pH value of 6.4, and then again for the molecule at a pH value of 7.4. Using these two values one then calculates: Tumor specificity factor = (Efficacy factor at pH 6.4) / (Efficacy factor at pH 7.4) 25 For a molecule containing 3 acid moieties, each having the same pKa value, 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. In this case the efficacy factor for the 3-acid onco 30 tool is: Efficacy factor = [(a 3 )/ ( a 3 + 3a 2 b + 3ab 2 + b 3 ) ] x 100 22 WO 2008/121126 PCT/US2007/021002 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 5 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) For cases where the acid moieties of the onco-tool have differing pKa values, as is the case for onco-tools containing mixed pH-switches, for a two-pH-switch 1o onco-tool one uses the Henderson-Hasselbalch equation to calculate the (a) value for one of the acid moieties (designated: a 1 ) and then calculates the (a) value for the other acid moiety (designated: a 2 ). Then, rather than the a 2 term of the binomial expansion constituting the portion of the molecules which are in the non-ionic cell penetrating form, instead (a1)(a 2 ) constitutes the portion of the onco-tool molecules 15 which are in the non-ionic cell-penetrating form. Likewise, for an onco-tool containing three acid moieties, each with a different pKa value, one uses (a1)(a 2 )(a 3 ) to calculate the portion of the onco-tool molecules which are in the non-ionic cell-penetrating form. 20 2. The pH-Switch Components 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 25 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 30 pH-switch structures used in onco-tools. a) Conventional pH-switches A conventional pH-switch contains a simple acid moiety, typically with a pKa 23 WO 2008/121126 PCT/US2007/021002 value in the range of about 4.7 to 5.1. Figure 3 shows a representative conventional pH-switch structure. In this particular 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 5 to increase lipophilicity and thereby enhance the cell-penetrating ability of the non ionic form of the molecule in which they are incorporated. b) Advanced pH-switches Earlier herein a multi-acid design strategy was described for achieving an 10 unprecedented increase in specificity for tumors containing acidic areas. Two ancillary strategies (adjusting lipophilicity and adjusting pKa), were also described which calculations suggest can be used to avoid incurring an undue decrease in the efficacy of such multi-acid onco-tool compositions. The strategy of adjusting the pKa of one or more of the acid moieties to within a fairly narrow range calculated to be 15 suitable for both high tumor specificity and adequate efficacy appears to be unique to onco-tools. Further, the 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 20 internal acid-specific H-bond. In regard to the means for adjusting the pKa of acid moieties of onco-tools, initially the inventor (James E. Summerton) predicted that formation of an internal acid-specific H-bond in a pH-switch component should favor the transition of the acid moiety of a pH-switch component to its free-acid form. This, in turn, was predicted to 25 result in a significant increase in the pKa value for that acid moiety. In regard to designing pH-switch structures capable of forming an internal H bond in an aqueous environment, experts in hydrogen bonding typically suggest that a lone hydrogen bond will not be stable in an aqueous environment because of competition with waters vast concentration of H-bond acceptor sites (110 Molar) and 30 H-bond donor sites (110 Molar). Instead, it is commonly believed that a stable non covalent interaction in aqueous solution requires a multiplicity of interactions selected from H-bonds, hydrophobic interactions, and electrostatic interactions. While this requirement for a multiplicity of non-covalent bonds appears to be 24 WO 2008/121126 PCT/US2007/021002 well established for inter-molecular interactions, the inventor postulated that, contrary to the conventional wisdom, it might be possible to devise compact structures which will form a single relatively stable pH-dependent acid-specific intra-molecular H-bond in aqueous solution, where that H-bond both increases the hydrophilicity/lipophilicity 5 differential between the two forms, and serves to significantly favor the free-acid form over the anionic form (ie., raise the pKa). The crucial question then was: could practical pH-switch structures be devised which would form such an internal acid specific H-bond in aqueous solution? After considerable experimentation, novel structures have been devised and 10 prepared and have been demonstrated to form the desired single pH-dependent internal acid-specific H-bond, and such structures are suitable for incorporation into onco-tools of the present invention. As initially predicted by the inventor, a number of such internal acid-specific H-bonds have now been demonstrated to significantly increase the pH at which the structure switches from its anionic form to its non-ionic 15 form, relative to the pH at which a similar acid moiety undergoes this transition. Further as predicted, such an intemal acid-specific H-bond also appears to significantly enhance the hydrophilicity/lipophilicity differential when the pH-switch goes from its anionic form to its non-ionic form. A pH-switch which has a structure designed to form an internal acid-specific 20 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. 25 i) acid-specific H-bond 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. When that acid moiety is in its free-acid form it must serve as the H-bond donor, and the proximal H 30 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". Conversely, a structure which can form both an internal H-bond when the acid 25 WO 2008/121126 PCT/US2007/021002 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 5 from its anionic hydrophilic form to its non-ionic lipophilic form. ii) minimal conformation freedom 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 10 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. 15 In addition to the foregoing 2 essential properties, it appears that at least one additional property, selected from the following three properties, is desirable to achieve formation of an internal H-bond in aqueous solution. iii) acid moiety insulated from inductive effects 20 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. 25 iv) partial shielding of H-bonding site 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. Based both on biochemical studies of enzyme catalytic sites and on the inventor's 30 extensive molecular modeling, the inventor postulated that the desired intra molecular 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. In the context of partial shielding from the solvent, titration studies comparing an advanced pH-switch 26 WO 2008/121126 PCT/US2007/021002 (structure a of Figure 4) and a corresponding structure lacking the three methyl groups, gave results which suggest that partial shielding of the H-bonding site from the solvent by a methyl and a methylene group of the pH-switch does indeed substantially favor the desired intramolecular H-bond. 5 v) low-barrier H-bond The term "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 10 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 15 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. Below is a summation of properties which have been found to be essential for 20 an advanced pH-switch component designed to form an internal acid-specific hydrogen bond: 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; 25 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; 30 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 to each other and are properly positioned and 27 WO 2008/121126 PCT/US2007/021002 oriented such that they are compatible with formation of an internal acid specific H-bond. c) Merged PH-switches 5 With the goal of generating very compact onco-tools, merged pH-switch structures have been devised wherein two acid H-bond donor moieties can simultaneously H-bond to a single H-bond acceptor moiety. In the context of onco tool design for high specificity such merged pH-switches count as two pH-switches because each contains two acid moieties which act relatively independently of each 10 other with respect to pH-mediated shifts between anionic and non-ionic forms. In such merged pH switches the very-electron-rich oxygen of an N-oxide is particularly suitable as the single H-bond acceptor moiety that serves to H-bond to two acid H-bond donor moieties. Figure 7 shows two such merged pH-switches, and Figure 13 shows onco-tools containing such merged pH-switches. 