JP2009536152A - Non-peptide molecules for detecting and treating tumors - Google Patents

Abstract

  One of the most common characteristics of malignant tumors is their acidity. Onco-tools are small non-peptide synthetic molecules that are designed to exploit this acidity for early detection and destruction of tumors. Each onco-tool is anionic at pH 7.4 and is hydrophilic, so it is repelled from the negatively charged surface of cells in normal tissue. Those non-ionic lipophilic forms that are designed to invade cells such as cells within the acidic region of the tumor when the onco-tool enters an acidic environment such as in a tumor. Convert to Prior to use of the onco-tool, the selected radioisotope is coupled to the onco-tool. When the radioisotope emits radiation that can be detected outside the body, the onco-tool can serve to detect the tumor. If the radioisotope emits radiation that is effective to kill cells, the onco-tool can serve to treat the tumor.

Description

  The present invention relates to a composition that is effective to be selectively captured in the acidic region of a tumor in a subject for purposes of detecting and treating the tumor.

Acidity: Almost universal properties available for tumors :
In 1930, the prominent physiologist Otto Warburg reported that one of the most universal properties of malignant tumors is their acidity. Subsequent studies have suggested that such acidity arises because new blood vessels must be induced in order for the tumor to grow to about 1 mm in diameter. Because tumor-causing blood vessels are poorly spaced and abnormal, areas of the tumor more than tens of microns away from such capillaries are generally hypoxic (oxygen deficient). Cells in such hypoxic regions of the tumor die or are converted to glycolytic metabolism that results in their lactate excretion. Due to insufficient circulation in the tumor, excreted lactic acid accumulates in the interstices between cells in the hypoxic region of the tumor. This can cause a pH of from the lowest 6.0 in the region farthest from the capillary to about 7.0 near the capillary. Incidentally, the pH of the gap in normal tissue is strictly adjusted to about 7.3 to pH 7.5.

Resting cells in the acidic region of the tumor: a major unexamined challenge in cancer treatment
As mentioned above, tumor acidity generally increases with increasing distance from tumor capillaries. Nearly normal pH tumor cells close to the capillaries show a high metabolic rate and fast cell division, whereas tumor cells in higher acid regions that are further away from the capillaries have a lower metabolic rate, Split slowly or not at all. Compared to rapidly dividing tumor cells, slowly dividing and non-dividing tumor cells called resting are substantially more resistant to cytotoxic agents such as radiation and toxic chemicals.

  Traditional cancer therapies (radiation and chemotherapy) have been selected primarily for their ability to kill rapidly dividing cells while preserving the slow and non-dividing cells typical of most normal tissues. Such conventional cancer therapies are quite effective at killing rapidly dividing tumor cells at near normal pH close to capillaries. Not surprisingly, however, these same therapies are rather ineffective against slow dividing and non-dividing resting cells in the more acidic areas of the tumor. Thus, conventional cancer therapies primarily kill well-oxygenated rapidly dividing tumor cells while preserving resting cells in the hypoxic / acidic region of the tumor. This early killing of rapidly dividing cells leads to tumor remission, while killed cells are disposed of by the body's normal cleansing process. However, during this purification process, most of the surviving treatment-resistant resting cells in the acidic region of the tumor gradually recover sufficient oxygen, nutrient uptake and waste disposal, which is a high metabolic rate and rapid cell Allows to return to division. This neoplasia of previously resting tumor cells leads to a possible relapse that is responsible for most deaths from cancer.

Traditional efforts to exploit the hypoxic / acidic region of the tumor for treatment :
The hypoxic / acidic properties of tumors have been known for 75 years and it has long been speculated that such properties could be used for therapy. However, until recently, most successful efforts to exploit these properties focused on hypoxia. Specifically, substances have been developed that exhibit considerable cytotoxicity in hypoxic cells while at the same time exhibiting minimal cytotoxicity in normoxic cells. One such substance has entered the clinical trial phase.

  Compared to exploiting hypoxia in tumors, much less effort was focused on exploiting tumor acidity. One unsuccessful approach was based on the finding that the acidic pH in the tissue acts to sensitize those cells to thermal damage. However, efforts to take advantage of this acid-mediated heat sensitivity of the cells have resulted in disappointing results.

  Another approach related to acidity in tumors is that low pH in tumors ionizes weakly basic cytotoxic substances, thereby making them membrane impermeable and thus into cells in the more normal pH region Based on the fact that it leads to a selective reduction of the invasion of a large number of such weakly basic substances into the acidic area of the tumor compared to the invasion of such substances. In this regard, rather than attempting to exploit the low pH in the tumor, a means to partially deionize, thereby increasing the penetration of such weakly basic cytotoxic substances into the cells of the tumor As such, efforts were focused on raising the pH in the tumor. Such efforts have had some success.

  Another approach that is more closely related to the present invention relies on the fact that a low pH in the tumor gap causes partial deionization of the weakly acidic cytotoxic agent chlorambucil (shown in FIG. 1). . As a result of the lower degree of ionization of this well-known weakly acidic cytotoxic drug in the acidic region of the tumor, it should exhibit increased invasion and thus greater cytotoxicity in cells in the acidic region of the tumor. Expect it to be. This expectation was tested by Kozin et al., Who made tumors in tumor-bearing mice more acidic using established methods (see Non-Patent Document 1). They reported that, as expected, a decrease of about 0.3 pH units in the tumor was consistent with a modest improvement of 1.7 times the tumor growth delay caused by the weak acid (pKa5.8) chlorambucil. . In the conclusion of their paper, they said, “As far as we know, CHL (chlorambucil) is the only clinical therapeutic that is a weak acid with an appropriate pKa ≦ 6.5. Provide a basis for the design of new and potent drugs that exhibit weak acid properties and the contribution of diffusion to intracellular uptake, and as shown here, between such compounds and modulators of radiation and / or pH gradients. The combination offers an additional opportunity to maximize the therapeutic response. "

  In the aforementioned paper, the authors provide a basis for the design of new weak acid cytotoxic anticancer drugs that can demonstrate high efficacy, with their results showing a modest increase in the efficacy of the off-the-shelf cytotoxic drug chlorambucil However, there is no guidance on the likely molecular structure of such drugs, and no guidance on how to go about incorporating both weak acid and cytotoxic functions into the appropriate drug structure. There is no guidance on what specific properties are desirable, no guidance on how to design, manufacture or test such weak acid cytotoxic drugs, and such cytotoxic drugs No guidance was given on how to apply or use it. A more serious limitation in our proposal that novel weak acid cytotoxic drugs with improved efficacy can be designed based on the chlorambucil model is designed to selectively penetrate such drugs The resting cells in the acidic region of the tumor being treated are particularly resistant to conventional cytotoxic drugs because they are resting. Thus, conventional cytotoxic drugs such as chlorambucil can make a strong case that their use is insufficient to kill resting cells in the acidic area of the tumor.

  Instead, Summerton (inventor of onco-tool) determines what is necessary to effectively destroy resting cells in the acidic region of the tumor, and determines those treatment-resistant resting tumor cells. Argues that it is a specially designed drug that has a particularly destructive effect on cell viability that is sufficient to disrupt it. Combined with this requirement of destructive effects on cell viability is the simultaneous requirement that such drugs be largely eliminated from cells in normal tissue and at the same time be effectively delivered into tumor cells .

Background technology :
Unlike the proposal of Kozin et al., Which uses conventional weak acid cytotoxic drugs to take advantage of the acidity of the tumor, Dr. James Summerton develops a novel peptide composition specifically designed to take advantage of the difference in pH between normal tissue and the acidic region of the tumor in order to provide a safer and more effective therapy for a wide range of tumors Started. These large peptide compositions (generally having a mass in the range of 2000-4000 daltons) are referred to as “embedder and transporter peptides”. Their structure and methods of use to detect and treat tumors are disclosed in pending US Pat. No. 5,057,086 and US Pat. No. 5,037,028 issued Nov. 7, 2006 (both inventors Dr. James E. Summerton). Has been. Although such embedder and transporter peptides were found to penetrate tumors in tumor-bearing mice fairly selectively, nonetheless, experimental results from mouse studies showed that such peptides were more desirable than desired. Suggests achieving low specificity and efficacy. They also appeared to undergo excessive reuptake in the kidney. This is undesirable in diagnostic applications and unacceptable in therapeutic applications.

  Considering the limitations of embedders and transporter peptides, Summerton is an onco-tool that aims to achieve significantly improved specificity, efficacy and safety compared to previous embedders and transporter peptides. The quest to come up with an entirely new class of small non-peptide drugs called later began. These small non-peptide onco-tools, including the present invention, are the subject of Dr. James E.M. It was disclosed only in Patent Document 3 filed on March 30, 2006 by Summerton and in Patent Document 4 and Patent Document 5 filed on June 7, 2006.

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  An object of the present invention is to provide non-peptide compositions and methods for detecting and treating tumors containing acidic regions.

An onco-tool that takes advantage of tumor acidity for early detection and destruction of tumors :
The onco-tools of the present invention are a new class of molecules designed to detect and treat tumors. Each onco-tool contains two or more pH converting components that readily convert to an anionic hydrophilic form at higher pH and a nonionic lipophilic form at lower pH. Each onco-tool also includes a cargo component that is effective to bind to a selected radioisotope suitable for serving the onco-tool diagnostic or therapeutic role. Onco-tools consist of relatively small non-peptide synthetic molecules having a mass (not counting the mass of radioisotopes) generally in the range of about 300-1500 daltons.

  At neutral pH in normal tissues, onco-tools exist in their water-soluble negatively charged form that is designed to be repelled from the negatively charged surface of cells and easily excreted by the kidneys. . Conversely, when onco-tools enter the acidic area of the tumor, some of the onco-tool molecules convert to their non-ionic lipophilic forms that are designed to rapidly enter nearby cells. Thus, when a person is injected with a dose of onco-tool, if the person has a tumor that is greater than about 1 mm in diameter, a portion of the injected volume will quickly penetrate into the cells in the acidic region of the tumor, The remaining amount is excreted by the kidneys. In other words, the onco-tool is designed to have the important properties of a) repelled from cells in normal tissue, b) trapped within the acidic region of the tumor, and c) not trapped within the acidic region of the tumor. Onco-tools are designed to be quickly removed from the body via the kidneys.

  Prior to use of the onco-tool, the selected radioisotope is coupled to the onco-tool. The combined radioisotope plays an onco-tool diagnostic or therapeutic role. The diagnostic role is to report the presence of an onco-tool within the tumor to a detector outside the body. The therapeutic role is to destroy the tumor.

Onco tools for diagnosis :
Due to the large variation between tumors, it is routine enough for a wide range of tumor types to be treated early enough (preferably before metastasis, manifesting symptoms, or causing severe organ damage) Detection was an unachieved goal of medicine. Many of the difficulties in routine early detection of tumors are common to most or all tumors and identify certain characteristics that can be used effectively for routine and available early detection of tumors It was in. Onco-tools with appropriate radioisotopes (eg, gamma rays or positron emitters) that emit signals that can be easily detected outside the body, combined with modern imaging techniques, are very early in most or all tumor types. It is designed so that it can be detected safely.

  The diagnostic onco-tool is designed to be widely used for routine annual physical examination to allow very early detection of essentially all tumors larger than the microscopic size. Thus, a single diagnostic onco-tool could serve as an excellent and comprehensive alternative to a wide range of current tumor diagnostic methods such as breast mammograms, Papanicolaou smears, prostate examinations and colonoscopy There is. In addition, many tumor types then require pain, organ dysfunction, or some other common symptom that requires an exhaustive and very expensive diagnostic procedure to identify the root cause of the problem Or it should be understood that at present, it cannot be detected by routine methods until a very late stage of development manifested by a series of symptoms. By the time such a tumor is detected, it is often too advanced to wish for a successful treatment (at least with currently approved therapies). In this regard, diagnostic onco-tools are likely to have an entirely new capability for routine early detection of such difficult-to-diagnose tumors.

Onco tools for treatment :
The therapeutic onco-tool is designed to take advantage of the acidic properties of the tumor to provide safe and effective destruction of the tumor. For optimal results, a combination of two onco-tool formulations should be used. One formulation is radioactive that emits short path length high linear energy transfer radiation such as ultra high energy alpha particles that are effective in killing adjacent radioresistant resting cells in the acidic region of the tumor (see FIG. 2). Has an isotope. Other formulations have a radioisotope that emits energy beta particles in the mid-path length effective to kill more far-radiosensitive rapidly dividing tumor cells near the capillaries. When used in combination, these two formulations are designed to effectively kill the entire tumor with little or no damage to other cells in the body. As a result, therapeutic onco-tools are designed to be effective against all cell types in most or all tumors and are therefore much less toxic and much more effective with little or no recurrence Expected to be a tumor therapy.

Definition of terms :
The terms used in this specification have the following specific meanings unless otherwise specified.

  The terms “comprising”, “having”, “containing” and “including” and other forms are equivalent in meaning, and the next of any one of these terms It does not mean that an item or items in the list is an exhaustive list of such items or items, or is limited to only one or more items listed It is intended that a limited range is not defined in that it does not mean.

  The words “consisting of”, “consisting of” and other forms are equivalent in meaning, and the item or items that follow any one of these terms are such one item. Alternatively, it is an exhaustive list of a plurality of items, and it is intended that a limited range is defined in the sense that it is limited to only one item or a plurality of items listed.

  pH switch component—an onco-tool structural component capable of undergoing a pH-mediated transition between an anionic hydrophilic form at higher pH and a non-ionic lipophilic form at lower pH.

  Advanced pH switch component-pH switch component designed to form internal acid-specific hydrogen bonds. The advanced pH switch has the following characteristics.

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) containing a carboxylic acid moiety directly attached to the aliphatic ring structure;
c) the carboxylic acid moiety is separated from the attached electron withdrawing group by at least two carbons;
d) i) part of an aliphatic ring structure,
ii) directly bonded to an aliphatic ring structure;
iii) comprising an H-bond acceptor moiety selected from the group consisting of those bonded to an aliphatic ring structure through one atom;
e) the H bond acceptor moiety has a structure that, in its non-ionic form, does not serve as an H bond donor moiety;
f) The carboxylic acid moiety and the H-bond acceptor moiety are positioned in close proximity and appropriately positioned and oriented to be compatible with the formation of internal acid-specific H bonds.

  Cargo component—A structural component of an onco-tool that serves to bind to a radioisotope that is effective in reporting the presence of an onco-tool or is effective in killing cells. The cargo component can exist as a precursor form ready to bind to a radioisotope or as a final form containing a radioisotope.

  Precursor form cargo component—a cargo component that has a structure that can be easily bound to a selected radioisotope but is not yet bound to a radioisotope. An onco-tool with this precursor form of the cargo component is suitable for long-term storage.

  Final form cargo component—a cargo component comprising at least one bound radioisotope. An onco-tool with this final form of the cargo component is suitable for delivery to a subject for the purpose of detecting and / or killing tumors containing acidic regions.

  Onco-tool-a non-peptide composition comprising at least two pH switch components and at least one cargo component.

