BRPI0710026A2 - precursor structure, compositions and methods for detecting and treating acidic areas in an individual - Google Patents

Abstract

<B> <UM> PRECURSOR STRUCTURE, COMPOSITIONS AND METHODS TO DETECT AND TREAT ACID AREAS IN AN INDIVIDUAL <D> <MV> One of the many universal characteristics of the malignant tumor is its acidity. Oncological instruments are small synthetic molecules of non-peptides designed to explore acidity in the early detection and destruction of a tumor. Each cancer instrument has a structure that is anionic and hydrophilic at pH 7.4 and thereby repels negative cell-charged surfaces in normal tissues. When an oncological instrument associates with an acidic environment, such as a tumor, a fraction of the molecules of oncological instruments changes to their non-ionic lipophilic form which is designed to associate with cells, such as cells in acidic areas of a tumor . Prior to the use of an oncological instrument, a selection of radioactive isotope is linked to the oncological instrument. If the radioactive isotope emits radiation that can be detected outside the body, then the cancer instrument can be used to detect tumors. If the radioactive isotope emits radiation that is effective in destroying cells, then the cancer instrument can be used to treat tumors.

Description

PRECURSING STRUCTURE, COMPOSITIONS, AND METHODS FOR DETECTIONING AND ACTING ACID AREAS IN AN INDIVIDUAL

RELATED PATENT APPLICATIONS & MERCHANTABILITY

This order is a deposited PCT request

35 USC 363 and claims priority based on US Utility Patent Application No. 11 / 395,487, filed March 30, 2006, and US Utility Patent Application No. 11 / 449,495 and 11 / 449,508, both filed June 7, 2006 .

These related utility patent applications are incorporated herein by reference and form part of this application. If any conflict arises between the disclosure of the invention in this PCT application and those in the related utility patent applications, disclosure in this PCT application shall govern. In addition, any U.S. patents, U.S. patent applications, and other documents, hardcopy or electronic copy, quoted or referred to in this application are incorporated herein by reference and are part of this application. TECHNICAL FIELD

This invention relates to compositions effective to be selectively isolated in detective acidic areas within an individual for the purpose of detecting and treating tumors.

BACKGROUND OF THE INVENTION

Acidity: an almost universal exploitable feature: In 1930, the famous physiologist Otto Warburgrelated that one of the most universal malignant features is its acidity. Subsequent research suggests that such acidity arises because in order for a tumor to grow larger than approximately 1 millimeter in diameter, it must induce new blood vessels. Because tumor-inducing blood vessels are poorly spaced and abnormal, areas larger than a few ten microns away from such capillaries usually become hypoxic (oxygen deficiency). Cells in such hypoxic areas of tumors either die or change glycolytic metabolism - resulting in lactic acid excretion. Because of poor tumor circulation, this excreted lactic acid accumulates in the interstitial space between cells in the hypoxic areas of the tumor. This can result in a pH as low as 6.0 in the most distant areas of the capillaries, up to approximately pH 7.0 near the capillaries. For comparison, the pH in the interstitial space in normal tissues is tightly regulated between approximately pH 7.3 and pH 7.5.

Inactive cells in acidic areas of tumors: the inadequate challenge in cancer therapy:

As noted above, the characteristic acidity of tumors generally increases with the growing distance from tumor capillaries. While tumor cells at near-normal pH in close proximity to the capillaries have high metabolic rates and rapid cell division, those tumor cells in more acidic areas farther from the capillaries have lower metabolic rates and divide slowly or moderately. Compared to rapidly dividing tumor cells, these slow dividing and non-dividing tumor cells, referred to as inactive, are substantially more resistant to cell-damaging agents such as comoradiation and toxic chemicals.

Since conventional cancer therapies (radiation and chemotherapy) have been largely selected for their ability to destroy rapidly dividing cells while sparing the divisionless, slow-dividing cells typical of most normal tissues, such conventional cancer therapies are reasonably effective in destroying the cells. tumor cells that rapidly divide at near normal pH near the capillaries. Not surprisingly, those same therapies are somewhat ineffective against slow-dividing inactive cells without division in the most acidic areas of a tumor. Consequently, conventional cancer treatments predominantly destroy well-oxygenated, rapidly dividing tumor cells while sparing inactive cells. in the hypoxic / acidic areas of this tumor. This initial destruction of the rapidly dividing cells causes the tumor to remit while those destroyed cells are discarded by normal body cleansing processes. However, during the course of this cleansing process, treatment-resistant resistant surviving native cells quite often slowly regain access to adequate oxygen, nutrients, and waste disposal, eventually allowing them to revert to elevated taxametabolic and rapid cell division. Previously inactive tumor cell stereovescence generally leads to the dreaded relapse that is responsible for most cancer deaths.

Previous efforts to explore hypoxic / acidic areas for therapy:

The hypoxic / acidic properties of tumors have been known for over 75 years and it has long been speculated that such properties could be exploitable for therapy. However, until recently the most successful efforts to exploit these properties have focused on hypoxia. Specifically, substances have been developed to exhibit minimal cytotoxicity in normoxic cells while exhibiting considerable cytotoxicity in hypoxic cells. One such agent has achieved progress in the clinical trial stage.