15 d) Adiust lipophilicity of PH-switch In addition to assuring that an acceptable percent of onco-tool molecules are in their cell-penetrating form at the pH in acidic areas of tumors, it is also necessary that the cell-penetrating form be sufficiently lipophilic that it can readily enter cell 20 membranes. Stated differently, two key factors which determine how effectively an onco-tool is sequestered in acidic areas of a tumor are: 1) the efficacy factor, which is the percent of the onco-tool molecules which are in their non-ionic cell-penetrating form at pH 6.4 (achievable in tumors); and, 2) the lipophilicity of that non-ionic form, which determines its propensity to partition from the acidic aqueous extra-cellular 25 medium into the lipophilic cell membrane of nearby tumor cells. Figure 8 shows related pH-switch structures which vary substantially in their lipophilicity. Initially it was useful to obtain a quantitative assessment of how increasing lipophilicity of the non-ionic form of the pH-switch affects its partitioning properties (ie., how it partitions between an aqueous phase and a lipophilic phase such as 30 octanol or a cell membrane). To this end, mathematical modeling was carried out to calculate the pH-dependent transition between forms of a weak-acid substance which exists at high pH in an anionic water-soluble form, [A-], and which converts at a lower pH to a non-ionic lipophilic form which has some aqueous solubility, 28 WO 2008/121126 PCT/US2007/021002 (HAaqueous], and which can also become water-insoluble/lipid soluble, denoted by [HAoctanol], by virtue of partitioning into a lipophilic phase such as octanol or a cell membrane. Results from that modeling indicated that the pH of the mid-point of the 5 transition between the anionic form, [A-], and the non-ionic forms, [ HAaqueous] plus [ HAoctanol ], progressively increases as the octanol/water partitioning coefficient, P, of the non-ionic free-acid form is increased. However, as noted earlier it should be appreciated that incorporating excessive lipophilic moieties as the sole means for increasing the rate of entry of the 10 nonionic form into cells can be counter productive. This is because when a very high lipophilicity is generated solely by incorporation 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 is large surface and the hydrophilic moieties are well separated from this lipophilic surface. In cases where onco-tool is sequestered in both tumor and normal tissues the result will be high background signal in the diagnostic application, and significant toxicity in the therapeutic application. 20 3. Cargo Component a) Structural requirements The cargo component is a structural component of an onco-tool which serves 25 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 30 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 29 WO 2008/121126 PCT/US2007/021002 radioisotopes are killing cells of the tumor. iii) 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 5 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 1o specificity. Based on these requirements for the cargo component, it appears that radiohalogens selected from F, Br, I and At constitute the best radioisotope types, and that a vinyl group or a single unsaturated ring or single aromatic ring will best serve for binding the radiohalogen. Figure 9 illustrates several representative cargo 15 components in both their precursor forms and in their radioisotope-containing final forms ready for diagnostic or therapeutic use. b) Precursor and final forms While a large number of different structures have been reported in the nuclear 20 medicine field which are suitable for binding a wide variety of radioisotopes, many such structures are inappropriate for use in onco-tools because they would dominate the onco-tool's hydrophilicity/lipophilicity properties. Figure 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 25 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. 39, page 4277 (1998); Miyake & Yamamura, Chemistry Letters, pages 981 - 984 (1989); Marshall & Bourbeau, Tetrahedron Letters, Vol. 44, pages 1087 - 1089 (2003); and, Corriu, 30 Geng, & Moreau, J. Org. Chem. Vol. 58, page 1443 (1993). 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: 30 WO 2008/121126 PCT/US2007/021002 Radioimmunotherapy of Cancer, Ed. by Abrams and Fritzberg, Pub. by Marcel Dekker, Inc. (2000). Procedures for preparing other suitable cargo components and incorporating a radiohalogen to give the final form are described in: Zalutsky et al., Proc. Nat. Acad. Sci. USA, Vol. 86, Pages 7149 - 7153 (1989); and, Vaidyanathan & 5 Zalutsky, Nature Protocols, Vol. 1, Pages 1655 -1661 (2006). Reactions are also shown for preparing a preferred vinyl silane moiety which has been found to be stable to harsher synthetic conditions than vinyl tin moieties. Procedures for preparing vinyl silane moieties are modeled after a report by Stamos, Taylor, and Kishi in Tett. Left., Vol. 37, page 8647 (1996), and modeled after 10 synthetic procedures developed by Patrick McDougal at Reed College (personal communication). Schemes b and c of Figure 18 show procedures for converting such vinyl silanes to their radioisotope-containing final forms. Procedures for displacing the silane moiety with a halogen are modeled after reports by: Hartig, Krohn, Hirshman in Anal. Biochem. Vol. 144, page 441 (1985); Mason, Smith, 15 Danzo, Clanton, in Journal of Labeled Compd. Radiopharm., Vol. 31, page 729 (1992); Miller, McGarvey, in Journal of Org. Chem., Vol. 43, page 4424 (1978); Kalbalka, Mereddy, Green, in Labeled Compd. Radiopharm., Vol. 49, page 11 (2006); Kassiou, et. al., in Joumal of Labeled Compd. Radiopharm., Vol. 43, page 339 (2000); and, reactions developed by Patrick McDougal at Reed College 20 (personal communication). c) Selection of radioisotope cargo It should be noted that it is the bound radioisotope of the onco-tool which determines the application of that onco-tool. If the bound radioisotope emits a signal 25 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. Furthermore, if the onco-tool contains a radioisotope, such as lodine-131, which both emits a signal which is readily 30 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. When the selected radioisotope which is to be bound to an onco-tool has a 31 WO 2008/121126 PCT/US2007/021002 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. Alternatively, if a radioisotope is used which has a moderately 5 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 10 be used. i) for detecting tumors In onco-tools used for detecting tumors one has considerable latitude in selecting the radioisotope which is to generate the signal suitable for detection 15 outside the subject. Several 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 20 that onco-tool in its final ready-to-use form rapidly shipped to the end user. Radioisotope Half-life lodine-123 13 hours Bromine-76 16 hours 25 Bromine-77 57 hours lodine-124 100 hours lodine-131 194 hours ii) for treating tumors 30 The mean spacing between capillaries in tumors is about 280 microns (but only 160 microns in normal tissues). Thus, 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 32 WO 2008/121126 PCT/US2007/021002 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 5 concentration with increasing pH closer to capillaries. Thus, 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 10 400 hundred microns from the highest concentrations of sequestered onco-tools. To meet this challenge of killing tumor cells up to several hundred microns from the sequestered onco-tools a phenomenon referred to as a crossfire effect is exploited. In the case of a therapeutic onco-tool this requires the use of a radioisotope whose emission is quite effective for killing cells and has a sufficiently 15 long path length that it reaches the fast-dividing tumor cells in higher-pH regions near capillaries. 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 20 combination with radioisotopes with shorter path length emissions. In the context of killing the fast-dividing tumor cells near capillaries, such tumor cells are most sensitive to killing at the cell cycle stage wherein the DNA is being replicated. Thus, maximum cell killing can best be achieved when the sequestered radioisotopes continue to emit for a period of time sufficient for the 25 target cells to go through one or more cycles of DNA replication. This calls for a radioisotope with a half-life in the range of at least several hours up to several days. The table below shows a variety of beta-emitting radioisotopes which are suitable for use in therapeutic onco-tools. 30 Radioisotope Half-life Iodine-132 2.3 hours Bromine-83 2.4 hours Iodine-135 7 hours 33 WO 2008/121126 PCT/US2007/021002 lodine-1 30 12 hours lodine-1 33 21 hours Bromine-82 36 hours Iodine-131 194 hours 5 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 10 achieve such killing via a crossfire effect will need to be determined empirically. In this context, it is likely that the onco-tool's very high specificity for tumors (many fold higher than current tumor therapies) 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 15 such as in the bone marrow and lining the intestinal track - such damage commonly being the dose-limiting factor in current tumor treatments. Because 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 20 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-21 1, for 25 decisively killing the more-radiation-resistant quiescent cells in acidic areas. Alpha particles emitted by Astatine-21 1, 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 30 devastate even a non-dividing (quiescent) cell. For comparison, a beta particle deposits only about 0.02 million electron volts of ionizing energy while traversing a single cell diameter. 34 WO 2008/121126 PCT/US2007/021002 4. Onco-Tool Structures a) Structural requirements 5 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 io 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. 