  Diagnostic onco-tool—an onco-tool that contains a radioactive isotope effective to report the presence of an onco-tool within the tumor to a detector outside the subject undergoing diagnostic treatment.

  Therapeutic onco-tool—An onco-tool that contains a radioactive isotope effective to kill cells.

  Dual Radioisotope Onco-Tool Treatment Strategy-A treatment strategy to treat a tumor with at least two onco-tools, one onco-tool emitting high linear transition energy radiation and the other onco-tool Contains beta-ray radioactive isotopes.

  Interstitial space-a region of tissue or tumor outside the vascular bed and outside the cells.

  Tumor acidic area-the area of the tumor with a pH of the gap less than 7.0.

  Physiological conditions—an aqueous solution having a temperature in the range of 20 ° C. to 40 ° C. and a sodium chloride concentration of about 0.13 M to 0.17 M.

  Significant portion-over about 1%.

  Anionic hydrophilic form—a form having at least one negative charge and an octanol / water partition coefficient of less than 1.

  Nonionic lipophilic form-a form that has no ionic charge and has an octanol / water partition coefficient greater than 1.

  Effectiveness factor-the proportion of onco-tool molecules that are in their non-ionic form at pH 6.4.

  Specificity factor—ratio of (ratio of non-ionic onco-tool molecules at pH 6.4) divided by (ratio of non-ionic onco-tool molecules at pH 7.4).

  Effective for reporting presence-a radioisotope that generates a signal that radiation, such as gamma rays or positrons, can easily be detected outside the subject's body.

  pH value at the midpoint of the transition between the hydrophilic form of the pT-substance and the lipophilic form of the substance, the transition between the two forms being saline (or approximately equivalent with some buffer) Measured with the substance present at 5 mmol in the product. The pT value is similar to the substance's pKa value, except that the pT value is affected by both the substance's pKa and the substance's lipophilicity. The pT value accounts for carboxyl-containing materials where the non-ionic form is significantly lipophilic and thus may fall out of the aqueous solution during titration or partition into the nonpolar phase during the partitioning test. Useful for. In addition, pT values are measured in the presence of approximately physiological concentrations of sodium chloride, and thus better reflect the properties of the test substance in the in vivo environment.

  Radioisotope that emits high linear energy transfer radiation—radioisotope that emits alpha particles or emits Auger and / or Coster-Kronig electrons.

  Inventor-James E.M. Summerton, Ph. D.

Summary of disclosure :
A. Molecular design of onco-tools pH switch components a) Adjust pH switch lipophilicity b) Incorporate novel acid-specific internal H-bonds into pH switches c) Incorporate multiple pH switch components into onco-tools Cargo Component a) Structural Requirements b) Precursor and Final Form c) Radioisotope Volume Selection i) For Tumor Detection ii) For Tumor Treatment Onco-tool structure a) Structural requirements b) Onco-tool with 2 pH switch c) Onco-tool with 3 pH switch d) Onco-tool with 4 pH switch B. Composition, testing and optimization of ingredients and onco-tools Preparation of a structure comprising a representative pH switch a) Simple pH switch b) Advanced pH switch c) Advanced pH switch designed to form a low barrier H bond 2. pH switch test Preparation of typical onco-tool and conversion to final form a) Onco-tool with simple pH switch b) Onco-tool with advanced pH switch c) High pH designed to form low barrier H-bonds 3. Onco-tool with switch Onco-tool testing and optimization a) Iterative optimization process: synthesis and testing in cultured cells i) Prepare onco-tool sets with different lipophilicity ii) Invasion into cultured cells at pH 6.4 and pH 7.4 Iii) Repeat the above steps as needed b) Evaluate in vivo fate in normal mice c) Evaluate in vivo fate in tumor bearing mice d) Preclinical and clinical studies How to use an onco-tool Diagnosis method 2. Treatment methods a) Single radioisotope method b) Double radioisotope method 3. Comprehensive methods for detecting and treating tumors 4. Strategies for dealing with micrometastases 5. Treat to lower the pH in the tumor for increased efficacy and specificity. 6. Use multiple onco-tools with different pKa to increase efficacy and specificity. Treat to increase urine pH to prevent kidney damage 8. Monitor and lavage the bladder for increased safety

Embodiments for carrying out the invention :
A. Onco-Tool Molecular Design The onco-tools that make up the present invention are a novel class of molecules designed for tumor detection and treatment. Each onco-tool includes two or more pH switch components that readily undergo a pH-mediated transition between an anionic hydrophilic form at higher pH and a non-ionic lipophilic form at lower pH. . Each onco-tool also includes a cargo component that is effective to bind to a selected radioisotope suitable for serving the onco-tool diagnostic or therapeutic role.

1. If the pH switch component onco-tool is to achieve sufficient specificity for the acidic region of the tumor, the onco-tool will be charged in an aqueous solution at pH 7.4 where almost all of the onco-tool molecules are negatively charged to the cell membrane. It must have a structure that exists in a negatively charged form (anionic form) that lacks affinity for the surface. Conversely, to achieve sufficient efficacy at the pH present in the acidic region of the tumor, a significant portion (greater than 1%) of the onco-tool molecule is in a non-ionic lipophilic form that readily penetrates the cell membrane. Should be converted. A major challenge in designing an onco-tool is that a sufficient portion of the onco-tool molecule is anionic and hydrophilic within the very limited pH difference available between normal tissue and the acidic region of the tumor. To devise a structure that undergoes a transition between a natural form and a non-ionic lipophilic form. This pH difference is generally only about 0.5-0.7 pH units, but a simple step to substantially increase this pH difference between normal tissue and the acidic region of the tumor (herein It should be understood that can be easily performed. Such a step can increase the pH difference between normal tissue and the acidic region of the tumor, generally to 1.0 pH units or more.

  To address the highly demanding molecular design challenges inherent in the use of acidity in tumors, the inventors have devised pH switch structures containing weak acid moieties (generally carboxylic acids) that each have a negative charge at pH 7.4. . This essential weak acid moiety then exhibits substantial hydrophilicity in its non-ionic free acid form, so that to achieve sufficient efficacy, when the onco-tool enters the acidic area in the tumor, it quickly Two molecular design strategies were used to provide sufficient lipophilicity for proper membrane penetration. These two design strategies, used alone or in combination, can also play an important role in dramatically increasing the onco-tool specificity for the acidic region of the tumor when combined with the third design strategy. . The physical and mathematical basis for these three molecular design strategies, two of which are considered specific to onco-tools, is described below.

  a) Adjust the lipophilicity of the pH switch by incorporating multiple lipophilic moieties to partially defeat the inherent hydrophilicity of the free acid moiety. In this regard, how the increase in lipophilicity of the non-ionic free acid form of the pH switch affects its partitioning properties (ie how it partition between the aqueous phase and the lipophilic phase such as octanol or cell membrane) It is first useful to obtain a quantitative assessment of To that end, mathematical modeling is performed to exist in the anionic water-soluble form [A-] at high pH, have some water solubility at lower pH [HAsol], and precipitate or oil separation from aqueous solution. Forms of weak acid substances that convert to non-ionic lipophilic forms, which can also be water insoluble / lipid soluble, represented by [HAinsol] by (oiling) or by partitioning into lipophilic phases such as octanol or cell membranes The pH dependent transition between was calculated. These three forms of interconversion as well as important equations used for mathematical modeling are shown in FIG.

  The results from this modeling shown in FIG. 4 show that the pH at the midpoint of the transition between the anionic form [A−] and the nonionic form [HAsol plus HAinsol] is the octanol / water partition coefficient of the nonionic free acid form. It shows that P increases gradually as P increases. However, such an increase in the pH of the transition is only after the free acid form has reached an effective lipophilic threshold to initiate precipitation or oil separation, or when there is an apolar phase that allows partitioning. apply. Example 1 describes a titration experiment that illustrates this mathematically predicted effect of the lipophilicity of the acid form on the pH-dependent transition between the anionic and nonionic forms.

  However, it should be noted that incorporating excess lipophilic moieties as the only means for increasing non-ionic lipophilicity can be counterproductive. This is because when the very high lipophilicity occurs only by the incorporation of an excessive number of lipophilic moieties, even the anionic form of the pH switch can begin to have affinity for the cell surface, as well as normal tissues. This is to provide capture of the onco-tool in the tumor. This can be a particular problem when the lipophilic part forms one large surface and the hydrophilic part is sufficiently away from the lipophilic surface. If the onco-tool is also captured in normal tissue, the onco-tool will cause high background in diagnostic applications and significant toxicity in therapeutic applications.

  b) Incorporating novel acid-specific internal H-bonds into the pH switch to expand the difference in solubility between hydrophilic and lipophilic forms.

  In the previous section, rather conventional strategies have been described to improve onco-tool invasion into cells in the acidic region of the tumor, thereby increasing its effectiveness. The strategy is to incorporate lipophilic groups into the pH switch component. This simple strategy is commonly used for what is called “rational drug design”. This has been widely applied in attempts to improve the efficacy and specificity of cancer chemotherapeutic drugs. However, since there is only a fairly small difference in pH value between normal tissue and the acidic area of the tumor, the inventors devise additional strategies to increase onco-tool penetration into the acidic area. I planned. This was expected to increase the specificity of the onco-tool for the tumor. To this end, the inventor has substantially reduced the anionic and nonionic lipophilicity of the pH switch by inventing a structure that is effective in forming pH-dependent internal acid-specific hydrogen bonds. It was assumed that it could be increased to The inventor further noted that such internal H-bonds raise the pH at which the pH switch transitions between its anionic and non-ionic forms, thereby entering cells in the acidic region of the tumor. It was assumed that more onco-tools would be available in non-ionic form.

  Its predicted H-bond-mediated increase in lipophilicity in the non-ionic form and a concomitant increase in hydrophilicity in the anionic form indicate that in aqueous environments such internal H-bonds are some of the free acid forms (probably two ) Based on the expectation to replace bound water. For illustration, FIG. 5 a) shows the expected water of hydration directly H-bonded to standard carboxyls in the anionic and nonionic free acid forms. FIG. 5 b) shows the corresponding expected water of hydration directly H-bonded to the anionic and nonionic free acid forms of the structure effective to form the desired internal acid-specific H bond. Clearly, standard carboxyls and carboxyls that are effective in forming internal H-bonds lose their counterions as they transition from the anionic form to the nonionic free acid form. However, while standard carboxyls are expected to have the same number (probably four) of water directly H-bonded to the anionic and nonionic free acid forms, the inventor forms internal H-bonds. For the carboxyls that can, there is a net loss of some (probably two) direct H-bonded waters when moving from the anionic form (probably five H-bonded waters) to the non-ionic free acid form. Yes, we believe that this loss of some H-bonded water will substantially expand the hydrophilic / lipophilic difference between the two forms. If the inventor is able to form such internal acid-specific H-bonds in an aqueous environment, the loss resulting from some hydration water will rapidly invade cells in the acidic region of the tumor. -Expected to significantly increase the ability of the tool, while also significantly increasing onco-tool rejection by cells in normal tissues. In summary, the incorporation of such internal acid-specific H-bonds enables onco-tool specificity by differentially changing the number of water molecules bound to each of the two forms of the pH switch that are its components. And was expected to increase both effectiveness.

  The inventors also note that the formation of an internal acid-specific H bond in the pH switch component favors the transfer of the carboxyl moiety of the pH switch component to its free acid form, resulting in a significant increase in the pKa value of the carboxylic acid moiety. Predicted (and now demonstrated) it should be. As detailed in the next section, such an increase in the pKa of the carboxyl moiety dramatically increases the specificity of the onco-tool for the tumor by incorporating two or more pH switches into the on-cotool. Desirable in relation to increasing strategy.

  With respect to the design of pH switch structures that can form internal H-bonds in an aqueous environment, conventional insights in hydrogen bond specialists indicate that single hydrogen bonds are H-bond acceptor sites (110 moles) and H-bond donor sites (110 It is generally supported that it is not stable in an aqueous environment due to competition with enormous concentrations of water in (mol). Instead, it is generally believed that stable non-covalent interactions in aqueous solutions require a variety of interactions selected from H-bonds, hydrophobic interactions and electrostatic interactions.

  While this requirement for diversity of non-covalent bonds appears to be well established for intermolecular interactions, the inventor believes that, contrary to conventional insights, the H bond is hydrophilic between the two forms. Single, relatively stable pH dependence in aqueous solution that increases the difference in sexual / lipophilic properties and acts to favor the free acid form relative to the anionic form (ie, raises the pKa) It was postulated that it would be possible to invent a compact structure that forms intramolecular H-bonds. The critical question at that time was whether an actual pH switch structure could be invented that would form such an internal H bond in aqueous solution.

  After considerable experimentation, it was demonstrated to form the desired single pH-dependent internal H-bond, and a novel structure suitable for incorporation into the onco-tool of the present invention has now been invented. As the inventor first predicted, such internal H-bonds appear to significantly increase the hydrophilic / lipophilic difference as the pH switch transitions from its anionic form to its non-ionic form. . Some such internal acid-specific H-bonds markedly compare the pH at which the structure converts from its anionic hydrophilic form to its non-ionic lipophilic form compared to the pH at which a similar simple carboxyl moiety undergoes this transition. It has also been demonstrated to increase.