Compared to the exploitation of hypoxia in tumors, they have been much less focused on exploiting the acidity of tumors. An unsuccessful approach was based on the observation that acidic pH in tissues acts to sensitize these tissues to thermal damage. However, efforts to exploit this acid-mediated thermal sensitivity of cells have yielded disappointing results.

Another approach to acidity is not the fact that low pH in weakly based cytotoxic ionizing tumors and thus gives them a waterproof membrane, which in turn results in preferential reduction of entry of a number of such weakly based agents. in acidic areas of tumors relative to the entry of such agents into cells in areas of more normal umpH. In this regard, prior to this attempt to exploit low pH in a tumor, efforts were preferably focused on raising the pH in tumors as a means of partially deionizing and thereby enhancing the entry of such weak base cytotoxic agents into tumor cells. Such efforts have found some success.

Yet another approach that relates more closely to the present invention has relied on the fact that low pH in the interstitial space of tumors will cause partial deionization of the weak acid cytotoxic agent chlorambucil (shown in Figure 1). As a consequence of the lower degree of deionization of this known weak acid cytotoxic drug in acidic areas of tumors, I would only hope that it could show increased entry and therefore greater cytotoxicity in cells in acidic areas of tumors. This expectation was tested by Kozin et al., In which they used established methods to make more acidic tumors in tumor-bearing mice (Cancer Research Vol. 61, pages 4740 -4743 (2001)). They reported that, as predicted, a reduction of approximately 0.3 unit pH in the tumors coincided with a modest 1.7-fold improvement in the growth of the weak acid (pKa5,8) chlorambucil-induced tumor. At the conclusion of their work, they wrote: "To our knowledge, CHL (chlorambucil) is the only clinical therapy that is a weak acid with the appropriate pKa ≤ 6.5. This study thus provides an analysis for the design of potent new drugs that exhibit properties. similar to weak acid and to which diffusion contributes to intracellular absorption. As also shown here, the combined use of such compounds with radiation and / or pH gradient modulators provides additional opportunities for maximizing re-therapeutic. "

In the aforementioned work, the authors noted that their results showed modest intensification in the efficacy of the readily available cytotoxic chlorambucil drug, providing rational analysis for designing new weak acid cytotoxic anticancer drugs which may show high efficacy - but no guidance has been given to the expected molecular structures for such drugs, neither guidance was given on how to continue to integrate both weak acid function and cytotoxic function into an appropriate drug structure, nor was any guidance given on what specific properties are desirable, nor any guidance was given on how to continue designing, prepared to test such cytotoxic drugs. weak acid, no guidance has been given regarding the applications or methods of use of such cytotoxic drugs. The most serious limitation in the authors' proposal that new improved-potency weak acid drug-induced drugs could be designed based on the chlorambucil model is inactive cells in acidic tumor areas, where such drugs are designed to preferentially insert, are particularly resistant to conventional cytotoxic drugs because of them. Thus, a striking case can be made that conventional drug-toxicants, such as chlorambucil, are unsuitable for destroying inactive cells in acidic areas of tumors for which they have been proposed to be used.

Instead of Summerton (inventor of cancer instruments) claiming that what is actually required to destroy inactive cells in acidic areas of tumors are specially designed drugs that have a particularly devastating impact on cell viability to decisively destroy those inactive treatment-resistant tumor cells. Coupled with this requirement for a devastating impact on cell viability is the requirement that such drugs be effectively distributed within tumor cells while being largely excluded from cells in normal tissues.

TECHNICAL BACKGROUND

In contrast to Kozin et al.r's proposal to use conventional weak acid cytotoxic drugs to exploit tumor acidity, several years ago Dr. James Summerton, inventor of cancer instruments, began the development of new peptide compositions explicitly designed to exploit the differential. between normal tissues and acidic regions of tumors to provide safer and more effective treatments for a large scale of tumors. Those large peptide compositions (typically with masses ranging from 2,000 to 4,000 daltons) are referred to as "carrier peptides and binders". Their structures and methods used for detecting and treating tumors are disclosed in pending U.S. Patent Application No. 11 / 069,849 and U.S. Patent No. 7,132,393 filed November 7, 2006 - Dr. James E. Summerton the inventor. While such binding and transporting peptides have been found to insert tumors into mice with satisfactory electivity, nevertheless experimental results from mouse studies suggest that such peptides achieve less specificity and efficacy than desired. They also appear to be subject to improper kidney resorption - which is undesirable in diagnostic applications and unacceptable in therapeutic applications.

Because of the limitations of the transporter peptides and binders, Summerton subsequently undertook a quest to design an entirely new class of small non-peptide drugs called oncological instruments in order to considerably improve the specificity, efficacy and safety relative to the prior transporter peptides and binders. These small non-peptide imaging instruments comprising the present invention were disclosed by the present inventor, Dr. JamesE. Summerton, only in U.S. Patent Application No. 11 / 395,487 filed March 30, 2006, and U.S. Patent Applications No. 11 / 449,495 and 11 / 449,508 both filed June 7, 2006.