15 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 20 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. b) Onco-tools with 2 PH-switches 25 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. 30 Structures b, c, d, and e in Figure 10 show onco-tools, each of which contains two advanced pH-switches. 35 WO 2008/121126 PCT/US2007/021002 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. However, the 3-pH-switch onco-tools have the merit of affording appreciably higher 5 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. d) Onco-tools with 4 pH-switches 10 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. However, the 4-pH-switch onco-tools have the potential for even higher specificity factors, potentially ranging from about 400 to about 4000. Figure 12 illustrates two representative onco-tools, each of which contains four advanced pH 15 switches, and which satisfy the key structural requirements for onco-tools. e) Onco-tools with merged PH-switches Two onco-tools containing merged pH-switches are shown in Figure 13. The use of such merged pH-switches can afford very compact onco-tool structures which 20 can also be fairly easy to synthesize. f) Onco-tools with mixed pH-switches 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 25 versatile method for adjusting the efficacy and specificity of the onco-tool. It can also provide a simple means for incorporating the cargo component. B. Synthesis And Testing Of Components And Onco-Tools 30 1. Preparation of representative pH-switches The first step in developing an onco-tool is to prepare and assess the 36 WO 2008/121126 PCT/US2007/021002 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. a) Conventional pH-switches 5 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 component to 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. b) Advanced pH-switches Figure 15 shows a few of the many possible synthetic routes which can be is used to make advanced pH-switches of the types shown in Figure 4. In addition, synthetic procedures for preparing key precursors (see Figure 15) used in making several advanced pH-switches have been reported in the following sources: 1) Goldman, Jacobsen, and Torssell, Synthesis in the Camphor Series. Alkylation of Quinones with Cycloalkyl Radicals. Attempted Synthesis of Lagopodin 20 A and Desoxyhelicobasidin. Acta Chemica Scandinavica B 28 (1974) 492 -500; and, 2) Brian Thomas Connell, Synthesis and Evaluation of a New Camphor-Derived Lactam as a General Chiral Auxiliary for the Asymmetric Diels-Alder and Aldol Reactions. Thesis submitted to the Dept. of Chemistry, University of Rochester, Rochester, New York (1995). Furthermore, Yongfu Li at GENE TOOLS, LLC has 25 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). c) Merged pH-switches 30 Figure 15 also shows a synthetic scheme for making representative merged pH-switches (structure a in Figure 7, where R is a hydrogen). 37 WO 2008/121126 PCT/US2007/021002 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 5 for preparing the precursor forms of several variations of preferred cargo components. 3. Preparation of representative onco-tools and conversion to final form 10 a) Synthesis of onco-tools with cargo component in precursor form 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. 15 b) Conversion of onco-tool to final form by adding radioisotope 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. 20 Figure 19 shows two representative onco-tools in their final radioisotope containing form. 4. Testing pH-switches 25 a) Titration Simple titration assays of pH-switches provide both pKa values and useful information about the pH-dependent solubility properties of pH-switch structures. In such titrations it is recommended that deareated water be used in order to avoid 30 interference by dissolved carbonic acid (pKa 6.37). Typically 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 38 WO 2008/121126 PCT/US2007/021002 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 5 conventional carboxylic acid (propionic acid, pKa 4.87) in order to validate one's procedures and equipment. It should be noted that when 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 10 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. When the low-pH form of the pH-switch remains soluble during the course of the titration (ie., its concentration remains below its aqueous solubility limit) the first 15 derivative plot will appear fairly symmetrical about the minimum corresponding to the pKa value. However, in cases where the low-pH form of the pH-switch oils out or precipitates out during the course of the titration (because the aqueous solubility limit of its non-ionic form has been exceeded) the above first-derivative plot can appear seriously skewed and during the titration as the pH corresponding to the pKa value is 20 approached one typically observes light scattering (if oiling out) or precipitate formation on each addition of HCI. In such cases it is recommended that one carry out a second titration in methanol/water, 1:1 by volume. In titrations in this solvent one generally avoids exceeding the solubility limit for the non-ionic form of the pH switch and so the pH-switch remains soluble throughout the titration, and the first 25 derivative plot appears symmetrical about the minimum corresponding to the pKa value. However, it should be appreciated that titrations in methanol/water, 1:1 by vol. give pKa values about 1 pH unit above the true pKa in water. Therefore, one should also run a control titration in the same solvent using propionic acid, and then use the difference between its measured pKa value in methanol/water and its known 30 pKa value in water (pKa = 4.87) to calculate a corrected pKa value in water for the pH-switch being assessed. It should also be noted that advanced pH-switches which do not form a low barrier type H-bond, such as structure a of figure 4, often appear to be largely 39 WO 2008/121126 PCT/US2007/021002 internally-H-bonded when in their non-ionic form when the solvent is methanol/water, 1:1 by vol. However when the solvent is water only, it appears that much of the non ionic form exists in the non-internally-H-bonded form. In contrast, for many advanced pH-switch types designed to form a low-barrier type H-bond it appears that 5 the non-ionic form of the pH-switch exists predominantly in the H-bonded form in both methanol/water mixtures, and in water only. In particular, the non-ionic forms of structures b, c, d, and f appear to exist predominantly in their internally-H-bonded form in water. 10 b) Partitioning In selecting the 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 is 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 20 which has partitioned into the aqueous phase. For the more lipophilic pH-switches, such as structure d of Figure 4, the non ionic form of the pH-switch will partition predominantly into the n-octanol phase. For the more hydrophilic pH-switches, such as structures b and f of Figure 4, the non ionic form of the pH-switch will partition in substantial part into the aqueous phase. 25 However, it should be appreciated that 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 11 C DMO which has been used for years in the nuclear medicine field for detecting acidic areas in tumors. In octanol/water partitioning studies this weak-acid substance in its 30 non-ionic form partitions almost exclusively into the aqueous phase, but that same non-ionic form is nonetheless still capable of penetrating cell membranes at a rate sufficient to serve as a diagnostic for acidic areas of tumors. 40 WO 2008/121126 PCT/US2007/021002 5. Testing onco-tools a) Assess sequestering in cultured cells at PH 6.4 and PH 7.4 5 It is recommended that each new onco-tool initially 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. As described in Example 1, a preferred biological system for such initial testing comprises mammalian cells cultured in serum-containing medium buffered at pH 7.4 to emulate 10 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. In the other culture well the onco-tool is in medium buffered at pH 7.4. After incubating 15 minutes at 37 deg. C, the onco-tool 15 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 20 the cells at pH 6.4, but minimally sequestered by the cells at pH 7.4. In the above test if the onco-tool shows excessive entry into cells at pH 7.4 this suggests that its lipophilicity is too high and should be reduced - generally by removing lipophilic groups. Conversely, if the onco-tool shows inadequate entry into cells at pH 6.4 this suggests that its lipophilicity is probably too low and should be 25 increased by adding more lipophilic groups. Alternatively, 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. 