  A pH switch having a structure designed to form an internal acid-specific H bond is referred to as an “advanced pH switch”. Results from molecular modeling and extensive experimental studies suggest that the following three properties are essential for an advanced pH switch to form an internal acid-specific H bond that is tolerably stable in aqueous solution: is doing.

i) The acid-specific H-bond structure must include a carboxyl moiety that is positioned in close proximity to the H-bond acceptor moiety for H-bond formation. When the carboxyl moiety is in its free acid form, it must serve as an H-bond donor, and the proximal H-bond acceptor moiety can only serve as an H-bond acceptor in its non-ionic form. And must be unable to serve as an H-bond donor. The inventor has called the H bond formed by such a structure an “internal acid-specific H bond”. FIG. 6a shows a typical pH switch structure capable of forming only internal acid-specific H bonds. Conversely, FIG. 6b) is similar but allowed to form an internal H bond when the carboxyl is in its anionic form and an internal H bond when the carboxyl is in its non-ionic form. Indicates a structure that cannot. The inventor has referred to this double H-binding ability as an “internal non-acid specific H-bond” and the inventor's experimental results indicate that such non-acid-specific H bonds are the desired lipophilic form of the acid form Does not increase the pH of the anionic hydrophilic form to its nonionic lipophilic form, indicating that it is unacceptable. Example 2 describes that the structure in FIG. 6a) forms an internal acid-specific H bond under slightly acidic conditions in a one-to-one mixture of water and methanol—anionic form and free acid. A significant increase in the pH of the transition between forms (ie, an increase in the pKa value of the carboxyl moiety). Conversely, the very similar structure in FIG. 6b) designed to form a non-acid specific H bond in this same example is between the anionic form and the non-ionic form under these conditions. It also shows that the pH of the transition is not increased.

ii) Minimal conformational freedom between H-bonding moieties Carboxyls that serve as H-bond acceptor and H-bond donor moieties remain in close proximity to each other with structures having minimal conformational freedom It should be. This limited conformational freedom can be achieved using a suitable ring structure. Molecular modeling and experimental studies suggest that 4-, 5- and 6-membered aliphatic rings are preferred for this purpose. FIG. 7 a) shows a representative 4-membered, which allows only limited conformational freedom between the H-bond donor (OH of the carboxylic acid) and an appropriately positioned H-bond acceptor moiety. 1-membered and 6-membered aliphatic ring structures are shown. Conversely, FIG. 7 b) shows a somewhat similar structure whose acyclic structure is unacceptable to allow excessive conformational freedom between the H-bond donor and H-bond acceptor moieties. . Although the structure with minimal conformational freedom between H-bonding moieties in a 1: 1 mixture of methanol and water in Example 3 can result in a significant increase in the pKa value of the carboxyl moiety, Similar structures with substantially greater rotational degrees of freedom in between do not result in a corresponding increase in the pKa value of the carboxyl moiety and therefore probably cannot form stable internal acid-specific H-bonds under these conditions State that.

iii) The carboxylic acid to serve as a carboxyl H-bond donor moiety insulated from the inducing effect of the electron withdrawing group is separated from any attached electron withdrawing group by at least 2, preferably 3 or more carbons. Should. This avoids an excessive decrease in the pKa value of the carboxyl due to the induced effect of the electron withdrawing group. The molecular structure shown in FIG. 8a) and their corresponding pKa values show how increasing the number of aliphatic carbons can progressively insulate the carboxyl from the pKa lowering effect of the phenyl group. FIG. 8b) shows a structure where the attractive effect of the adjacent amide moiety clearly causes an excessive decrease in the pKa of the carboxylic acid moiety. In Example 4, even when there is minimal conformational freedom between the H-bond donor and acceptor moiety, if there is insufficient carboxyl insulation from the attraction effect, the anionic form and the free acid It states that the pH of the transition between forms is too low to be useful for pH switches.

  In addition to the above three essential properties, at least one additional property selected from the following two properties may be desirable to achieve internal H-bond formation in aqueous solution.

iv) Lipophilic groups that partially shield H binding sites The main challenge in forming a single H bond in an aqueous environment is the huge concentration of competitive H bond donors and H bond acceptors including ambient water This H bond is preferentially formed in the presence of. Based on biochemical studies of enzyme catalytic sites and the inventors' extensive molecular modeling, the inventors have found that if the H-binding sites are partially shielded from bulk water by the presence of lipophilic groups, the desired molecule It was assumed that internal H bonds would be more advantageous. In connection with partial shielding from the solvent, FIG. 9 shows two related structures. In FIG. 9 a), the H binding sites are completely open to the solvent. Conversely, the corresponding H binding sites in FIG. 9b) are partially shielded from the solvent by the adjacent methyl on one side and by the adjacent methylene on the other side. The synthesis and titration studies detailed in Example 5 have obtained results suggesting that such partial shielding of the H-bonding sites from the solvent favors the desired intramolecular H-bonding.

v) Low-barrier H-bond The term “low-barrier H-bond” is a non-covalent bond formed between an H-bond donor moiety and an H-bond acceptor moiety, where the two isolated moieties have a pKa value of each other As used herein to mean within about 2 pH units. It should be noted that this definition includes what hydrogen can also be interpreted as an internal salt closer to the acceptor moiety than to the donor moiety. Such low barrier H bonds are generally found to be exceptionally strong and can therefore be quite advantageous for the desired intramolecular H bonds in the pH switch. FIG. 10 a) and FIG. 10 b) show various H-bonding moieties suitable for forming low barrier H-bonds in pH switches. FIG. 10 c) shows some typical pH switch structures designed to form low barrier type H-bonds. Example 6 describes the synthesis and testing of structures i and iii) in two representative pH switches (FIG. 10b) and experimental evidence of the formation of the desired low barrier H bond in aqueous solution.

  The following is a summary of properties found to be suitable for advanced pH switch components designed to form internal acid specific hydrogen bonds.

a) comprising an aliphatic ring structure selected from the group consisting of 4-membered, 5-membered and 6-membered rings,
b) containing a carboxylic acid moiety directly attached to the aliphatic ring structure;
c) the carboxylic acid moiety is separated from the bonded electron withdrawing group by at least two carbons;
d) comprising an H-bond acceptor moiety selected from the group consisting of:
i) part of an aliphatic ring structure ii) directly bonded to the aliphatic ring structure, and iii) bonded to the aliphatic ring structure via one atom e) an H-bond acceptor moiety Has a structure that does not serve as an H-bond donor moiety in the non-ionic form, and f) the carboxylic acid moiety and the H-bond acceptor moiety are positioned in close proximity to each other, making them compatible with the formation of internal acid-specific H bonds Properly positioned and oriented.

c) Incorporating multiple pH switch components into each onco-tool as a novel strategy to dramatically increase the specificity of the onco-tool for the tumor without leading to undue loss of onco-tool effectiveness .
The two design strategies described in the previous two sections, used alone or in combination, are effective to penetrate the cell membrane at the pH present in the acidic region of the tumor, and thus provide a pH switch structure that provides sufficient efficacy Satisfy the requirement to generate However, such a design strategy provides only a slight specificity advantage over conventional cancer therapies such as chlorambucil. In this regard, it should be understood that for most cancer therapies currently used, it is their poor specificity that is generally the basis of many treatment failures for patients. That is, the current cancer therapy is estimated to have a specificity of about 2-8. This means that they are generally about 2 to 8 times more damaging to cancer than to normal tissue. Unless the inventor can devise any effective strategy to substantially increase the specificity for the tumor, a similar low level of specificity and corresponding low response rate would result in onco-tool therapy. He was seriously aware that he would be bothered. Thus, the inventor set out on a search several years ago to devise some way to substantially increase the specificity of onco-tools. That exploration led to the third and most essential onco-tool design strategy described below.

  The pKa of an acid is the pH of an aqueous acid solution in which half of the acid molecules are anionic and half are nonionic. Knowing the pKa of the acid and the pH of its aqueous solution, the following Henderson-Hasselbal equation is used to show the anionic form [H-] and non-ionic form [HA] present in the solution: Can be calculated: (pH = pKa + log [A −] / [HA]).

  Due to the simple application of the Henderson-Hessel bulk formula, the pH in the gap in normal tissue and the pH in the acidic region of the tumor differ by only 1.0 pH units (ie, about pH 6.4 and normal tissue achievable in the tumor). In the case of pH 7.4), the theoretical maximum specificity compared to normal tissue, which can be shown for the acidic region of the tumor, appears to be less than 10 Is done. In other words, if there is a pH difference of only 1.0 between normal tissue and tumor, a simple application of the Henderson-Hasselbulk formula can prevent the invasion of conventional weak acid-containing drugs into cells in the acidic region of the tumor. It appears that the rate can be about 10 times less than the rate of entry of the drug into cells in normal tissue.

  If this upper limit of about 10 times applies to onco-tool specificity, in diagnostic applications, this insufficient level of specificity is particularly important for very small early tumors whose detection results in the best prognosis for the patient. It can generate a large background signal from normal tissue that overwhelms most or all of the signal from the tumor that it was trying to detect for the case. Similarly, in therapeutic applications, such insufficient specificity levels can result in considerable toxicity in the patient, possibly to the extent that the patient dies (this is also the case with similar insufficient specificity levels). Commonly occurs with current chemotherapy).

  In order to overcome the problem of limited specificity, the inventor has found an onco--beyond an apparent theoretical maximum of 10 that would be caused by a small pH difference between normal and tumor tissue. A new design strategy was devised to dramatically increase the specificity of the tool. In essence, the strategy is to incorporate two or more pH switches into a single onco-tool, where the carboxyl of each pH switch significantly affects the ionization of one carboxyl to its neighboring carboxyls. Sufficiently far from the carboxyl of its adjacent pH switch (possibly more than about 5 angstroms away). Conversely, all of the pH switches of a multi-pH switch onco-tool have to be turned on by the onco-tool to penetrate its lipophilic interior, which is required for the onco-tool to be trapped in the acidic area of the tumor. Before approaching and then breaking through the negatively charged outer surface of the cell membrane, all of the component pH switches should be close enough that they must be in their non-ionic form. It is estimated that all carboxyls of the multi-pH switch onco-tool should be at a distance of about 15-20 angstroms or less from each other to fully meet this latter requirement.

  FIG. 11 a) shows various anion and non-ion forms of an onco-tool that abstractly includes a 2 pH switch component, and FIG. 11 b) shows an onco-tool that also includes three pH switch components. Figure 3 shows various anionic and non-ionic forms of the tool. When properly designed, only this non-ionic form of the onco-tool can efficiently contact and penetrate the cell membrane, and all other forms, including at least one anionic portion, are electrostatic. It should be understood that it is the concentration of the non-ionic form that is of particular interest as it is repelled from the surface of the anion cells by the force of the force.

  When using this multi-pH switch design strategy, the expected effectiveness of a given onco-tool using the pKa value of the carboxyl portion of the pH switch used in the onco-tool and the number of those pH switches in the onco-tool And the specificity factor can be calculated. In these calculations, the effectiveness factor is the percentage of onco-tool molecules that are non-ionic at pH 6.4. This factor gives a measure of how efficiently the onco-tool enters cells in the acidic area of the tumor. The specificity factor is the ratio of (% non-ionic form at pH 6.4) divided by (% non-ionic form at pH 7.4). This coefficient is a measure for the relative rate of onco-tool invasion into cells in the acidic region of the tumor (pH 6.4) relative to the rate of onco-tool invasion into cells in normal tissue (pH 7.4). give. For these calculations, for the pH switch initially selected using the Henderson-Hassel bulk equation, what percentage is pH 6.4 (can be achieved in the acidic region of the tumor) and pH 7.4 (present in normal tissue). To calculate whether it is non-ionic. The binomial expansion is then used to calculate the percentage of onco-tool molecules with all of their component pH switches in the non-ionic form.

  FIG. 12 shows the pKa values of a) those compositions pH switch of a) a composition containing 1 pH switch, b) an onco-tool containing 2 pH switch, c) an onco-tool containing 3 pH switch, and d) an onco-tool containing 4 pH switch. 2 shows a plot of computational effectiveness and specificity coefficient as a function of. These calculated effectiveness and specificity factors indicate that compositions containing a single carboxyl moiety, such as chlorambucil, have rather poor specificity for selective entry into cells in the acidic region of the tumor (specificity factor is always less than 10). Is essentially present. However, by incorporating two or more pH switches into a single onco-tool, a calculated practical effectiveness factor (more than about 5% of the non-ionic onco-tool at pH 6.4) and pKa Appropriate adjustment of the values (see previous section on internal acid-specific H-bonds) can increase the onco-tool specificity factor many times over that possible with a single carboxyl composition. It is suggested. In particular, with an appropriate pKa value, the specificity factor should be at least 50 for onco-tools with 2 pH switches and at least 200 for onco-tools with 3 pH switches and over 900 for onco-tools with 4 pH switches. Can do.

  As can be seen from FIG. 12, there is an inverse relationship between the effectiveness factor and the specificity factor. Thus, as the pKa of the pH switch increases, the effectiveness factor increases, but the specificity factor decreases. Since both reasonable efficacy and high specificity are desirable in cancer therapy, it is useful to consider what pKa values may provide a reasonably optimal balance between efficacy and specificity. . Based on the calculated values in b) of FIG. 12, a pH switch with a pKa of 5.9 provides a desirable balance of effectiveness (6%) and specificity (60) for on-co-tools containing 2 pH switches. Seem. Furthermore, the calculated values in FIG. 12 c) give a desirable balance of effectiveness (5.8%) and specificity (277) for on-co-tools in which a pH switch with a pKa of 6.2 includes a 3 pH switch. I suggest that. Still further, the calculated value in d) of FIG. 12 shows that a pH switch with a pKa of 6.4 provides a desirable balance between effectiveness (6.3%) and specificity (915) for an on-co-tool that includes a 4 pH switch. Suggest to bring.

  In this regard, as mentioned in the previous section, incorporating an internal acid-specific H bond into the pH switch would result in such dramatically improved specificity in these multiple pH switch onco-tools. It should be understood that it provides a suitable means of adjusting the pKa value of the pH switch to an appropriate range to obtain.

  The foregoing considerations and calculations are to optimize the pKa value of the onco-tool pH switch as a means to achieve the desired balance between efficacy and specificity (by incorporating internal acid-specific H-bonds). It was in focus (FIGS. 11 and 12). However, it should also be understood that increasing the onco-tool partition coefficient (ie, lipophilicity) has a similar effect on the balance between the effectiveness and specificity of the multi-pH switch onco-tool. This is because, as shown in FIG. 4, such an increase in lipophilicity also serves to raise the pH at which the onco-tool can enter the lipophilic phase such as the cell membrane. Thus, in this multi-pH switch strategy to increase the specificity of the onco-tool for the tumor, either by adjusting the lipophilicity of the onco-tool or by adjusting the pKa of the pH switch (internal acid specific H binding The balance between efficacy and specificity can be adjusted by adjusting the lipophilicity of the onco-tool and the pKa of the pH switch component.

  At this point, these calculated specificity values of the multi-pH switch on co-tool selectively enter the lipophilic phase (such as octanol or cell membrane) at the pH (pH 6.4) that can be achieved in the acidic region of the tumor. It is appropriate to ask whether it actually reflects its onco-tool's ability to sufficiently avoid entering such a lipophilic phase at normal tissue pH (pH 7.4).

  In order to address this question, a representative composition containing the two pH switch components shown in FIG. 13 a) was synthesized. After synthesis, the composition was partitioned between n-octanol and aqueous buffer ranging from pH 5.6 to pH 8.0. The result of this distribution experiment is plotted in FIG. These results show that at pH 6.4 (which can be achieved in tumors) half of this composition was partitioned in the octanol phase, but at pH 7.0 only about 2% was partitioned in the octanol phase and at pH 7.2 the composition. None of the objects were detected in the octanol phase.

  As suggested by the calculated values plotted in FIG. 12, these partition results are also present in the non-ionic form (ie, octanol) at the pH at which the majority of the compositions containing the two pH switch components are present in the acidic region of the tumor. It is also suggested that the composition can be almost completely converted to its anionic form (ie buffer soluble) at the pH present in normal tissues. . This provides experimental support for the usefulness of the new strategy of incorporating multiple pH switch components into the onco-tool as a means to dramatically increase the specificity of the onco-tool for the acidic region of the tumor.

  Various exemplary multi-pH switch on co-tool structures are described in A. Shown in item 3.

2. Load component
a) Structural requirement cargo component is the structural component of the onco-tool that serves to incorporate a radioisotope whose radiation is effective to report the presence of the onco-tool or to kill the cell. The cargo component should meet the following three design requirements:

    i) The precursor form of the cargo component should be effective to incorporate the selected radioisotope easily and efficiently with minimal manipulation.

    ii) the radioisotope bound to its final form of the cargo component remains so bound during the diagnostic procedure or throughout the course of therapy in which the radiation from the radioisotope kills the tumor cells. Should be.

    iii) The final form of the cargo component, including the bound radioisotope, should be such that it is sufficiently small and does not unduly affect the pH-dependent hydrophilic / lipophilic properties of the onco-tool. In other words, if the final form of the cargo component contributes to excessive hydrophilicity, it inhibits onco-tool non-ionic entry into the cells in the acidic region of the tumor, thereby increasing efficacy. May decrease. Conversely, if the final form of the cargo component contributes to excessive lipophilicity, it can cause excessive capture in normal tissue, thereby reducing specificity.

  Based on these requirements for cargo components, the radiohalogen selected from F, Br, I and At is the best radioisotope type, and the vinyl group or monounsaturated ring or monoaromatic ring is the best to which the radiohalogen is attached. It seems to play a role. FIG. 14 shows a number of expected cargo components of the precursor form selected and the final radioisotope-containing form ready for diagnostic or therapeutic use.

b) If the selected radioisotope to be bound to the precursor and final form onco-tool has a short half-life, the precursor form of the onco-tool is manufactured, transported, stored and then diagnosed or treated It is often desirable to apply a radioisotope load just before delivering the onco-tool to the subject to be treated. A number of different structures suitable for combining various radioisotopes have been reported in the nuclear medicine field, but many such structures are unsuitable for use in onco-tools. FIG. 14 shows some selected cargo components of their precursor forms and their final forms that meet the unique requirements for use in onco-tools. FIG. 15 shows a synthetic scheme for producing two such cargo components in precursor form. These syntheses use important reactions for adding a trialkyltin moiety already described (see, for example, Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4, and Non-Patent Document 5). FIG. 15 also shows a simple procedure for converting previously described (see Non-Patent Document 6) precursor forms to their final radioisotope-containing forms. Procedures for producing other suitable cargo components and incorporating radioisotopes to obtain the final form are described elsewhere (see, eg, Non-Patent Document 7 and Non-Patent Document 8).

c) Radioisotope volume selection It should be noted that it is the combined radioisotope of the onco-tool that determines the application of the onco-tool. If the bound radioisotope emits a signal that can be easily detected outside the body, the onco-tool can serve to detect tumors containing acidic regions. Conversely, if the radioisotope has a radioactive substance that is effective in killing the cell, the onco-tool can serve for the treatment of tumors that contain acidic regions. In addition, the onco-tool emits a signal (eg, gamma rays) that can be easily detected outside the body, and a radioactive isotope such as iodine 131 that has a radioactive material (eg, beta particles) effective to kill the cell. If included, the onco-tool can serve for diagnosis and treatment of tumors containing acidic regions.

i) In the onco-tool used to detect tumors for tumor detection, there is considerable tolerance in selecting radioisotopes that generate signals suitable for detection outside the subject's body. There are several radioisotopes that have advantageous properties for diagnostic applications:
Radioisotope half-life Fluorine 18 1.8 hours Bromine 75 1.7 hours Bromine 76 16 hours Bromine 77 2.4 days Iodine 123 13 hours Iodine 124 4 days Iodine 125 60 days Iodine 126 13 days Iodine 131 8 days

ii) Successful treatment of tumors for tumor treatment faces two challenges. One challenge is to completely kill all therapeutically sensitive rapidly dividing tumor cells at near normal pH close to tumor capillaries. Another less accessible issue is the complete killing of all treatment-resistant resting tumor cells in the acidic region of the tumor. A therapeutic onco-tool was first devised only as a means to kill treatment-resistant resting cells in the acidic region of the tumor. This is designed so that the onco-tool is captured only in the acidic area of the tumor, so such onco-tool must be used in conjunction with more conventional cancer therapies such as radiation or chemotherapy. Rather, conventional therapies would play a role in killing treatment-sensitive rapidly dividing tumor cells in more neutral areas of the tumor closer to the capillaries (because there are few onco-tools in such areas) This is because of the assumption.

  More recently, the inventor can destroy the entire tumor using only onco-tools, thereby avoiding the need for combined treatment with conventional cancer therapies that are more toxic and less specific. A heavy radioactive isotope strategy was devised. This dual radioisotope strategy requires the use of at least two onco-tool formulations that are effective in killing cells.

  Onco-tool formulations used to kill therapy-resistant resting tumor cells kill all of the nearby radiation-resistant resting tumor cells on which the therapeutic onco-tool containing the radioisotope is placed or Therefore, it should contain radioactive isotopes that emit high linear energy transfer radiation.

  One option for achieving this demanding requirement is that the energy released is highly effective in killing even cells that are highly resistant to most radiation. The use of radioisotopes that emit alpha particles that emit large amounts of energy over short distances (several cell diameters). The best radioisotope for this purpose appears to be astatine 211, a radiohalogen. This radioisotope is generated from natural bismuth 209 in a medium energy cyclotron equipped with an alpha particle beam. Astatine 211 has a half-life of 7.2 hours and has been shown to destroy cells within about 50-80 micron path length (several cell diameters) of emitted alpha particles. It emits two alpha particles with an energy of 45 million electron volts. Only a few such alpha emitters can kill even the most radioresistant quiescent tumor cells.

  Another class of radioisotopes that emit high linear energy transfer radiation are those that emit auger and / or coster-chronic electrons. Such radioisotopes can cause destructive damage to cellular DNA when placed in cells to be killed and are therefore extremely cytotoxic when localized in close proximity to the cell nucleus. Indicates. Such radioisotopes that are particularly suitable for therapeutic onco-tools include bromine 77 (half-life 57 hours) and iodine 123 (half-life 13 hours).

Onco-tool formulations used to kill therapeutically sensitive rapidly dividing tumor cells that are closer to the capillaries emit particles that release enough energy along their pathways to kill the therapeutically sensitive rapidly dividing tumor cells. Should contain one or more radioisotopes. More importantly, those that emit particles kill the therapeutically sensitive rapidly dividing tumor cells in the more neutral areas of the tumor where they can range from the acidic area where the onco-tool is captured to several hundred microns. It must have a sufficiently long path length that is effective for cells from the location where the onco-tool is captured to a few hundred microns. Beta-radioactive isotopes best meet this requirement. Although there are many beta-radioactive isotopes that can be used in this dual isotope therapy strategy, the following radiohalogens have advantageous properties for this application, including average path lengths greater than 600 microns .
Radioisotope half-life bromine 82 36 hours Bromine 83 2.4 hours Iodine 126 13 days Iodine 130 12 hours Iodine 131 8 days Iodine 132 2.3 hours Iodine 133 21 hours Iodine 135 7 hours

  The onco-tool is used only to kill resting cells in the acidic area of the tumor, and is used in conjunction with conventional cancer therapies effective to kill rapidly dividing tumor cells in other areas of the tumor with higher pH Compared to therapy, this new dual radioisotope onco-tool therapy strategy using only onco-tool to kill the entire tumor is simpler, less expensive and much more toxic Will provide a lower and much more effective treatment.

3. Onco-tool structure
a) Structural requirements Each onco-tool must contain two or more pH switch components. In order to achieve substantially greater (at least 20 or more, preferably 40 or more) specificity than is generally provided by current cancer therapies (specificity is generally estimated to be in the range of about 2-8) This “two or more” requirement is mandatory. In addition, each onco-tool exists almost completely in the anionic hydrophilic form at pH 7.4, but a significant portion (preferably 1% or more) is trapped in the acidic region of the tumor at pH 6.4. It must have a structure that can be transferred to a non-ionic lipophilic form that is effective for this purpose. Each onco-tool also includes a radioactive isotope that is effective to bind to a radioisotope, or suitable for reporting the presence of an onco-tool and / or suitable for killing a cell. Must be included.

  The following three sections describe an onco-tool that includes two, three and four pH switches. For such onco-tool structures, the selected combination of ingredients generally requires optimization of the R group in order to achieve sufficient effectiveness and the desired balance of effectiveness and specificity. Procedures for such optimization of the R group are described later in this document in section B and the figures and examples associated with this section.

b) Onco-tools containing 2 pH switches These onco-tools have the simplest structure and are generally easiest to synthesize. They generally have a specificity factor that is potentially in the range of about 20 to about 80. FIG. 16 shows various representative 2pH switch onco-tools that meet the important structural requirements of the onco-tool. FIG. 16a shows an onco-tool that includes two simple pH switches. FIG. 16b shows an onco-tool that includes two advanced pH switches. FIG. 16c shows an onco-tool that includes two advanced pH switches designed to form a low barrier H bond.

c) Onco-tool with a 3 pH switch An onco-tool with a 3 pH switch is more complex and generally more difficult to synthesize than the 2 pH switch onco-tool described above. However, the 3pH switch on co-tool has the advantage of providing a considerably higher specificity factor potentially in the range of about 100 to about 500. FIG. 17 shows various representative 3pH switch onco-tools that meet the important structural requirements of the onco-tool. FIG. 17a) shows an onco-tool that includes three advanced pH switches, and FIG. 17b) shows an onco-tool that includes three advanced pH switches designed to form low barrier H-bonds.

d) Onco-tool with a 4 pH switch An onco-tool with a 4 pH switch is more complex than the 3pH switch onco-tool described above and is generally more difficult to synthesize. However, the 4pH switch on co-tool has the potential for a higher specificity factor, potentially in the range of about 400 to about 2500. FIG. 18 shows two representative onco-tools that include four advanced pH switches designed to meet the important structural requirements of the onco-tool and form low barrier H-bonds.

B. Composition, testing and optimization of ingredients and onco-tools
1. Preparation of structures containing representative pH switches Before beginning the development of new onco-tool types, various pH switch types, as well as variations of the R group, initially serve to impart a wide range of lipophilicity to the structure. It is generally desirable to prepare in-mold variants and evaluate their properties.

a) Simple pH Switch FIG. 19 shows a typical synthesis route for an onco-tool structure including a simple pH switch with lipophilicity adjusted by changing the R group.

b) Advanced pH Switch FIG. 20 shows a synthetic route of a structure including an advanced pH switch type having lipophilicity adjusted by changing the R1 group.

c) Advanced pH Switch Designed to Form Low Barrier H Bonds FIGS. 21 and 22 show synthetic pathways for structures containing many advanced pH switch types designed to form low barrier H bonds.

  FIG. 21 shows useful amine ester and ketone ester intermediates for pH switch preparation and onco-tool preparation. Synthetic procedures for the preparation of important core components of such intermediates have already been reported (see, for example, Non-Patent Document 9 and Non-Patent Document 10).

  FIG. 22 shows representative synthetic pathways for converting several such intermediates into various pH switches designed to form internal acid specific low barrier H bonds.

2. pH Switch Testing Simple titration tests provide useful information regarding the pH-dependent dissolution properties of pH switch structures as well as their pKa values. The titration procedure used in Example 7 to test the pH switch component is described. FIG. 23 shows representative results of various past titrations.

  FIG. 23a shows a typical titration curve obtained by titrating simple carboxylic acid (butyric acid) and a high pH switch (an acid amide derivative of camphoric acid) in methanol / water (1: 1 volume ratio). On one plot, the same titration results are plotted in the form of a first derivative that gives more information.

  FIG. 23b shows a typical titration curve when titration was performed in water and each species was present at a concentration of 33 millimolar. Titration results are shown for three related 2 pH switch on co-tools that differ only in the R group (but contain stable iodine instead of radioactive iodine). Titration results also show related 3 pH switch structures where the R group is methyl.

  FIG. 23c shows a titration curve plotted as the first derivative for an advanced pH switch designed to form a low barrier H bond. This titration was carried out in water and a pH switch containing an N-oxide / acid structure derived from camphoric acid was present at a concentration of 5 millimolar.

  FIG. 24 shows an amide / acid advanced pH switch derived from camphoric acid, an N-oxide / acid advanced pH switch designed to form a low barrier H bond derived from isonipocotic acid, and derived from camphoric acid. Experimentally of three pH switch structures including an N-oxide / acid high pH switch designed to form a low barrier H bond (also shown as structure i in FIG. 10c and as shown in FIG. 23c). The determined pKa value is shown.

3. Preparation of typical onco-tool and conversion to final form
a) Onco-tool with a simple pH switch FIG. 19 shows a synthetic route for preparing a representative onco-tool with a simple pH switch.

b) Onco-tool including an advanced pH switch FIG. 25 shows a synthesis scheme of a representative onco-tool including an advanced pH switch. FIG. 25a shows an onco-tool whose two pH switches are joined by a diacyl hydrazide structure. FIG. 25b shows an onco-tool whose two pH switches are joined by a diamide structure.

c) Onco-tool containing an advanced pH switch designed to form a low-barrier H bond Figure 26 shows a representative onco-tool synthesis containing an advanced pH switch designed to form a low-barrier H bond. A scheme is shown.

  FIG. 26a shows a synthetic scheme for a 2pH switch on co-tool in which a single N-oxide moiety serves as an H-bond acceptor moiety for two carboxylic acid H-bond donor moieties. FIG. 26b shows a synthetic scheme for a 2pH switch on co-tool where both nitrogens of the hydrazine moiety serve as H-bond acceptor moieties for the two carboxylic acid H-bond donor moieties. FIG. 26c shows a synthetic scheme for a 2pH switch on co-tool where the cyanomethylamine moiety serves as the H-bond acceptor moiety for the carboxylic acid H-bond donor moiety. FIG. 26d shows a synthetic scheme for a 4 pH switch on co-tool where each N-oxide moiety serves as an H-bond acceptor moiety for two carboxylic acid H-bond donor moieties.

  In the foregoing synthesis of an onco-tool comprising an advanced pH switch designed to form a low barrier H bond, a ketone / ester intermediate such as structures b, d and f in FIG. When reductively alkylating an amine / ester that is cis to an ester moiety such as structures a, c and e in FIG. 21, the resulting 2 pH switch product generally comprises two isomers, one of which Note that both pH switch components are cis for amines and esters, and other products are one of the pH switch components is cis for the amine and ester moieties, and one of the pH switch components is trans. Should. In such cases, it is desirable to purify the desired cis / cis product and discard the cis / trans product. One way to accomplish this is to take advantage of the fact that the trans pH switch component is unable to form an internal acid specific low barrier H bond. As a result, its pKa is generally about 5.0 in aqueous solution. In contrast, cis pH switch components are generally capable of forming the desired internal acid specific low barrier H bond, thereby increasing the pKa of the included carboxyl moiety to about 6.0 or higher. As a result, after cleaving the ester moiety to obtain the carboxyl moiety, partitioning between an organic phase such as tetrahydrofuran, t-butyl methyl ether or dichloromethane and an aqueous phase buffered to about 6.0 provides the desired The cis / cis form should generally be easily separated from the undesired cis / trans form. In such a distribution system, the desired cis / cis form is generally preferentially distributed in the organic phase, while the undesired cis / trans form should be preferentially distributed in the aqueous buffer phase.

  The same basic separation strategy can be used to separate the desired full cis form of the 4pH switch on co-tool of FIG. 26d from multiple forms including at least one transformer pH switch.