30 b) Assess disposition in normal mice The above cell culture assays are relatively fast, simple, provide quantitative results, and are amenable to initial testing of a substantial number of onco-tool structures. However, it should be appreciated that this initial cell culture screening 41 WO 2008/121126 PCT/US2007/021002 system does not perfectly emulate the true complexity of a living subject. Accordingly, it is desirable to next take the most promising onco-tool structures and test them in living mammals. As a first step in such animal testing, it is useful to test these onco-tool structures in normal mice (pre-treated to assure that their urine is 5 slightly basic; see Section C.7. below), as described in Example 2. Such tests in normal mice allow one to discard any onco-tools which are found to exhibit an excessive affinity for normal tissues. c) Assess disposition in tumor-bearing mice 10 While the above relatively simple tests in normal tumor-free mice allow one to discard those onco-tools which have an excessive affinity for normal tissues, the more decisive test for a prospective onco-tool structure is to test it in tumor-bearing mice, as described in Example 3. Briefly, this entails injecting (preferably intravenous) each of the lodine-131-containing onco-tools into several tumor-bearing 15 mice, and then waiting a suitable period of time (a minimum of 1 hour up to a maximum of 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. One then terminates the mice and excises the major organs and any obvious tumors, followed by quantitation of the radiation emissions from the excised organs, 20 tumors, and remaining carcass. 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 25 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. 30 In the event the onco-tool is excreted by the kidneys so quickly that little onco tool has time to perfuse through and become sequestered within the tumors, still another pre-treatment may be desirable in order to improve efficacy. This entails pre-treating with probenecid, a drug which reduces the rate of excretion by the 42 WO 2008/121126 PCT/US2007/021002 kidneys by about two to three fold - as described later herein in Section C.5. d) Preclinical and clinical testing Procedures for testing radioisotope-containing substances in live animals, 5 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. 10 C. Methods Of Using Onco-Tools 1. Diagnostic Method 15 Because 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 20 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 25 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 30 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 43 WO 2008/121126 PCT/US2007/021002 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. It is expected that typically in subjects 5 who do not have tumors most of the onco-tool will be excreted in less than 24 hours, and possibly in less than about 1 hour. 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. Alternatively, the rate of excretion of onco-tool can be decreased by pre-treatment with Probenecid, as described later herein in Section io 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. With modem imaging equipment, such as 2-D gamma ray 15 scanners, 3-D SPECT scanners, and PET scanners, tumors should show up as an obvious radioisotopic hot spot at the site of each tumor. 2. Therapeutic Method 20 Because 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 25 for both veterinary medicine and human medicine. The therapeutic methods generally include, but are not limited to the following. a) Single-radioisotope method If a subject has been found to have one or more tumors, those tumors can be 30 treated with a single therapeutic onco-tool containing a radioisotope effective to kill cells. 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 44 WO 2008/121126 PCT/US2007/021002 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 5 intravenous injection. In this therapeutic method 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 10 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). b) Dual-radioisotope method 15 As noted in earlier herein, radioisotopes which have high linear energy transfer emissions (such as alpha particles) 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 20 dividing cells in less acidic areas of tumors near capillaries - because such areas will be relatively devoid of sequestered onco-tool. Conversely, 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 25 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. 30 Accordingly, 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 45 WO 2008/121126 PCT/US2007/021002 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. 5 3. 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 10 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 15 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. Stated differently, if 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 20 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 25 obscure a tumor in or near the kidneys. In the case of a therapeutic onco-tool this entry of onco-tool into cells lining the proximal tubules is expected to cause killing of the cells lining the proximal tubules - effectively rendering the kidneys non-functional. Luckily, there are methods well known in the medical arts for raising the pH of a subject's urine for sufficient time to carry out diagnosis or therapy with onco-tools. 30 By using a suitable drug to maintain the urine at a slightly basic pH during the period when most of the non-sequestered onco-tool is being excreted by the kidneys, that onco-tool excreted in the urine will remain in its anionic cell-repelling form and be safely passed from the body by urination. One safe and effective substance for 46 WO 2008/121126 PCT/US2007/021002 rendering the urine basic is the carbonic anhydrase inhibitor drug, Acetazolamide. Thus, 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 pre-treat the subject with this drug, or other substance effective to raise and maintain the pH of the urine 5 at a pH above about 7.4. 4. Treat to decrease PH in tumors for increased efficacy and specificity 10 Both the efficacy and the specificity of an onco-tool for acidic areas of tumors is a strong function of the pH differential between the acidic areas of the tumor and normal tissues. Thus, if one can selectively reduce the pH in acidic areas of tumors even further than is found in the natural condition - without concomitant reduction of the pH in normal tissues, this will afford greater sensitivity in diagnostic applications is and greater efficacy and specificity in therapeutic applications of onco-tools. Over the past half century a number of treatments have been reported to alter the pH in tumors - some causing the pH to increase and some causing the pH to decrease. In the context of diagnostic and therapeutic onco-tools, it is the treatments that selectively decrease the pH in tumors, without a concomitant 20 reduction in the pH in normal tissues, which are of interest. Following are three such treatments that have been reported to selectively reduce the pH in tumors. a) It has long been known that introduction of glucose into tumor-bearing animals acts to reduce the pH in the interstitial space in hypoxic areas of the tumors, typically for a period of about 2 to 3 hours - while having little or no effect on the pH 25 of the interstitial space in normal tissues (Naeslund & Swenson (1953) Acta Obstet. Gyneocol. Scand. 32, 359 - 367). Additional intake of glucose by mouth can significantly extend the length of time during which the pH in tumors remains so reduced. b) The pH in acidic areas of tumors can also be further reduced by treating 30 with the mitochondrial inhibitor, meta-iodobenzylguanidine, again apparently without undue effect on the pH in normal tissues (Jahde et. al., (1992) Cancer Research 52, 6209 - 6215). It has also been reported that the pH in acidic areas of tumors can be further 47 WO 2008/121126 PCT/US2007/021002 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). c) Still further, the pH in acidic areas of tumors can be further reduced by vasodilator drugs which are routinely used to treat persons with hypertension 5 (Adachi and Tannock (1999) Oncology Research 11, 179 - 185). Such drugs are probably effective because the abnormal vasculature of tumors generally lacks vasoconstrictor nerve fibers. Therefore, when a subject is given a vasodilator drug, resistance to blood flow is unchanged in tumors but decreases in normal tissues. These differential effects of the vasodilator result in a significantly greater blood flow 10 through normal tissues and a concomitant reduction in blood flow through the tumor. In turn, this reduced blood flow through the tumor probably reduces the washout of the lactic acid produced by tumor cells in hypoxic areas of the tumor - resulting in the observed vasodilator-mediated drop in the pH in acidic areas of the tumor. Such treatments, but preferably a combination of two or more such treatments 15 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. 20 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. 25 5. Treat to decrease rate of excretion by kidneys for increased efficacy At neutral pH an onco-tool carries one or more negative charges and is very hydrophilic, and so it can be excreted very rapidly by the kidneys. As a consequence, sequestering of an onco-tool in the poorly perfused acidic areas of a 30 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. Luckily, a simple inexpensive drug (Probenecid) has long been available 48 WO 2008/121126 PCT/US2007/021002 which can substantially slow the kidneys' rate of excretion of small highly water soluble substances (see: Butler in Nature Vol. 438, page 7064 (2005); Vlasses et al., in Antimicrobial Agents Chemother. Vol. 17, page 847 (1980)). Thus, pre-treatment of the subject with Probenecid before administering the onco-tool can serve to 5 substantially slow (by several fold) the rate at which the onco-tool is cleared through the kidneys. This, in turn, can allow substantially more of the administered dose of onco-tool to be sequestered in any tumors which are present in the subject. This effectively reduces the dose (and hence cost) of onco-tool which needs to be administered. 