4). Onco-Tool Testing and Optimization The following information sources serve as a starting point for designing and developing effective and specific onco-tools. The effectiveness and specificity plot in FIG. 12 provides guidance on the desired pKa value of the pH switch component as a function of the number of pH switch components included in the selected onco-tool. FIG. 4 provides guidance on the effect of changes in the onco-tool non-ionic partition coefficient, generally adjusting the partition coefficient by changing the R group lipophilicity of the pH switch component. Furthermore, B. above. 1. Term and B.I. 2. The synthesis of the various expected pH switch types and their R-group variants as described in the titration tests after those pH switches described in the section will give the appropriate pH switch components of the expected onco-tool. Provide useful information to choose. Finally, FIG. 14 shows a number of cargo components that are suitable for incorporation into onco-tools.

  Although the above information on component parts provides a useful basis for the design and development of effective and specific onco-tools, nonetheless, those component parts are expected to contain radioisotopes Once incorporated into an onco-tool, it is generally necessary to optimize the onco-tool structure to achieve sufficient effectiveness and the desired balance of effectiveness and specificity.

a) Iterative optimization process: synthesis and testing in cultured cells . A preferred optimization process requires:

i) An optimization process for preparing a set of onco-tools with different lipophilicity incorporates selected components that exhibit a wide range of lipophilicity because the set of onco-tools contains different R1 and R2 groups. Start by preparing a set of expected onco-tool structures. Each onco-tool in the set should also include a suitable radioisotope such as iodine 131 that allows easy detection and quantification in biological systems.

ii) each of those expected onco-tool structures that assess invasion into cultured cells at pH 6.4 and pH 7.4 , the major biological environment in which the expected onco-tool is encountered in living subjects and It should be subjected to testing in a relatively simple biological system exposed to the structure. As described in Example 8, a preferred biological system for such initial testing is buffered at pH 7.4 to mimic normal tissue and pH 6.4 to mimic the acidic region of the tumor. Mammalian cells cultured in serum-containing medium buffered with In summary, two different wells of cultured cells are exposed to a given onco-tool for 1 hour. In one culture well, the onco-tool is placed in a medium at pH 6.4. In other culture wells, the onco-tool is placed in the medium at pH 7.4. After 1 hour incubation at 37 ° C., the medium containing the onco-tool is removed, the cells are washed thoroughly with medium of the same pH, and then the radioisotopes retained by the cells are counted to obtain two pHs. A measure of the relative amount of onco-tool captured under each of the conditions.

  Preferred onco-tool structures are those that are maximally captured by cells at pH 6.4 but minimally captured by cells at pH 7.4.

  The first application of the two optimization steps above is acceptable efficacy (related to the amount captured at pH 6.4) and specificity (captured at pH 6.4 / captured at pH 7.4). It should generally be useful to identify the lipophilicity of a reasonably narrow range of onco-tools that give a value (related to the ratio of quantities). Alternatively, such an initial test would rather have insufficient onco-tool components selected, and different pH switch components and / or different cargo components should be used for the expected onco-tool structure. May be useful to show that there is.

iii) Further optimization cycles within progressively narrower structural ranges that repeat the above steps as needed may lead to even better onco-tool activity.

b) The above cell culture test to assess in vivo fate in normal mice is relatively quick, simple and yields quantitative results, so it applies to the first test of many expected onco-tool structures it can. However, it should be recognized that this first cell culture screening system does not completely mimic the actual complexity of living subjects. Therefore, it is desirable to then select a reasonable number of the most promising onco-tool structures, identify them in the iterative optimization step above, and test them in living mammals. As a first step in such animal studies, as described in Example 9, these promising onco-tool structures were pretreated to ensure that normal mice (those urines were slightly basic) , See section C.7 below). Such a test in normal mice makes it possible to discard onco-tools that are found to show excessive affinity for normal tissue.

c) The above relatively simple test in normal tumor-free mice to assess body fate in tumor-bearing mice allows the onco-tool with excessive affinity for normal tissue to be discarded, but the expected onco- A more critical test of the tool structure is to test it in tumor bearing mice as described in Example 10. In summary, this involves injecting (preferably intravenously) each of the onco-tools containing iodine 131 into several tumor-bearing mice, and then at a dose that is not trapped in the mouse tissue and / or tumor. Wait for an appropriate period (about 5-24 hours) to allow normal excretion by some kidneys. The mice are then sacrificed and the major organs and obvious tumors removed, followed by quantification of radiation emissions from the removed organs, tumors and remaining cadaver parts.

  Onco-tools perform best when using two auxiliary methods. One such method entails pretreating the mice to prevent reuptake of onco-tools into the cells of the lining of the proximal tubule of the kidney. This reuptake is described below in C.I. 7. As described in the section, it is blocked by making the urine slightly basic. Other methods require pre-treating the mice to further increase acidity (lower pH) in the hypoxic / acidic region of the tumor. Three pretreatments for this purpose are described later in this document. 5). Described in the section. A combination of at least one, and preferably two or three such pretreatments should be used to modulate the tumor microenvironment to best match the effective and specific onco-tool activity.

d) Preclinical and clinical trials Procedures for testing radioisotope-containing substances in living animals, including humans, are well known in the nuclear medicine field and particularly in the subfield of radioimmunotherapy. Such known methods are described below in C.I. It can be easily adapted to onco-tool testing by incorporating the onco-tool method described in the section.

C. Method using onco-tool
1. Diagnostic methods Onco-Tools exploit the acidity that is a nearly universal property of tumors, so Onco-Tools detect most or all types of tumors ranging in size from nearly microscopic to very large Should be effective. The following methods using onco-tools to detect tumors are suitable for many research applications and veterinary and human medicine. The diagnostic method generally includes, but is not limited to, the following steps.

Step 1 . Diagnosis by contacting the precursor form of the onco-tool with an appropriate radioisotope that is effective to report its presence in the tumor to a detector outside the body of the living subject or that already contains such a radioisotope The final form of the diagnostic onco-tool is prepared by obtaining the final form of the onco-tool directly from the supplier.

Step 2 . A diagnostic onco-tool is generally administered into a subject by intravenous injection.

Step 3 . Wait for an appropriate period of time (eg, about 10 to about 50 minutes) to be captured in the acidic area of the tumor where onco-tools may be present. By waiting for additional time (several hours) for excretion via most kidneys of that portion of the onco-tool dose that was not captured in the acidic area of the tumor, significantly increased sensitivity can be obtained. Waiting for this additional time helps to significantly reduce the background signal from normal tissue and allows the detection of fairly small tumors. In this regard, if the majority of the onco-tool injection volume is excreted by the kidney over several hours and only one or more tumors are present, only significant retention of the onco-tool occurs. It is expected that in subjects who generally do not have a tumor, most of the onco-tool will be excreted in less than 24 hours and perhaps less than about 4 hours. The rate of excretion of the uncaptured onco-tool can be increased by increasing the subject's fluid intake, especially when the fluid contains a diuretic.

Step 4 . The final step in the diagnostic method is to subject the subject with a device suitable for detecting radiation from the radioisotope component of the onco-tool to assess whether significant onco-tool has been captured by the one or more tumors. Is to scan. With modern imaging devices such as gamma scanners and PET scanners, tumors appear as distinct radioisotope hotspots at each tumor site.

2. Treatment methods Onco-Tools take advantage of acidity, an almost universal property of tumors, so Onco-Tools treat most or all types of tumors ranging in size from nearly microscopic to very large Should be effective. The following methods using onco-tools to treat tumors are suitable for many research applications and veterinary and human medicine. Treatment methods generally include, but are not limited to, the following steps.

a) If a single radioisotope subject is found to have one or more tumors, those tumors will contain a single therapeutic oncogene containing a radioisotope effective to kill the cells. Can be treated with tools. The treatment method generally includes the following two steps, but is not limited thereto.

Step 1 . A final form of the therapeutic onco-tool containing a radioactive isotope effective to kill the cells is prepared. This is done by contacting the precursor form of the onco-tool with the appropriate radioisotope, or by obtaining the final form of the therapeutic onco-tool that already contains such a radioisotope from the supplier. be able to.

Step 2 . The therapeutic onco-tool is generally administered to the subject by intravenous injection.

  In this therapy, a radioactive isotope effective to kill cells can be one whose radiation has a high linear energy transfer, such as alpha particles, or which emits beta particles. The particular advantage of using a radioisotope with a high linear energy transfer emitter, such as the alpha-radioactive isotope astatine 211, is significantly more effective than the beta-radioactive isotope in killing resting cells in the acidic region of the tumor It is to be. However, for using onco-tools that include such radioisotopes, those emissions generally have a fairly short path length (eg, 80 microns or less). It may therefore be necessary to use such onco-tools in combination with conventional cancer therapies to ensure the destruction of rapidly dividing tumor cells in a more normal pH region close to the capillaries. In addition, the preferred alpha emitter, At-211, has a short half-life (7 hours) and therefore transports this radioisotope or an onco-tool containing this radioisotope a significant distance from the location of origin. Can be a problem. In addition, At-211 must be generated in a high-end cyclotron with an alpha beam capable output, and there are currently only a relatively small number of facilities where such devices are available.

  Another single radioisotope method involves preparing a therapeutic onco-tool that contains beta radioisotopes. Although beta radiation is significantly less effective than alpha radiation in killing resting cells in the acidic region of the tumor, this lower effectiveness of beta radiation can be compensated using much larger doses. This much higher dose has some loss of specificity, but nevertheless has the potential to produce an acceptable therapeutic outcome due to the exceptionally high level of specificity to tumors that can be achieved with onco-tools. In this regard, iodine 131, a beta emitter, has several useful properties: That is, it is available in large quantities at a reasonable price and has a relatively long half-life (8 days), so it can be transported before use and stored for a reasonable period of time, with a medium average path length (approximately 900 microns) Sufficient to kill cells throughout the tumor, including rapidly dividing tumor cells in the region closer to the neutral pH near the capillaries where the captured onco-tool is relatively lacking It is.

b) Double Radioisotope Method As previously mentioned herein, radioisotopes with high linear energy transfer emissions are optimal for killing radioresistant resting cells in the acidic region of the tumor. Onco-tools that contain such radioactive isotopes, although the short path length of such radioactives is to kill rapidly dividing cells in the distal region of the tumor near the capillaries where the pH is closer to neutrality Conversely, it is invalidated. The reason is that such areas are relatively lacking of captured onco-tools.

  Conversely, onco-tools containing beta-radioactive isotopes are relatively ineffective against tumor radioresistant resting cells, but the pH is low even when the onco-tool is present only in the acidic region of the tumor. It can be quite effective in killing radiosensitive rapidly dividing cells in areas of the tumor that are closer to neutrality. This is because the larger path lengths of beta particles allow them to reach and kill more sensitive tumor cells.

  Thus, a preferred method of treating tumors is to use a combination of two therapeutic onco-tools, one onco-tool for killing proximal radioresistant resting cells in the acidic region of the tumor Containing a radioisotope having a high linear energy transfer emitter (preferably selected from astatine 211, bromine 77 and iodine 123), another onco-tool is relatively lacking in the captured onco-tool Includes beta-radioactive isotopes for killing distal radiosensitive rapidly dividing tumor cells in higher pH regions near capillaries.

3. Comprehensive method for detecting and treating tumors The onco-tool is a highly desirable property that it can detect and treat most or all types and sizes of tumors ranging from nearly microscopic to very large There is. The diagnostic and therapeutic onco-tools can be essentially the same (generally differing only in the radioisotopes involved), so generally if a given onco-tool structure is effective for detecting a tumor The same onco-tool structure (generally differing only in the radioisotopes involved) should be effective in treating the same tumor. These special properties of the onco-tool facilitate a comprehensive method of detecting tumors in living subjects and subsequently treating the tumors so detected. This comprehensive method is suitable for both veterinary and human medicine. It includes, but is not limited to:

a) Tumor detection
Step 1 . The first step is to prepare a final form of a diagnostic onco-tool that contains a radioisotope effective to report the presence of an onco-tool within the tumor to a detector outside the living subject.

Step 2 . The next step is to administer the diagnostic onco-tool to the subject, typically by intravenous injection.

Step 3 . The subsequent step is to wait for an appropriate period of time to be captured in the acidic area of any tumor where an onco-tool may be present. This step may also include waiting for additional time for most of the onco-tool dose not captured in the acidic area of the tumor to be excreted through the kidney. During this period, the subject may be given a liquid, particularly a liquid containing a diuretic, to increase excretion of that portion of the dose of onco-tool that was not captured in the acidic area of the tumor.

Step 4 . The final step in the detection process is to use a device suitable for detecting onco-tool radioisotope emissions to assess whether significant onco-tool has been captured by one or more tumors. Is to scan. With modern imaging devices such as gamma scanners and PET scanners, tumors should appear as distinct radioisotope hotspots at each tumor site.

  If one or more tumors are detected in step 4, proceed to treat the detected tumors.

b) Treatment of detected tumor
Step 5 . One or more therapeutic onco-tools are provided to treat the detected tumor. This may be a single onco-tool containing beta emitting radioisotopes, but one contains radioisotopes that emit high energy transfer radiation and the other contains beta emitting radioisotopes, two or It is generally preferred to provide more onco-tools.

Step 6 . One or more prepared onco-tools are generally administered to the subject by intravenous injection.

4). Strategies to address micrometastasis When tumors reach a substantial size (such as 1 cm or more), the tumors are released from the parent tumor by a single tumor cell or a very small aggregate of tumor cells. The metastasis generally begins when the released cells can colonize at a remote site in the body. These cell colonies, called micrometastases, can grow into new offspring tumors. The difficulties this poses for onco-tool treatment methods are the time of formation of micrometastasis and the size of such micrometastasis to develop its own acidic region (diameter about 1 mm) In the period between the time to the time, those sub-millimeter progeny tumors cannot generally be detected or killed by onco-tools. Thus, parent tumors containing acidic regions can be detected and destroyed by onco-tool, but progeny micrometastases less than about 1 mm in diameter remain with onco-tool treatment, eventually resulting in recurrence ( Such recurrence may not occur for several years after the first onco-tool therapy).

  Strategies to solve this micrometastasis problem exist and grow to a size (generally about 1 mm in diameter) where the micrometastasis develops an acidic region that may remain with initial treatment. To wait for a period after the first onco-tool therapy (steps 5 and 6 in section 3 above) sufficient. Conversely, their progeny micrometastasis should not grow to a much larger size (probably about 1 cm in diameter) where they also initiate metastasis. Once the micrometastasis that may have escaped initial onco-tool therapy has entered this appropriate size range (large enough to include the acidic region but not large enough to initiate metastasis), the onco-tool The detection and treatment process (repeating steps 1-6 in the third section above) is performed again.