10 6. Monitor bladder for improved safety Because of the potential harm to the kidneys by therapeutic onco-tools if the is kidneys are excreting acidic urine, when onco-tools are used for treating tumors it may 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 20 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. 25 7. Flush bladder for improved detection and safety Over the course of one to a few hours much of the injected dose of an onco tool will be excreted by the kidneys and stored in the bladder until voided by 30 urination. For the case of an onco-tool diagnostic application, this buildup of radioactive onco-tool in the bladder can obscure the presence of tumors in close proximity to the bladder. For the case of a therapeutic onco-tool the buildup of radioactive onco-tool in the bladder has the potential of damaging the bladder. If 49 WO 2008/121126 PCT/US2007/021002 buildup of radioactive onco-tool in the bladder proves to be a significant problem, it can be substantially reduced by using an inflow/outflow catheter to irrigate the bladder from the time the onco-tool is injected until such time as most of the injected dose has been cleared from the patient's body. 5 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). This serves to assure the safety of the medical 1o personnel attending the subject, and it largely precludes inadvertent contamination of lavatories and other areas by radioactive onco-tool voided by the subject. 8. Use multiple onco-tools varying in pKa to increase efficacy Reported experimental measurements of the acidity in tumors have shown a 15 gradient of pH ranging from about 7.0 close to tumor capillaries and decreasing with increasing distance from tumor capillaries, with values as low as about 6.0 having been reported for areas most distant from tumor capillaries. For tumors having a broad range of pH values there is the potential that therapeutic onco-tools with too high of a pKa may rapidly enter cells in mildly-acidic areas close to tumor capillaries 20 to such an extent that inadequate perfusing onco-tool remains to reach the lowest pH areas of the tumor most distant from the capillaries. Alternatively, 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 25 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. The rationale for using such a combination of onco-tools is that the onco-tool with the higher pKa may be largely 30 sequestered in areas close to capillaries - leaving little available for reaching areas of lower pH further from capillaries Conversely, the onco-tool with the lower pKa should be poorly sequestered in areas of higher pH near capillaries and so remain available to diffuse into areas of lower pH further from capillaries where it can then 50 WO 2008/121126 PCT/US2007/021002 be effectively sequestered. Thus, together such a combination of low-pKa and high pKa onco-tools should better achieve complete destruction of the tumor. 5 9. Comprehensive Method For Detecting And Treating Tumors 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 io 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 15 comprehensive method is suitable for both veterinary medicine and human medicine. It includes, but is not limited to, the following steps. a) Detecting tumors Step 1 The first step it to provide a diagnostic onco-tool in its final form 20 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 25 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 30 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. 51 WO 2008/121126 PCT/US2007/021002 With modern 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. In the event one or more tumors are detected in step 4, one then proceeds to 5 the treatment process. b) Treating detected tumors Step 5. To treat the detected tumor, one or more therapeutic onco-tools are provided. While this can be a single onco-tool containing a beta-emitting 10 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. 15 Step 6. The one or more provided onco-tools are delivered into the subject generally by intravenous injection. 10. Strategy For Dealing With Micro-Metastases 20 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 25 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. Thus, while the larger parent tumor containing acidic areas are 30 amenable to detection and destruction 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. 52 WO 2008/121126 PCT/US2007/021002 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). However, those progeny 5 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. Once 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), 10 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 is 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. 20 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. It seems likely that such a second treatment should have a high probability of completely destroying any progeny tumors which might have escaped, in the form of micro metastases, during the course of the initial onco-tool treatment - thereby completely 25 clearing the patient of the original tumor and all its progeny. EXAMPLES: 30 Example 1. Cell culture test system for onco-tools It is recommended that initially 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 53 WO 2008/121126 PCT/US2007/021002 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 5 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. 10 Before the experiment, Hela 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. Further, 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 15 minutes. After the incubation, 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. Next, 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 20 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. 25 Example 2. Testing onco-tools in normal mice 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. However, this initial assessment should next be followed up for the most promising onco-tool structures with tests in 30 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. Next, a suitable quantity of lodine-1 31-containing onco-tool in phosphate-buffered saline should be injected, preferably intravenous such as into 54 WO 2008/121126 PCT/US2007/021002 the tail vein. Thereafter 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. The main purpose of this testing in normal mice is to identify any onco-tools 5 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. 10 Example 3. Testing onco-tools in tumor-bearing mice While the above relatively simple tests in normal tumor-free mice allow one to discard those onco-tools which have an excessive affinity for normal tissues, the 15 more decisive test for a prospective onco-tool structure is to test it in tumor-bearing mice. In such experiments it is recommended that one first pre-treat the 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 20 the pH in tumors. Such substances include: i) glucose (Naeslund & Swenson (1953) Acta Obstet. Gyneocol. Scand. 32, 359 - 367); ii) the mitochondrial inhibitor, meta-iodobenzylguanidine (Jahde et. al., (1992) Cancer Research 52, 6209 - 6215); and, iii) vasodilator drugs routinely used to treat persons with hypertension (Adachi and Tannock (1999) Oncology Research 11 179 - 185). 25 After an appropriate period of time following such pre-treatments, the tumor bearing mice are injected (preferably intravenous) with the lodine-131-containing onco-tool. 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 30 organs and any obvious tumors excised. Each organ and tumor and the remaining carcass is then counted in a gamma counter. In regard to pre-treating the mice, it is likely that onco-tools will work best when both types of pre-treatments are used. One type which serves to prevent re 55 WO 2008/121126 PCT/US2007/021002 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 which serves to further increase the acidity (reduce the pH) in hypoxic/acidic areas of tumors. Preferably a combination of two or three such pre-treatments to selectively reduce 5 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. Further, in the event the onco-tool is excreted so quickly by the kidneys that insufficient onco tool is sequestered by the tumors, an additional pre-treatment with Probenecid may also prove beneficial for increasing efficacy - by virtue of slowing excretion of the 10 onco-tool by the kidneys and thereby allowing greater amounts of the onco-tool to be sequestered in acidic areas of the tumors. The above presents a description of the best mode contemplated for the 15 compositions and methods of the present invention, and of the manner and process of making and using such compositions and methods in such full, clear, concise, and exact terms as to enable any person skilled in the art to which they pertain to make and use the compositions and methods. These compositions and methods are, however, susceptible to modifications and alternate constructions from the illustrative 20 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 25 out and distinctly claim the subject matter of the disclosed compositions and methods. 56

Claims (41)

1. A composition containing an advanced pH-switch component, said advanced pH switch component comprising: 5 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 10 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 is are positioned and oriented such that they are compatible with formation of an internal acid-specific H-bond.

2. The composition of Claim 1, wherein the aliphatic ring structure is a 5-membered ring. 20

3. The composition of Claim 2, wherein the H-bond acceptor moiety is directly linked to the aliphatic ring structure and is an N,N-dialkyl-N-trifluoroethylamine moiety.

4. The composition of Claim 1, wherein the stand-alone form of the H-bond acceptor 25 moiety of the advanced pH-switch component has a pKa value in the range of approximately 3.0 to approximately 6.5.

5. The composition of Claim 1, wherein the H-bond acceptor moiety of the advanced pH-switch component is selected from the group consisting of: 30 (a) a cyanomethyl amine; (b) a trifluoroethyl amine; (c) an alkoxy amine; (d) a hydrazine; 57 WO 2008/121126 PCT/US2007/021002 (e) an N-oxide; (f) an imidazole; (g) an aniline; (h) an amide; 5 (i) a phosphoramide; (j) a thiophosphoramide; (k) a urea; and, (1) an ether. 10

6. A composition containing a merged pH-switches component, comprising two acid H-bond donor moieties positioned and oriented to simultaneously H-bond to a single H-bond acceptor moiety.

7. The composition of Claim 6 wherein the single H-bond acceptor moiety is the 15 oxygen of an N-oxide.

8. An onco-tool composition comprising: (a) at least two pH-switch components, each pH-switch component containing an acid moiety and being effective to undergo a pH 20 mediated transition between an anionic hydrophilic form at a higher pH and a non-ionic cell-penetrating form at a lower pH; and, (b) at least one cargo component selected from the group consisting of: i) a structure effective to bind a radioisotope, and ii) a structure which contains a radioisotope. 25

9. The onco-tool composition of Claim 8, which includes at least two dissimilar pH switch types.

10. The onco-tool composition of Claim 8, wherein at least two of the pH-switch 30 components share a single H-bond acceptor moiety thereby creating a merged pH switches component.

11. The onco-tool composition of Claim 8, further comprising a radioisotope selected 58 WO 2008/121126 PCT/US2007/021002 from the group consisting of: (a) a radioisotope effective to report the presence of the composition; (b) a radioisotope effective to kill cells; and, (c) a radioisotope effective to report the presence of the composition 5 and effective to kill cells.

12. The onco-tool composition of Claim 8, where the radioisotope is a radiohalogen.

13. The onco-tool composition of Claim 8, which has a calculated tumor specificity 10 factor greater than 20.

14. The onco-tool composition of Claim 8, wherein at least one of the acid moieties of the pH-switch components has a pKa value in the range of 5.0 to 6.8.

15 15. The onco-tool composition of Claim 8 where at least one of the pH-switch components is an advanced pH-switch component, said advanced pH-switch component comprising: a) an aliphatic ring structure selected from the group consisting of a 4 membered ring, a 5-membered ring, and a 6-membered ring; 20 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 25 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. 30

16. A diagnostic onco-tool composition for detecting a tumor containing acidic areas, comprising: (a) at least two pH-switch components containing an moiety and 59 WO 2008/121126 PCT/US2007/021002 effective to undergo a pH-mediated transition between an anionic hydrophilic form at a higher pH and a non-ionic cell-penetrating form at a lower pH; and, (b) at least one radioisotope whose emission from within a tumor in the 5 subject can be detected outside the subject.

17. The diagnostic onco-tool composition of Claim 16, wherein the radioisotope is a radiohalogen. 1o

18. A therapeutic onco-tool composition for treating a subject having a tumor containing acidic areas, comprising: (a) at least two pH-switch components containing a carboxyl moiety and effective to undergo a pH-mediated transition between an anionic hydrophilic form at a higher pH and a non-ionic cell 15 penetrating form at a lower pH; and, (b) at least one radioisotope whose emission is effective to kill cells.

19. The therapeutic onco-tool composition of Claim 18, wherein the radioisotope is a radiohalogen. 20

20. The therapeutic onco-tool composition of Claim 18, wherein the radioisotope emits a beta particle.

21. The therapeutic onco-tool composition of Claim 18, wherein the radioisotope 25 emits an alpha particle.

22. A combination of therapeutic onco-tool compositions of Claim 18, comprising: (a) at least one first onco-tool composition containing a first radioisotope which emits an alpha particle; and, 30 (b) at least one second onco-tool composition containing a second radioisotope which emits a beta particle. 60 WO 2008/121126 PCT/US2007/021002

23. A diagnostic method for detecting in a subject a tumor containing acidic areas, the method comprising the steps of: (a) providing a diagnostic onco-tool composition of Claim 11; (b) introducing the diagnostic onco-tool composition into the subject; 5 (c) waiting for approximately 10 minutes to approximately 48 hours; and, (d) scanning the subject with equipment effective to detect emissions from diagnostic onco-tool composition remaining in the subject. io

24. The diagnostic method of Claim 23, further comprising the step of: pre-treating the subject with at least one substance selected from the group consisting of: (a) a first substance effective to increase the pH of the subject's urine; (b) a second set of substances effective to decrease the pH in tumors; 15 and, (c) a third substance effective to slow the rate of onco-tool excretion by the kidneys.

25. The diagnostic method of Claim 24, comprising the step of: 20 pre-treating the subject with at least one substance selected from the group consisting of: (a) Acetazolamide effective to increase the pH of the subject's urine; (b) Glucose, meta-lodobezylguanidine, and Hydralazine effective to decrease the pH in tumors; and, 25 (c) Probenecid effective to slow the rate of onco-tool excretion by the kidneys.

26. A therapeutic method for treating tumors in a subject, the method comprising the steps of: 30 (a) providing a at least one therapeutic onco-tool composition of Claim 18; and, (b) introducing at least one of the therapeutic onco-tool compositions into the subject. 61 WO 2008/121126 PCT/US2007/021002

27. The therapeutic method of Claim 26, further comprising the step of: pre-treating the subject with at least one substance selected from the group consisting of: 5 (a) a first substance effective to increase the pH of the subject's urine; (b) a second set of substances effective to decrease the pH in tumors; and, (c) a third substance effective to slow the rate of onco-tool excretion by the kidneys. 10

28. The diagnostic method of Claim 27, comprising the step of: pre-treating the subject with at least one substance selected from the group consisting of: (a) Acetazolamide effective to increase the pH of the subject's urine; (b) Glucose, meta-lodobezylguanidine, and Hydralazine effective to 15 decrease the pH in tumors; and, (c) Probenecid effective to slow the rate of onco-tool excretion by the kidneys.

29. The therapeutic method of Claim 27, further comprising the step of: 20 irrigating the bladder of the subject following introducing the therapeutic onco-tool composition into the subject.

30. A therapeutic method for treating tumors in a subject, the method comprising the steps of: 25 (a) providing a combination of the therapeutic compositions of Claim 22; and, (b) introducing the therapeutic compositions into the subject.