  The complicating factor in the above strategy is that the tumor exhibits a wide range of growth rates, and therefore how long it takes for micrometastasis that may have escaped the initial therapy to reach a size that includes the acidic region. Is difficult to predict. Therefore, a sensible course is that micrometastasis escapes destruction in the first onco-tool therapy, and then such micrometastasis grows to a size sufficient to generate an acidic region, followed by repeated diagnosis. Continue the diagnostic steps 1-4 above for a sufficient period of time (probably 4-6 years) to actually confirm whether it is detected in one of the treatments (repeat of steps 1-4 above) Repeat at appropriate intervals (perhaps once a year). If one of the repeated diagnostic procedures detects one or more tumors, the patient is treated again according to steps 5 and 6 above. Such a second treatment completely destroys any progeny tumor that may have been escaped in the form of micrometastasis during the first onco-tool treatment, thereby removing the first tumor and all its progeny. It is likely that there is a high potential for complete removal from the patient.

5). The effectiveness and specificity of the onco-tool for the acidic area of the tumor treated to reduce the pH in the tumor for increased efficacy and specificity is due to the strong pH difference between the acidic area of the tumor and normal tissue It is a function. Thus, if the pH in the acidic region of the tumor can be selectively reduced without being accompanied by a concomitant decrease in pH in normal tissue, than in natural conditions, this is more than in onco-tool diagnostic applications. This will result in greater sensitivity and greater effectiveness and specificity in therapeutic applications.

  Over the past half century, many treatments have been reported to change the pH in tumors (some increase the pH and some decrease the pH). In the case of diagnostic and therapeutic onco-tools, the treatment is to selectively lower the pH in the tumor without the concomitant reduction in pH in the normal tissue in question. The following are three such treatments that have been reported to selectively reduce the pH in tumors.

  a) Introduction of glucose into tumor bearing animals reduces the pH of the gap in the hypoxic region of the tumor, generally during a period of about 2-3 hours, with little or no effect on the pH of the gap in normal tissues It has been known for many years (see, for example, Non-Patent Document 11). Further oral ingestion of glucose can significantly extend the time that the pH in the tumor stays so reduced.

  b) The pH in the acidic region of the tumor can be further reduced by administration of the mitochondrial inhibitor metaiodobenzylguanidine again without apparently having an inappropriate effect on the pH in normal tissues (eg, non- (See Patent Document 12).

  It has also been reported that the pH in the acidic region of the tumor can be further reduced to 0.7 pH units by combining glucose and metaiodobenzylguanidine (see, for example, Non-Patent Document 13).

  c) Furthermore, the pH in the acidic region of the tumor can be further reduced by vasodilators routinely used to treat people with hypertension (see, for example, Non-Patent Document 14). Since the abnormal vasculature of tumors generally lacks vasoconstrictor muscle cells and their nerve fibers, such agents are likely to be effective. Thus, when a vasodilator is administered to a subject, resistance to blood flow does not change in the tumor but decreases in normal tissue. These different effects of vasodilators result in significantly greater blood flow through normal tissue and a concomitant reduction in blood flow through the tumor. In turn, this low blood flow through the tumor is likely to reduce the washout of lactate produced by tumor cells in the hypoxic region of the tumor and the observed vasodilator-mediated pH drop in the acidic region of the tumor. It is thought to bring.

  Such a treatment to further lower the pH in the acidic region of the tumor, preferably a combination of two or more such treatments, increases onco-tool capture in the now more acidic region of the tumor, It should also result in an increase in the area of the tumor that is sufficiently acidic to capture the onco-tool. Both of these effects play a role in increasing the effectiveness and specificity of the onco-tool.

  Making the tumor more acidic also makes it possible to use an onco-tool that includes a pH switch with a lower pKa (which can lead to a significant increase in the specificity of the onco-tool).

6). Reported experimental measurements of acidity in tumors using multiple onco-tools with different pKa's to increase efficacy and specificity show a pH gradient ranging from about 7.0 close to tumor capillaries and A decrease with increasing distance from the tumor capillaries was shown, with a value as low as about 6.0 reported for the region farthest from the tumor capillaries. For tumors with a wide range of pH values, a therapeutic onco-tool with too high pKa will leave insufficient onco-tool to reach the lowest pH region of the tumor farthest from the tumor capillaries. To a certain extent, there is the possibility of rapidly invading cells in a slightly acidic area close to the tumor capillaries. Alternatively, therapeutic onco-tools with too low pKa may be trapped too poorly in slightly acidic areas closer to the capillaries in the tumor, thereby leading to poor treatment of such areas There is sex. One strategy to deal with the wide pH range within the tumor is to use a combination of two or more onco-tools, where one onco-tool with a higher pKa is closer to the capillaries. Other onco-tools that are maximally effective in the higher pH region and that have a lower pKa are those that are maximally effective in the lower pH region of the tumor farther from the capillaries. The rationale for using such a combination of onco-tools is that onco-tools with higher pKa can be mainly trapped in the region close to the capillaries, reaching the lower pH region farther from the capillaries. This means that there is almost nothing available for use. Conversely, onco-tools with lower pKa are poorly trapped in the higher pH region near the capillaries, and thus lower pH farther from the capillaries that can effectively capture them. It remains available for diffusion into the region. Thus, when taken together, such a combination of low pKa and high pKa onco-tools should better achieve complete destruction of the tumor.

7). One of the functions of the kidney that is treated to raise the urinary pH to prevent kidney damage is to maintain the pH in the body very close to 7.4. To perform this function, the kidneys can excrete urine from moderately basic to fairly acidic. In this process, the substance is filtered from the blood in the kidney glomeruli, after which the substance (dissolved in the urine) passes through the proximal tubules, where the important components excreted in the glomeruli are the proximal tubules It is reabsorbed by cells on the inner layer surface. When urine is acidic at this location, the onco-tool in this acidic environment binds to cells on the inner surface of the proximal tubule of the kidney so that the onco-tool enters the tumor cells from the acidic extracellular environment. And has the ability to penetrate. In other words, if the excreted urine is sufficiently acidic, the onco-tool will convert to its non-ionic lipophilic form. If the conversion to the lipophilic form occurs in an area where the urine is in direct contact with the cell membrane, as is the case with cells on the inner lining of the proximal tubule of the kidney, the onco-tool will have such cells Can invade. In the case of a diagnostic onco-tool, such invasion of the onco-tool into cells on the inner lining surface of the proximal tubule can lead to an excess from the kidney that can obscure the tumor near or in the kidney Can cause a bad signal. In the case of a therapeutic onco-tool, such invasion of the onco-tool into the cells on the inner lining surface of the proximal tubule can kill the cells on the inner lining surface of the proximal tubule (this causes the kidney to Become nonfunctional).

  Fortunately, there are well known methods in the medical art for raising the pH of a subject's urine for a time sufficient to perform onco-tool diagnosis or treatment. By using appropriate drugs to maintain urine at a slightly basic pH during the period when the majority of uncaptured onco-tools are excreted by the kidney, the onco-tool excreted in urine is It stays in its anionic hydrophilic form and is safely discharged from the body by urination. One safe and effective substance for making urine basic is acetazolamide, a carbonic anhydrase inhibitor. Thus, a sensible course in any diagnosis or treatment with an onco-tool first increases the pH of this drug, or urine, to the subject about 1-2 hours before administering the onco-tool to the subject, and about 7.4. To administer other substances effective to maintain the above pH.

8). Use a micro pH probe at the end of the catheter to monitor the bladder for increased safety and if the kidney to be washed is excreting acid urine, due to potential harm to the kidney by a therapeutic onco-tool It may also be desirable to continuously monitor the pH of urine entering the bladder. The preferred period of such monitoring is from just before onco-tool injection to the time when most of the onco-tool that was not captured in the acidic area of the tumor was excreted by the kidney (generally about 3 to 6 hours). ). Such monitoring allows emergency intervention (such as injecting additional doses of acetazolamide) when newly excreted urine begins to drop below about pH 7.4.

  Over the course of several hours, much of the onco-tool injection is excreted by the kidneys and stored in the subject's bladder until it is excreted by urination. For the case of onco-tool diagnostic applications, this accumulation of radioactive onco-tool in the bladder can obscure the presence of a tumor proximate to the bladder. In the case of a therapeutic onco-tool, accumulation of radioactive onco-tool in the patient's bladder can damage the bladder. If accumulation of radioactive onco-tool in the bladder proves to be a significant problem, inflow / outflow from the time of onco-tool injection to the point where most of the injection volume is removed from the patient's body This can be substantially reduced by irrigating the bladder with a catheter.

  Lavage of the bladder during the period when the majority of the onco-tool dose is excreted by the kidneys, until the radioisotope is disrupted to a safe level (generally the 10 half-life of the radioisotope) It serves the additional purpose of being able to pass through a shielded storage container that can be included. This helps to ensure the safety of health care workers attending the subject and largely prevents inadvertent contamination of hand-washing rooms and other areas by radioactive onco-tools excreted by the subject.

(Example 1)
Lipophilic effect on the pH of the transition between ionic and non-ionic forms Sodium salt of propionic acid (hydrophilic carboxylic acid) and octanoic acid (lipophilic carboxylic acid) dissolved in water at a concentration of 33 mM with 5M HCl Titration. The midpoint of transition between salt and acid (pKa value) was 4.83 as expected for propionic acid, but was found to be surprisingly high for 5.5. The first derivative of the titration curve was symmetric for propionic acid but highly asymmetric for octanoic acid. Furthermore, during the octanoic acid titration, a small drop of octanoic acid separated from the solution.

  In contrast, when these same two acid salts are titrated in a semi-aqueous medium: methanol / water (1: 1 volume ratio), the midpoint of their transition is practically the same, and their The first derivative of the titration curve was now both symmetric. Finally, octanoic acid did not oil separate from solution in titration in methanol / water, a semi-aqueous medium.

  These results suggest that the surprisingly high midpoint of titration observed for octanoic acid in aqueous solution was solely due to the solubility effect shown in FIGS. The results further show that titration in a semi-aqueous solution methanol / water (1: 1 volume ratio) avoids these solubility effects on the titration curve.

(Example 2)
Comparison of acid-specific and non-acid-specific internal H-bonds with respect to the pH of the transition between ionic and non-ionic forms Figure 6a) (designed to form acid-specific internal H-bonds) and figure A 30 mM solution of the sodium salt of the compound shown in 6 b) (designed to form non-acid specific H bonds) was titrated with 5M HCl in aqueous solution. The first derivative of the titration curve of the compound in FIG. 6b) is symmetric and exhibits an expected pKa value of 4.82 (typical for simple carboxylic acids), and the apparent insoluble material generated during titration is Did not exist. In sharp contrast, the first derivative of the compound titration curve in Figure 6a) is very distorted, showing a surprisingly high value of pH 5.8 and a minimum, with the approximate midpoint of the titration being The pH was 5.6. In addition, there was a large amount of precipitate that formed with each addition of HCl since the pH dropped below 5.8.

  These dramatic differences in titration properties between compounds of approximately the same structure indicate that the structure in FIG. 6a) forms the predicted internal acid specific H bond, which is in ionic form and non-ionic Supports the inference that it acts to increase the pH of the transition between forms. Conversely, the structure in FIG. 6 b) may form an internal H bond, but since the H bond can be formed with the carboxylate form or the carboxylic acid form, such an H bond is It is not significantly advantageous in the non-ionic form compared to the ionic form, and therefore does not significantly change the pH of the transition between the two forms compared to what is observed for simple carboxylic acids such as propionic acid.

(Example 3)
Effect of low conformational freedom H-bonding on the pH of the transition between ionic and nonionic forms Titration in aqueous solution of the acyclic structure of Fig. 7 b) predicted an expected transition of 4.95 PH value (typical for simple carboxylic acids) and no precipitation of the acid form. In contrast, the titration of the five-membered ring structure of FIG. 7a) showed an unexpectedly high pH of 5.6 for the transition between the anionic and nonionic forms. Titration of the ring structure was accompanied by a large amount of precipitation when the pH dropped below about 5.8, and the shape of the titration curve was highly asymmetric.

  However, when these two compounds were titrated instead in semi-aqueous medium methanol / water (1: 1 volume ratio), there was no sign of insolubility and the plot of the first derivative of the titration curve was now symmetric. . For the acyclic compound shown in FIG. 7 b), the pH at the midpoint of titration was 5.8, whereas for the ring structure of FIG. 45.

  Thus, even in the absence of distortion of the titration curve due to precipitation of the lipophilic acid form, in a semi-aqueous titration experiment, for the cyclic compound in FIG. 7a, a substantial 0.65 pH at the midpoint of the titration curve. An increase in units is allowed. The most reasonable explanation for this seems to be that in this semi-aqueous solution, the internal acid-specific H bond is formed only in the case of a ring structure, and this H bond favorably moves the equilibrium to the acid form. It is.

(Example 4)
The importance of insulating the carboxyl from the electron-withdrawing effect of the linking group Due to the molecular modeling of structure 8b, the internal H-bond is between the almost complete shape of the H-bond and the H-bond donor and H-bond acceptor moieties. A very limited conformational freedom suggests significant advantages. Despite these factors favoring the formation of internal acid-specific H-bonds, the titration curve is less than 3.0 when the salt of the structure in FIG. 8 b) is prepared and titrated with HCl in aqueous solution. The pKa value was shown.

  This result suggests that the amide-induced effect (which propagates easily via an intervening double bond) probably causes a large decrease in the pKa of the carboxyl, and that the negative induced effect is that the internal acid-specific H bond is carboxylated. It is surmised that the positive effects that can be exerted on the increase in pKa are far exceeded.

(Example 5)
Advantage of partially shielding H-binding sites from aqueous solvent The two compounds in FIG. 9 were prepared and titrated in the semi-aqueous solvent methanol / water (1: 1 volume ratio). This semi-aqueous solvent was used for these titrations to prevent insoluble effects from affecting the titration results. In these experiments, the first derivative of the titration plot was symmetric and there was no visible sign of precipitation. Therefore, the purpose of avoiding insoluble effects was met.

  It was observed that structure (a) in FIG. 9 showed the midpoint of the titration curve at a pH of 5.6, while structure (b) in FIG. 9 showed a significantly higher midpoint of titration at pH 6.14. That is.

  Again, a possible explanation for the pH value of the significantly higher transition of structure (b) in these titrations is that in this semi-aqueous solution structure (b) acts to favorably move the equilibrium to the acid form. The acid-specific H bond is formed, but the closely related structure (a) does not form such an internal acid-specific H bond. Moderate shielding of the H binding site in structure (b) is an advantageous factor for the formation of H bonds in structure (b), while the lack of such shielding of the H binding site in structure (a) is the structure (a It appears to be the dominant cause of the apparent lack of internal H bonds in

(Example 6)
Preparation of a 2 pH switch structure designed to form an internal acid-specific low barrier H bond and titration of the methyl ester of isonipocotic acid reductively alkylated with 3-pentanone and then the resulting tertiary amine Was converted to N-oxide using metachlorobenzoyl peroxide. Finally, the ester was cleaved with aqueous sodium hydroxide to give structure iii in FIG. This product in sodium carboxylate form was titrated in water. The first derivative of the titration plot is one at pH 3.8 (approximate value predicted for the N-oxide moiety) and substantially higher than expected for the N-oxide or carboxylic acid moiety at pH 6.0. Two minimum values with other minimum values are shown. However, in light of the results in previous examples herein, the most likely explanation is that this pKa 6.0 value is an internal low barrier H bond between the carboxyl H bond donor and the N-oxide H bond acceptor. The internal H bond acts to shift the equilibrium towards the acid form, i.e. increase the pKa.

  The cis (3-amine-1-acid) derivative of camphoric acid is reductively alkylated with excess acetaldehyde, and the resulting tertiary amine product is converted to N-oxide with metachlorobenzoyl peroxide. Thus, the structure i in FIG. This product in sodium carboxylate form was titrated in water. The first derivative of the titration plot is one at pH 4.2 (approximate value predicted for the N-oxide moiety) and substantially higher than expected for the N-oxide moiety or carboxylic acid moiety. Two minimum values are shown, with other minimum values. Again, in light of the results from previous examples herein, the most likely explanation for this exceptionally high pKa value of 6.5 is that the internal low barrier H bond is a carboxyl H bond donor and an N-oxide. It is formed between the H bond acceptor and the internal H bond acts to move the equilibrium strongly towards the acid form, thereby increasing the pKa value.

  The above internal H-linked isonipocotic acid derivative has a considerably lower pKa value (6.0) than the somewhat similar internal H-linked succinic acid derivative (the H-bond donor and H-bond acceptor moieties are virtually the same in both structures) Despite that, it is worth noting. The most likely explanation for this considerable difference between pKa values is that the lowest energy conformation of a six-membered aliphatic ring, such as the isonipocotine structure above, is generally a chair conformation, and the chair conformation is This generally requires about 5 Kcal to convert to the twisted boat shape, which is the most stable conformation. Since the inventors' molecular modeling has shown that only the twisted boat conformation of the isonipocoti derivative forms an internal acid-specific H bond, the strength of this H bond is the conversion of the chair to the twisted boat conformation. It can be attenuated by energy costs.

  In contrast, the 5-membered aliphatic ring of camphoric acid derivatives can be easily and at low energy costs to move between different packer conformations, thus forming an internal acid-specific H bond. There is almost no energy cost to choose a suitable conformation. As a result, substantially all the energy of the H bond is available to dominate the acid form over the salt form and thus raise the pKa to an exceptionally high value of 6.5.

  The significance of the above is subtle to adjusting the pKa value of the pH switch fairly precisely in order to achieve the desired balance between effectiveness and specificity (the importance of which is shown in FIG. 12). It is to show that structural characteristics can be utilized.

(Example 7)
Titration procedure for testing pH switch structures
a) Aqueous titrations A new structure that may generally be suitable as a high pH switch component is their pT value, ie the pH value at the midpoint of their transition between anionic and nonionic forms. Evaluate first. This requires preparing a sodium salt form of this structure and dissolving it in 50 ml of 0.15 M NaCl (degassed to minimize carbonate content) at a concentration of 5 mM. Add a small magnetic stir bar and adjust the pH of the solution to about 9 with a small amount of 1M NaOH. While stirring, add 5 μl of 1M HCl and record the pH 1 minute after each HCl portion is added. If no pH switch precipitation or oil separation occurred during the titration, a plot of pH versus added HCl volume gives a normal titration curve. However, a much more useful representation of the titration results that makes it possible to obtain a fairly accurate measure of the pKa value is obtained by plotting instead the first derivative of this titration curve. This plots the values of _{(pH n + pH n + 1} ) / 2 on the X axis requires plotting the values of (pH _n -pH _{n + 1)} on the Y axis.

b) If the semi-aqueous titration pH switch precipitates or oil separates during titration, the titration curve is generally significantly distorted. In such cases, it is difficult to distinguish the solubility effect from the pKa effect at the midpoint of the transition between the anionic and nonionic forms of the pH switch. In such cases, solubility effects can be avoided by titrating in a semi-aqueous solvent in which both the salt and acid forms of the pH switch are completely soluble. A 1: 1 mixture of methanol and water generally serves this purpose. In this solvent, the first derivative of the titration curve becomes symmetrical again. For substances that cannot form stable internal H bonds, such as octanoic acid, the titration plot is a classic pKa value very similar to that observed for a completely water-soluble simple carboxylic acid such as propionic acid. Indicates. However, for materials designed to form stable internal acid-specific H bonds, such as the advanced pH structure described in the above example, the titration plot is symmetric in methanol / water, but nevertheless. First, the titration plot can show a substantially higher pKa value compared to a very similar structure that cannot form an internal acid-specific H bond, and this high pKa value is This suggests an internal acid-specific H bond that forms.

  Thus, in titration in saline, the total apparent increase in pH of the transition with asymmetry of the titration curve may be due to a combination of internal acid specific H-binding effects and solubility effects. However, when the titration is performed instead in a semi-aqueous solvent such as methanol / water, the internal acid-specific H bond is compared to the pKa of a very similar substance that cannot form an internal acid-specific H bond. The substantial increase in the pKa value of the material designed to form is mainly due to internal H bonds only and increased lipophilicity of the acid moiety that may be the result of the internal H bonds. Not due to However, to a certain extent, the less polar methanol / water solvent is inherently advantageous for the formation of internal H-bonds, and therefore, if it can occur anyway, when testing a new pH switch structure type, a completely aqueous solution It should also be understood that it is desirable to confirm the formation of internal acid specific H bonds by titrating in.

(Example 8)
Onco-tool cell culture test system It is recommended to first test each potential onco-tool structure in a relatively simple biological system, in which case the onco-tool is the main one encountered in living subjects. Biological environment and structure (mammals exposed to serum-containing medium buffered at pH 7.4 to mimic blood and normal tissue and buffered at pH 6.4 to mimic the acidic region of the tumor The onco-tool is exposed to (including cells in particular).

  Such a suitable test system requires the preparation of two preparations of isotonic medium. One should contain 10% serum and be strongly buffered to pH 7.4 with 50 mM Hepes buffer (pKa 7.5). Others should contain 10% serum albumin and be strongly buffered in 50 mM Bistris buffer (pKa 6.5). An onco-tool containing iodine 131 should be added to each medium at equal concentrations.

  Prior to the experiment, Hela cells should be grown to fusion in 12-well culture plates. The medium is then removed from the 4 wells of the cells and replaced with onco-tool containing medium buffered at pH 7.4. In addition, the media is removed from the other 4 wells of the cells and replaced with media containing onco-tool buffered at pH 6.4. The plate is then incubated for 1 hour at 37 ° C. After incubation, the onco-tool containing medium is removed and replaced with onco-tool free medium of the same pH, swirled briefly and removed. This washing procedure is repeated a total of 4 times. The cells are then lysed with 1 ml of detergent solution, the lysate solution removed, counted with a scintillation or gamma counter and a measure of the relative amount of onco-tool captured under each of the two pH conditions. Get.

  The preferred onco-tool structure is maximally captured by cells at pH 6.4 but only minimally captured by cells at pH 7.4.

Example 9
The onco-tool cell culture test system for testing onco-tools in normal mice is the first rapid and quantitative characteristic of the high-efficiency efficacy and specificity characteristics of a substantial number of potential onco-tools. Enable evaluation. However, this initial assessment should be followed by testing in living mice for the next most promising onco-tool structure. It is recommended that mice are first pre-administered with a carbonic anhydrase inhibitor, such as acetazolamide, so that the urine of the mouse remains basic over time. Next, an appropriate amount of an onco-tool containing iodine 131 in phosphate buffered saline should be injected, preferably intravenously, such as in the tail vein. The mice are then monitored at regular intervals over a period of up to about 24 hours (such as by placing them briefly under an appropriate gamma counter or gamma camera) to determine the rate of excretion of the labeled onco-tool.

  The main purpose of this test in normal mice is the excess in normal tissue, possibly due to excessive lipophilicity or possibly due to some structural element with unexpected affinity for normal tissue or certain specific organs etc. Identifying onco-tools to be captured. Onco-tools that are rapidly and completely excreted from normal mice and thus pass this preliminary animal test should then be tested in tumor bearing mice as described in the following examples.

(Example 10)
Onco-tool testing in tumor- bearing mice The above relatively simple test in normal tumor-free mice allows the onco-tool with excessive affinity for normal tissue to be discarded, but a promising onco-tool A more definitive test of the structure is to test it in tumor bearing mice.

  In such experiments, mice a) one or a combination of a) a carbonic anhydrase inhibitor such as acetazolamide to increase urine pH, and b) a substance effective to selectively reduce pH in the tumor. It is recommended to pre-administer first. Such substances include i) glucose (see, for example, Non-Patent Document 11), ii) mitochondrial inhibitor metaiodobenzylguanidine (see, for example, Non-Patent Document 12), and iii) to treat people with high blood pressure. And vasodilators used routinely (see, for example, Non-Patent Document 14).

  After an appropriate period after such pre-administration, tumor bearing mice are injected (preferably intravenously) with iodine-131 containing onco-tool. After an appropriate period of time (about 5 to 24 hours) to allow normal excretion by some kidney of a dose not captured in the tissue and / or tumor of the mouse, the mouse is sacrificed and the major organs and obvious The tumor is removed. Each organ and tumor and the remaining cadaver part are counted with a gamma counter.

  For mouse pretreatment, it should be emphasized that the onco-tool performs best when both types of pretreatment are used, and one type is directed to cells on the inner surface of the proximal tubule of the kidney. It helps to prevent re-uptake of onco-tools. This reuptake is prevented by making the urine slightly basic. Other types help to further increase acidity (lower pH) in the hypoxic / acidic region of the tumor. Combination of two or three such pretreatments to selectively reduce the pH in the tumor, preferably to adjust the tumor microenvironment to best match the effective and specific onco-tool activity Should be used.

  Above, the best mode anticipated for the compositions and methods of the present invention, and such terms in complete, clear, concise and accurate terms that will enable one of ordinary skill in the art to make and use the compositions and methods. A description of techniques and methods for making and using various compositions and methods is provided. However, these compositions and methods permit variations and alternative constructions from the above-described illustrative embodiments that are completely equivalent. Accordingly, it is not intended that the disclosed compositions and methods be limited to the specific embodiments disclosed. On the contrary, the intention is to express in detail the subject matter of the disclosed compositions and methods, and to include all modifications and variations that fall within the spirit and scope of the compositions and methods generally expressed by the claims that are explicitly claimed. Include alternative configurations.

FIG. 5 shows the weak acid cytotoxic chemotherapeutic drug chlorambucil in both anionic and nonionic forms. It is a figure which shows distribution of the acidity in a tumor. FIG. 5 shows pH-mediated and solubility-mediated transitions between forms. FIG. 3 shows a calculated titration curve as a function of lipophilicity of a nonionic acid form. a) shows the expected water of hydration, showing water molecules H-bonded to the normal carboxylic acid in its anionic and non-ionic forms, and b) H in its anionic form and within it. FIG. 4 shows the expected water of hydration showing water molecules H-bonded to a bound non-ionic carboxylic acid containing structure. a) shows acid-specific H-bonds in a structure that forms internal acid-specific H-bonds only in its non-ionic form, and b) non-acids in both its anionic and non-ionic forms It is a figure which shows the structure which forms a specific H bond. a) shows a representative ring structure suitable for an advanced pH switch including a 4-membered ring structure, a 5-membered ring structure, and a 6-membered ring structure, and b) shows an acyclic structure because of having an acyclic structure. It is a figure which shows the structure which cannot accept | permit to use as a switch. a) shows the insulation of the carboxyl moiety from the induced effect by increasing the number of carbons separating the carboxyl from the electron withdrawing group, and b) has insufficient insulation of the carboxyl from the induced effect. It is a figure which shows a structure. a) is a diagram showing a structure in which the H binding site is open to the solvent, and b) is a diagram showing a structure in which the H binding site is partially shielded from the solvent. FIG. 5 shows components of low barrier H bonds, a) is a carboxylic acid suitable for forming low barrier H bonds with a free-standing H bond acceptor moiety having a pKa value in the range of about 3.0 to 6.5. Figure 2 shows an H-bond donor containing an acid, b) shows a representative H-bond acceptor moiety suitable for forming a low-barrier H bond with a carboxylic acid donor, and c) is an internal acid specific. FIG. 2 shows an exemplary advanced pH switch designed to form a static low barrier H bond. FIG. 5 shows the statistical basis of increased specificity of a multi-pH switch onco-tool, a) shows the non-capture anion and tumor capture non-ion forms of an onco-tool containing a 2 pH switch, b ) Shows non-capture anion and tumor capture non-ion forms of an onco-tool containing a 3 pH switch. FIG. 4 shows the computational effectiveness and specificity factor of a multi-pH switch onco-tool, a) showing the computational effectiveness and specificity factor as a function of the pKa value of the structure containing the 1 pH switch, b ) Shows the computational effectiveness and specificity factor as a function of the pKa value of the onco-tool including the 2pH switch, and c) the computational effectiveness as a function of the pKa value of the onco-tool including the 3pH switch and FIG. 4 is a diagram showing the specificity factor, d) showing the computational effectiveness and the specificity factor as a function of the pKa value of the onco-tool including the 4 pH switch. Figure 2 shows a composition comprising two advanced pH switches, a) showing the structure of this composition, and b) n-octanol / buffer of this two pH switch composition as a function of pH. It is a figure which shows the plot of distribution. FIG. 4 is a diagram showing representative cargo components in their precursors and final form. FIG. 2 shows two synthesis routes for representative cargo components, including a simple procedure to convert selected precursor forms to their final forms. FIG. 2 is a diagram illustrating an exemplary 2pH switch on co-tool and showing two exemplary 2pH switch on co-tools including a simple pH switch. FIG. 2 illustrates a representative 2pH switch on co-tool and illustrates five representative 2pH switch on co-tools including an advanced pH switch. FIG. 2 illustrates a representative 2 pH switch on co-tool, showing 10 exemplary 2 pH switch on co tools including an advanced pH switch designed to form a low barrier H-bond, Structure 1 And 2 show two onco-tools in which the H-bond acceptor moiety of the pH switch is an N-oxide moiety, and each N-oxide moiety serves as an H-bond acceptor for two carboxylic acid H-bond donor moieties. FIG. FIG. 2 shows a representative 3pH switch on co-tool, a) shows two representative 3pH switch on co-tools including an advanced pH switch, and b) forms a low barrier H bond. FIG. 2 shows two representative 3 pH switch on co-tools that include an advanced pH switch designed to: FIG. 4 shows a representative 4 pH switch on co-tool, a) showing a 4 pH switch on co tool in which the H bond acceptor portion of the pH switch is an alkoxyamine moiety, and b) shows the pH switch H co tool. FIG. 4 shows a 4 pH switch on co-tool in which the bond acceptor moieties are N-oxide moieties, each N-oxide moiety serving as an H-bond acceptor for two carboxylic acid H-bond donor moieties. FIG. 4 shows the synthesis of an onco-tool containing a simple pH switch that can adjust lipophilicity by changing the R group. FIG. 3 shows a synthetic scheme for an advanced pH switch that can adjust lipophilicity by changing the R group. FIG. 2 shows amine ester and ketone ester intermediates useful for onco-tool synthesis. FIG. 4 shows a representative synthesis of a pH switch designed to form an internal acid-specific low barrier H bond, and a) shows the synthesis of a pH switch where the H bond acceptor is a cyanomethylamine moiety. And b) shows the synthesis of a pH switch in which the H-bond acceptor is an N-oxide moiety, and c) shows the synthesis of a pH switch in which the H-bond acceptor is a trifluoroethylamine moiety. . It is a figure which shows the titration result of various pH switch structures, and is the case at the time of performing titration in methanol / water of 1: 1 volume ratio about simple carboxylic acid (butyric acid) and advanced pH switch (acid amide derivative of camphoric acid) FIG. 6 shows a plot of the same data in the form of a normal titration curve as well as a first derivative giving more information. FIG. 5 is a diagram showing titration results of various pH switch structures. 2 pH switch on co-tool (including stable iodine instead of radioactive iodine) with different R groups and related 3 pH switch structures were titrated in water, and each chemical species It is a figure which shows the normal titration curve at the time of existing by 33 mmol concentration. FIG. 4 shows titration results for various pH switch structures, a titration curve plotted as the first derivative of an advanced pH switch designed to form a low barrier H bond (this titration is performed in water and from camphoric acid). PH switch containing a derivatized N-oxide / acid structure is present at a 5 millimolar concentration). An amide / acid high pH switch derived from camphoric acid shown in FIG. 23a, an N-oxide / acid high pH switch designed to form a low barrier H bond derived from isonipocotic acid, and camphor shown in FIG. FIG. 5 shows experimentally determined pKa values for three pH switch structures including N-oxide / acid high pH switches designed to form acid-derived low barrier H bonds. FIG. 2 shows a synthesis scheme of a representative onco-tool that includes an advanced pH switch, showing the onco-tool in which the two pH switches are linked by a diacyl hydrazide structure. FIG. 2 shows a synthesis scheme of a representative onco-tool that includes an advanced pH switch, showing the onco-tool with its two pH switches linked by a diamide structure. FIG. 2 shows a synthesis scheme of an exemplary onco-tool that includes an advanced pH switch designed to form a low barrier H bond, where a single N-oxide moiety is an H bond to two carboxylic acid H bond donor moieties. FIG. 2 shows a synthesis scheme of a 2pH switch on co-tool that serves as a receptor moiety. FIG. 2 shows a synthesis scheme of a representative onco-tool that includes an advanced pH switch designed to form a low barrier H bond, where both nitrogens of the hydrazine moiety are H bonds to two carboxylic acid H bond donor moieties. FIG. 2 shows a synthesis scheme of a 2pH switch on co-tool that serves as a receptor moiety. FIG. 2 shows a synthesis scheme of an exemplary onco-tool that includes a high pH switch designed to form a low barrier H bond, with the cyanomethylamine moiety as the H bond acceptor moiety relative to the carboxylic acid H bond donor moiety. FIG. 2 shows a synthesis scheme of a 2pH switch on co-tool that plays a role. FIG. 2 shows a synthesis scheme of a representative onco-tool that includes an advanced pH switch designed to form a low barrier H bond, each N-oxide moiety being an H-coupled receptor for two carboxylic acid H-bond donor moieties. FIG. 4 shows a synthesis scheme of a 4pH switch on co-tool that serves as a part.