31. The therapeutic method of Claim 30, further comprising the step of: 30 pre-treating the subject with at least one substance selected from the group consisting of: (a) a first substance effective to increase the pH of the subject's urine; (b) a second set of substances effective to decrease the pH in tumors; 62 WO 2008/121126 PCT/US2007/021002 and, (c) a third substance effective to slow the rate of onco-tool excretion by the kidneys. 5

32. The therapeutic method of Claim 30, further comprising the step of: irrigating the bladder of the subject following the introducing of the therapeutic composition into the subject.

33. A method for detecting in a subject a tumor containing an acidic area, and 10 treating any of the tumors detected, comprising the steps of: (a) providing a diagnostic composition effective to be sequestered in the acidic area of the tumor and containing a radioisotope which emits a signal that can be detected from outside the subject; (b) introducing the diagnostic composition into the subject; 15 (c) waiting approximately 10 minutes to approximately 48 hours; (d) scanning the subject with equipment effective to detect the signal emitted from the diagnostic composition; and, for the subject where the scan indicates the presence of a tumor; (e) providing at least one therapeutic onco-tool composition which is 20 selectively sequestered in the acidic areas of the tumor and containing a radioisotope effective to kill cells; and (f) introducing at least one of the therapeutic onco-tool compositions into the subject. 25

34. The method of Claim 33, further comprising the step of: pre-treating the subject with at least one substance selected from the group consisting of: (a) a first substance effective to increase the pH of the subject's urine; (b) a second set of substances effective to decrease the pH in tumors; 30 and, (c) a third substance effective to slow the rate of onco-tool excretion by the kidneys wherein, the pre-treating step is performed before the introducing of the 63 WO 2008/121126 PCT/US2007/021002 diagnostic composition into the subject and again before the introducing of the therapeutic onco-tool compositions into the subject.

35. The method of Claim 33, further comprising the step of: 5 irrigating the bladder of the subject after introducing the therapeutic onco-tool composition into the subject.

36. The method of Claim 33, further comprising the steps of: repeating steps (a) through (d) after a period of time estimated to be sufficient 10 for any micro-metasteses to grow into new tumors of sufficient size to contain acidic areas; and, repeating steps (e) and (f) if at least one tumor is detected in step (d).

37. A method for detecting and treating in a subject a tumor containing an acidic 15 area, comprising the steps of: (a) introducing into the subject a first substance effective to increase the pH of the urine in the subject; (b) introducing into the subject a second substance effective to decrease the pH in the tumor; 20 (c) providing a diagnostic composition capable of being selectively sequestered in the acidic area of the tumor and containing a radioisotope which emits a signal that can be detected from the exterior of the subject; (d) introducing the diagnostic composition into the subject; 25 (e) scanning the subject with a device effective to detect the signal emitted from the diagnostic composition; (f) providing at least one therapeutic composition capable of being selectively sequestered in the acidic areas of the tumor and containing a radioisotope effective to kill cells; and 30 (g) introducing at least one of the therapeutic compositions into the subject.

38. The method of Claim 37, wherein the diagnostic composition is an onco-tool. 64 WO 2008/121126 PCT/US2007/021002

39. The method of Claim 37, wherein the therapeutic composition is an onco-tool.

40. The method of Claim 37, wherein the diagnostic composition is an onco-tool and 5 the therapeutic composition is an onco-tool.