Claims (32)

A precursor structure that can be readily converted to a composition that is preferentially captured in the acidic region that may be present in a tumor in a living subject, wherein the composition is (i) an aqueous solution at pH 7.4 Exists in a primarily negatively charged water soluble form, but in the acidic region below pH 7.0, a significant portion of the molecules of the composition are effective to enter the lipophilic phase, which may be the cell membrane. Has the property of converting to a non-ionic form and (ii) can report its presence by emitting photons, gamma rays (high energy photons) or positrons and / or in the region where it is captured Containing cargo components that can cause cell damage,
The addition of radiohalogens can be converted into improved compositions that are preferentially captured by improved specificity in the acidic region,
(A) at least two acid moieties;
(B) at least one of the acid moieties is incorporated in a component capable of binding H to the interior having the following properties i) to iii):
i) a carboxylic acid moiety in which the acid moiety is directly bonded to the ring structure;
ii) the ring structure is non-aromatic and is selected from the group consisting of 4-membered, 5-membered and 6-membered rings;
iii) The ring structure also has an H-bond acceptor moiety having a pKa of less than 7 and a structure that cannot serve as an H-bond donor moiety in its non-ionic form, as an acceptor in an H bond Selected from the group consisting of atoms in which the role plays a direct bond to the ring structure and those bonded to the ring structure through one atom;
The carboxylic acid moiety and the H-bond acceptor moiety are positioned and oriented such that they are compatible with the formation of internal H-bonds;
(C) comprising at least one cargo component in a precursor form effective to bind easily and stably to a radiohalogen;
(D) Sulfonamide moiety (R ₁ SO ₂ NR ₂ R ₃ )
A precursor structure that can be easily converted into a composition that is preferentially trapped in the acidic region, characterized in that it comprises an improved precursor structure that lacks.   The improved precursor structure of claim 1, wherein at least one cargo component in the precursor form comprises a trialkyltin moiety. The best mode structure is

The improved precursor structure of claim 2, wherein: A composition that is preferentially trapped in an acidic region that may be present in a tumor in a living subject, (i) present in a water solution that is predominantly negatively charged in an aqueous solution at pH 7.4. In the acidic region below pH 7.0, a significant portion of the molecules of the composition has the property of converting to a non-ionic form effective to enter the lipophilic phase, which may be a cell membrane, (ii) Includes a cargo component that can report its presence by emitting photons, gamma rays (high energy photons) or positrons and / or cause cell damage in the area where it is captured, ,
Captured preferentially by improving specificity in the acidic region,
(A) at least two acid moieties;
(B) at least one of the acid moieties is incorporated in a component capable of binding H to the interior having the following properties i) to iii):
i) a carboxylic acid moiety in which the acid moiety is directly bonded to the ring structure;
ii) the ring structure is non-aromatic and is selected from the group consisting of 4-membered, 5-membered and 6-membered rings;
iii) The ring structure also has an H-bond acceptor moiety having a pKa of less than 7 and a structure that cannot serve as an H-bond donor moiety in its non-ionic form, as an acceptor in an H bond Selected from the group consisting of atoms in which the role plays a direct bond to the ring structure and those bonded to the ring structure through one atom;
The carboxylic acid moiety and the H-bond acceptor moiety are positioned and oriented such that they are compatible with the formation of internal H-bonds;
(C) A composition preferentially captured in the acidic region, characterized in that it comprises an improved composition comprising at least one cargo component comprising radiohalogen.   5. The improved composition of claim 4, wherein the at least one cargo component comprises a radiohalogen selected from the group of halogen forms consisting of fluorine, bromine, iodine and astatine. The H-bond acceptor moiety in the component capable of binding H to at least one interior is
(A) an amide,
(B) trifluoroethylamine,
(C) cyanomethylamine,
(D) alkoxyamine,
(E) N-oxide,
(F) imidazole,
(G) aniline,
6. The improved composition according to claim 5, characterized in that it is selected from the group consisting of (h) phosphoramide, and (i) urea. The component capable of binding H to at least one interior is

7. The improved composition of claim 6, having a structure selected from the group consisting of: The molecular structure is

The improved composition according to claim 7, characterized in that it is selected from the group consisting of: The best mode structure is

The improved composition according to claim 8, wherein: A composition for detecting the presence of an acidic region that may be present in a tumor in a living subject, wherein (i) it exists in an aqueous solution at pH 7.4, mainly in a negatively charged water-soluble form. In the acidic region below pH 7.0, it has the property that a significant portion of the molecules of the composition convert to a non-ionic form that is effective to enter the lipophilic phase, which may be a cell membrane in biological tissue. And (ii) a cargo component that can report its presence by emitting photons, gamma rays (high energy photons) or positrons from the region where it is captured,
(A) at least two acid moieties;
(B) at least one of the acid moieties is incorporated in a component capable of binding H to the interior having the following properties i) to iii):
i) a carboxylic acid moiety in which the acid moiety is directly bonded to the ring structure;
ii) the ring structure is non-aromatic and is selected from the group consisting of 4-membered, 5-membered and 6-membered rings;
iii) The ring structure also has an H-bond acceptor moiety having a pKa of less than 7 and a structure that cannot serve as an H-bond donor moiety in its non-ionic form, as an acceptor in an H bond Selected from the group consisting of atoms in which the role plays a direct bond to the ring structure and those bonded to the ring structure through one atom;
The carboxylic acid moiety and the H bond acceptor moiety are positioned and oriented such that they are compatible with the formation of internal H bonds.
And (c) i) gamma rays,
An improvement for detecting with improved specificity the presence of an acidic region comprising ii) a positron, and iii) at least one cargo component comprising a radioactive halogen emitting radiation of a type selected from the group consisting of positrons and gamma rays A composition for detecting the presence of an acidic region, characterized in that   The improved composition according to claim 10, wherein the radiohalogen is selected from the group consisting of bromine 76, bromine 77, iodine 123, iodine 124 and iodine 131. The structure of the best mode is

The improved composition of claim 11, wherein: (A) (i) present in an aqueous solution that is predominantly negatively charged in aqueous solution at pH 7.4, but in the acidic region below pH 7.0, a significant portion of the molecules of the composition is biological tissue Having the property of converting to a non-ionic form effective to enter the lipophilic phase, which may be a cell membrane in
(Ii) providing a composition comprising a cargo component capable of reporting its presence by emitting photons, gamma rays (high energy photons) or positrons from the region in which it is captured;
(B) introducing the prepared composition into a subject;
(C) waiting for a period ranging from 10 minutes to 48 hours;
(D) a method for detecting an acidic region in a subject, wherein the subject may be present in the tumor by evaluating the subject with a device effective to detect radiation from a cargo component of the prepared composition. Because
The improvement comprises an improved method of detecting an acidic region in a subject with improved specificity, and the prepared composition is the composition of claim 10, wherein the subject is a device effective for detecting gamma radiation. A method for detecting an acidic region in a subject, characterized in that   14. Improved method according to claim 13, characterized in that the improved method is used to assess a living subject for the presence of a tumor comprising an acidic region. Further comprising first treating the subject with (a) at least one substance effective to increase the urine pH of the subject, and (b) at least one substance effective to lower the pH in the tumor. The improved method of claim 13, wherein: At least one substance effective to raise the pH of the subject's urine is
The improved method according to claim 15, characterized in that it is selected from the group consisting of (a) carbonic anhydrase inhibitor acetazolamide, and (b) buffer sodium bicarbonate. At least one substance effective to lower the pH in the tumor is
(A) glucose,
16. The improved method of claim 15, characterized in that it is selected from the group consisting of (b) mitochondrial inhibitor metaiodobenzylguanidine, and (c) vasodilator captopril. A composition for treating an acidic region that may be present in a tumor in a living subject, (i) present in an aqueous solution that is primarily negatively charged in an aqueous solution at pH 7.4, In the acidic region below pH 7.0, it has the property that a significant portion of the composition's molecules convert to a non-ionic form effective to enter the lipophilic phase, which may be a cell membrane in biological tissue. (Ii) including a cargo component that can cause cell damage in the area in which it is captured,
(A) at least two acid moieties;
(B) at least one of the acid moieties is incorporated in a component capable of binding H to the interior having the following properties i) to iii):
i) a carboxylic acid moiety in which the acid moiety is directly bonded to the ring structure;
ii) the ring structure is non-aromatic and is selected from the group consisting of 4-membered, 5-membered and 6-membered rings;
iii) The ring structure also has an H-bond acceptor moiety having a pKa of less than 7 and a structure that cannot serve as an H-bond donor moiety in its non-ionic form, as an acceptor in an H bond Selected from the group consisting of atoms in which the role plays a direct bond to the ring structure and those bonded to the ring structure through one atom;
The carboxylic acid moiety and the H-bond acceptor moiety are positioned and oriented such that they are compatible with the formation of internal H-bonds;
For treating an acidic region with improved specificity comprising (c) i) alpha particles, and ii) at least one cargo component comprising a radiohalogen that emits a type of radiation selected from the group consisting of: A composition for treating acidic regions, characterized in that it comprises an improved composition.   19. The improved composition of claim 18, wherein the radioactive halogen emits alpha particles and is astatine 211. The best mode structure is

The improved composition according to claim 19, characterized in that   19. Improvement according to claim 18, characterized in that the radioactive halogen emits beta particles and is selected from the group consisting of bromine 82, bromine 83, iodine 130, iodine 131, iodine 132, iodine 133 and iodine 135. Composition. (A) (i) present in an aqueous solution that is predominantly negatively charged in aqueous solution at pH 7.4, but in the acidic region below pH 7.0, a significant portion of the molecules of the composition is biological tissue Having the property of converting to a non-ionic form effective to enter the lipophilic phase, which may be a cell membrane in
(Ii) providing a composition comprising a cargo component capable of causing cell damage in the area in which it is captured;
(B) a method of treating an acidic area in a subject by introducing the prepared composition into the subject, wherein the acidic area may be in a tumor;
19. Treatment of an acidic region in a subject, wherein the improvement comprises an improved method of treating an acidic region in a subject with improved specificity, wherein the prepared composition is the composition of claim 18. how to.   23. The improved method of claim 22, wherein the improved method is used to treat a living subject having one or more tumors that include an acidic region. Further comprising first treating the subject with (a) at least one substance effective to increase the pH of the subject's urine, and (b) at least one substance effective to decrease the pH in the tumor. The improved method of claim 22, characterized in that: At least one substance effective to raise the pH of the subject's urine is
25. The improved method of claim 24, selected from the group consisting of (a) carbonic anhydrase inhibitor acetazolamide, and (b) buffer sodium bicarbonate. At least one substance effective to lower the pH in the tumor is
(A) glucose,
25. The improved method of claim 24, selected from the group consisting of (b) mitochondrial inhibitor metaiodobenzylguanidine, and (c) vasodilator captopril.   24. The improved method of claim 22, wherein at least two compositions of claim 18 are prepared and introduced into a subject, wherein one prepared composition of claim 18 is a radioactive halogen astatine. 21. The method of claim 18, wherein the second prepared composition of claim 18 comprises a radioactive halogen that emits beta particles. The prepared composition of the best mode is

28. The improved method of claim 27, wherein:   28. The improved method of claim 27, wherein the improved method is used to treat a living subject having one or more tumors comprising an acidic region. Further comprising first treating the subject with (a) at least one substance effective to increase the pH of the subject's urine, and (b) at least one substance effective to decrease the pH in the tumor. 28. The improved method of claim 27, wherein: At least one substance effective to raise the pH of the subject's urine is
31. The improved method of claim 30, wherein the method is selected from the group consisting of (a) a carbonic anhydrase inhibitor acetazolamide, and (b) a sodium bicarbonate buffer. At least one substance effective to lower the pH in the tumor is
(A) glucose,
31. The improved method of claim 30, wherein the method is selected from the group consisting of (b) mitochondrial inhibitor metaiodobenzylguanidine, and (c) vasodilator captopril.

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