41. The method of Claim 37, further comprising the steps of: (a) waiting for the diagnostic composition to be sequestered in the acidic area of the tumor after introduction of the diagnostic 10 composition into the subject; (b) introducing into the subject an additional quantity of the first substance effective to increase the pH of the urine in the subject; and, (c) introducing into the subject an additional quantity of the second substance effective to decrease the pH in the tumor. 15 20 65 WO 2008/121126 PCT/US2007/021002 AMENDED CLAIMS received by the International Bureau on 22 July 2008 (22.07.08) 5 1. An improved acid-targeted composition of a type which 10 (a) is preferentially sequestered in acidic areas, which may be in tumors in a living subject, due to existing largely in a negatively-charged water-soluble form in aqueous solution at pH 7.4, but in acidic aqueous solution below pH 7.0 a substantial portion converts to a non-ionic form effective to enter a lipophilic phase, which may be a cell membrane, by virtue of containing at least two advanced 15 pH-switches, each of which is an internally H-bondable component having i) a non-aromatic 4-membered, 5-membered, or 6-membered ring, ii) a carboxylic acid moiety directly linked to the non-aromatic ring, iii) an H-bond acceptor moiety having a pKa of less than 7 and in its non-ionic form having a structure which cannot serve as an H-bond donor moiety and 20 where the atom that serves as the acceptor in an H-bond to the carboxylic acid moiety is directly linked to the non-aromatic ring structure or linked through one atom to the non-aromatic ring structure, and iv) the carboxylic acid and the H-bond acceptor moieties attached to the non-aromatic ring are positioned and oriented such that they are compatible 25 with formation of an internal H-bond, and (b) contains at least one cargo component having a stably-bound radiohalogen; wherein the improvement comprises: an acid-targeted composition having at least two advanced pH-switches of the 30 merged pH-switches type wherein the carboxylic acid moiety of a five-membered non aromatic ring and the carboxylic acid moiety of a six-membered non-aromatic ring are positioned to simultaneously H-bond to the same H-bond acceptor moiety. 35 66 WO 2008/121126 PCT/US2007/021002 2. The improved acid-targeted composition of Claim 1, wherein the best mode structure containing merged pH-switches is: O 5 I 124; 10 3. An improved acid-targeted composition of a type which (a) is preferentially sequestered in acidic areas, which may be in tumors in a living subject, due to existing largely in a negatively-charged water-soluble form in 15 aqueous solution at pH 7.4, but in acidic aqueous solution below pH 7.0 a substantial portion converts to a non-ionic form effective to enter a lipophilic phase, which may be a cell membrane, by virtue of containing at least two advanced pH-switches, each of which is an internally H-bondable component having i) a non-aromatic 4-membered, 5-membered, or 6-membered ring, 20 ii) a carboxylic acid moiety directly linked to the non-aromatic ring, iii) an H-bond acceptor moiety having a pKa of less than 7 and in its non-ionic form having a structure which cannot serve as an H-bond donor moiety and where the atom that serves as the acceptor in an H-bond to the carboxylic acid moiety is directly linked to the non-aromatic ring structure or linked 25 through one atom to the non-aromatic ring structure, and iv) the carboxylic acid and the H-bond acceptor moieties attached to the non-aromatic ring are positioned and oriented such that they are compatible with formation of an internal H-bond, and (b) contains at least one cargo component having a stably-bound radiohalogen; 30 wherein the improvement comprises: an acid-targeted composition having mixed pH-switches wherein at least two of its component advanced pH-switches differ from each other in structure. 67 WO 2008/121126 PCT/US2007/021002 4. The improved acid-targeted composition of Claim 3, wherein the best mode structure containing mixed pH-switches is: CF 3 O H( 'I*( 5 H*N - N O 1311 10 5. An improved precursor structure of a type that by addition of a radiohalogen to its cargo component can be readily converted to an acid-targeted composition which (a) is preferentially sequestered in acidic areas, which may be in tumors in a living 15 subject, due to existing largely in a negatively-charged water-soluble form in aqueous solution at pH 7.4, but in acidic aqueous solution below pH 7.0 a substantial portion converts to a non-ionic form effective to enter a lipophilic phase, which may be a cell membrane, by virtue of containing at least two advanced pH-switches, each of which is an internally H-bondable component having 20 i) a non-aromatic 4-membered, 5-membered, or 6-membered ring, ii) a carboxylic acid moiety directly linked to the non-aromatic ring, iii) an H-bond acceptor moiety having a pKa of less than 7 and in its non-ionic form having a structure which cannot serve as an H-bond donor moiety and where the atom that serves as the acceptor in an H-bond to the carboxylic 25 acid moiety is directly linked to the non-aromatic ring structure or linked through one atom to the non-aromatic ring structure, and iv) the carboxylic acid and the H-bond acceptor moieties attached to the non-aromatic ring are positioned and oriented such that they are compatible with formation of an internal H-bond, and 30 (b) contains at least one cargo component having a stably-bound radiohalogen; wherein the improvement comprises: a precursor structure having at least one cargo component which includes a vinyl silane moiety. 68 WO 2008/121126 PCT/US2007/021002 6. The improved precursor structure of Claim 5, wherein the best mode structure containing a vinyl silane is: 0 O O 5 0,. 7. An improved method of detecting acidic areas in a subject, where the acidic areas may 10 be in tumors, where such methods include introducing into the subject at least one acid-targeted composition which (a) is preferentially sequestered in acidic areas due to existing largely in a negatively charged water-soluble form in aqueous solution at pH 7.4, but in acidic aqueous solution below pH 7.0 a substantial portion converts to a non-ionic form effective to 15 enter a lipophilic phase, which may be a cell membrane, by virtue of containing at least two advanced pH-switches, each of which is an internally H-bondable component having i) a non-aromatic 4-membered, 5-membered, or 6-membered ring, ii) a carboxylic acid moiety directly linked to the non-aromatic ring, 20 iii) an H-bond acceptor moiety having a pKa of less than 7 and in its non-ionic form having a structure which cannot serve as an H-bond donor moiety and where the atom that serves as the acceptor in an H-bond to the carboxylic acid moiety is directly linked to the non-aromatic ring structure or linked through one atom to the non-aromatic ring structure, and 25 iv) the carboxylic acid and the H-bond acceptor moieties attached to the non-aromatic ring are positioned and oriented such that they are compatible with formation of an internal H-bond, and (b) contains at least one cargo component having a stably-bound radiohalogen that is effective to report the presence of the acid-targeted composition by emitting 30 a photon, gamma ray, or positron; wherein the improvement comprises: pre-treating the subject with a substance effective to decrease the excretion rate of the acid-targeted composition by the kidneys. 69 WO 2008/121126 PCT/US2007/021002 8. The improved method of detecting acidic areas in a subject of Claim 7, wherein the best mode substance used to decrease the excretion rate of the acid-targeted composition by the kidneys is the drug, Probenecid. 5 9. An improved method of treating acidic areas in a subject, where the acidic areas may be in tumors, where such methods include introducing into the subject at least one acid-targeted composition which (a) is preferentially sequestered in acidic areas due to existing largely in a negatively 10 charged water-soluble form in aqueous solution at pH 7.4, but in acidic aqueous solution below pH 7.0 a substantial portion converts to a non-ionic form effective to enter a lipophilic phase, which may be a cell membrane, by virtue of containing at least two advanced pH-switches, each of which is an internally H-bondable component having 15 i) a non-aromatic 4-membered, 5-membered, or 6-membered ring, ii) a carboxylic acid moiety directly linked to the non-aromatic ring, iii) an H-bond acceptor moiety having a pKa of less than 7 and in its non-ionic form having a structure which cannot serve as an H-bond donor moiety and where the atom that serves as the acceptor in an H-bond to the carboxylic 20 acid moiety is directly linked to the non-aromatic ring structure or linked through one atom to the non-aromatic ring structure, and iv) the carboxylic acid and the H-bond acceptor moieties attached to the non-aromatic ring are positioned and oriented such that they are compatible with formation of an internal H-bond, and 25 (b) contains at least one cargo component having a stably-bound radiohalogen that is effective to kill cells by emitting an alpha or beta particle; wherein the improvement comprises: pre-treating the subject with a substance effective to decrease the excretion rate of 30 the acid-targeted composition by the kidneys. 70 WO 2008/121126 PCT/US2007/021002 10. The improved method of treating acidic areas in a subject of Claim 9, wherein the best mode substance used to decrease the excretion rate of the acid-targeted composition by the kidneys is the drug, Probenecid. 5 10 15 20 25 30 71 WO 2008/121126 PCT/US2007/021002 Statement under Article 19(1) Opinions in ISR: 1. Compositions of the invention are not novel. 2. No inventive step was made because: a) compositions are not novel, and b) objective was not achieved. 3. Lacks industrial application. The International Search Report (ISR) contends the claimed compositions do not satisfy the novelty requirements of Art. 33(2) PCT. This finding was a consequence of the search being carried out without a key structural limitation being considered - that being the contested term "aliphatic". This is remedied by new replacement claims wherein the contested term is replaced by the non-controversial equivalent term "non-aromatic". The new replacement claims also incorporate added structural limitations disclosed in the specification, but not initially incorporated into the original claims. Therefore, compositions covered by the new claims are thoroughly differentiated from the purported prior art cited in the ISR. With these changes the new replacement claims fully meet the novelty requirement of Article 33(2) PCT. The ISR purported the claimed compositions lack an inventive step: (a) because they are not novel; and, (b) because there is no evidence any of the disclosed compositions "solve the underlying technical problem, namely the treatment of tumors." (a) Lack of novelty is rectified by the new replacement claims. (b) The underlying technical problem, contrary to the contention in the ISR, is that all prior art acid-targeted compositions afford poor specificity for acidic areas. The 72 WO 2008/121126 PCT/US2007/021002 compositions of the invention indeed solve this technical problem by providing improved specificity for acidic areas. Further, in an earlier PCT Patent Application PCT/US2007/008215 filed 30 March 2007 by the same inventor, which was incorporated into the present application by reference and made a part thereof, the Description and Figures of that earlier PCT Application exhaustively describe the theoretical basis and mathematical underpinning for compositions which achieve this improved specificity, as well as methods for synthesizing such compositions. Furthermore, Figures 13 and 23 of that earlier PCT Application show structures of several such compositions which were synthesized during development of the invention. Those figures also show results from experiments with those compositions which demonstrate to persons skilled in the art of acid targeted compositions that those compositions indeed achieve greater specificity for acidic areas than afforded by prior art acid-targeted compositions. The improvements in structures and methods of use for these acid-targeted compositions, which are disclosed and claimed in the current PCT Patent Application, build on those earlier syntheses and experimental testing. Thus, disclosed compositions and experimental results demonstrate that an inventive step (achieving improved specificity for acidic areas) has been made, as required by Article 33(3) PCT. The ISR contended that because the compositions of the invention require a radioisotope in order to report the composition's presence or to kill cells, therefore claimed compositions which are in their precursor form, lacking a radioisotope, thus lack industrial application. This reasoning is refuted by the tens of millions of dollars in annual sales of precursor forms (lacking radioisotopes) of radiodiagnostics and radiotherapeutics used in medical clinics worldwide - wherein the essential short-lived radioisotopes are subsequently attached shortly before use in patients. 73