SUMMARY OF THE INVENTION The object of the present invention is to provide non-peptide compositions and methods for detecting and treating tumors containing acidic areas. Oncological instruments explore tumor acidity for early detection and tumor destruction:

Cancer instruments of the present invention are a novel class of molecules designed for the detection and treatment of tumors. Each cancer instrument contains two or more pH selector components which readily convert between a higher pH anionic hydrophilic form and a lower pH nonionic lipophilic form. Each oncological instrument also contains a charge component which is effective for binding a selected radioactive isotope which is suitable for performing the diagnostic or therapeutic role of an oncological instrument. Cancer instruments consist of relatively small synthetic non-peptide molecules massastipically in the range of approximately 300 to 1500 daltons - not counting the mass of the radioactive isotope.

At the neutral pH of normal tissues, oncological instruments exist in their negatively charged water-soluble form which is designed to be repelled by negatively charged cell surfaces and readily excreted by the kidneys. Conversely, when oncological instruments insert into the acidic region of a tumor, a fraction of the oncological instrument's molecules change to their lipid-soluble nonionic form which is designed to readily insert into any small cell. Thus, when a dose of the cancer instrument is injected into a person, if that person has a tumor larger than approximately 1 millimeter in diameter, a dice injected fraction is designed to rapidly insert and remain within cells in acidic regions of the tumor, with the remainder of the dose. Excreted by the kidneys.Differently stated, oncological instruments are designed to have key properties: a) to be repelled from cells in normal tissues; (b) be isolated within the acidic areas of tumors; and (c) any cancer instrument not isolated in an acidic area of a tumor is designed to be rapidly eliminated from the body through the kidneys.

Prior to the use of an oncological instrument a selected radioactive isotope is interconnected with this oncological instrument. This unidored radioactive isotope fulfills the diagnostic or therapeutic role of the instrument. The diagnostic role is to report the presence of the cancer instrument within a tumor to a forced body detector. The therapeutic role is to destroy the tumor.

Diagnostic Oncological Instruments:

Because of the large variability between tumors, routine detection of a large range of tumor types at a sufficiently early stage at which they can be successfully treated (preferably before they are metastasized, show obvious symptoms or cause serious organ damage) has long been unsatisfactory time of medicine. Much of the difficulty with early routine tumor detection has been to identify some property which is common to most or all tumors, and which can effectively be exploited by routine and early available tumor detection. An oncological instrument conducting an appropriate radioactive isotope (eg, a gamma or positron emitter) which emits a readily detectable signal outside the body, combined with modern imaging technologies, is designed to safely provide very early detection of most or all tumor types. .

A diagnostic cancer instrument is designed to be widely used in routine annual physical examinations to provide very early detection of essentially all tumors larger than size microscopic. Thus, a single diagnostic diagnostic instrument can serve as a superior and comprehensive replacement for an extensive range of current tumor diagnostic procedures, such as mammograms, Pap smears, prostate exams, and echo-scans. In addition, it should be appreciated that many types of tumors currently cannot be routinely detected until they reach a somewhat late stage of development in which they manifest as pain, organ malfunction or some other general symptom or set of symptoms, which then require diagnostic procedures. exhaustive and very expensive to identify the root cause of the problem. Until such a tumor is detected it is often advanced to any successful treatment expectation (at least currently approved therapies). In this regard, diagnostic cancer instruments offer the promise of an entirely new capability for the early detection of such difficult-to-diagnose tumors. Therapeutic cancer instruments:

Therapeutic cancer instruments are designed to exploit the characteristic acidity of tumors to provide safe and effective tumor destruction. For optimal results a combination of two cancer instrument formulations should be used. One formulation leads to a radioactive isotope which emits radiation of high linear energy transfer during a short path, such as a very high energy alpha particle, that is effective in destroying radiation-inactive cells in close proximity to acidic tumor areas (see Figure 2). The other formulation carries an isotope radioactive which emits a medium energy beta particle during an effective medium pathway to destroy rapidly dividing radiation sensitive cells farther away from capillary arteries. When used in combination, these two formulations are designed to effectively destroy the entire tumor, with little or no damage to other cells in the body. As a result, therapeutic cancer instruments are designed to be effective in the majority against every cell type or in all tumors, and thus offer the far less effective and much more effective treatment of tumors with little or no relapse.

DETAILED DESCRIPTION OF THE INVENTION BRIEF DESCRIPTION OF THE DRAWINGS: Figure 1 shows weak acid chlorambucil cytotoxic chemotherapeutic in its anionic and nonionic forms.

Figure 2 illustrates the distribution of acidity in the others.

Figure 3 illustrates the mediated pH-to-solubility transitions mediated between forms.

Figure 4 shows titration curves calculated as lipophilicity function of the nonionic acid form.

Figure 5a illustrates the expected hydration waters, showing H-bridged water molecules to a conventional carboxylic acid in its ionic and nonionic forms.

Figure 5b illustrates the expected hydration waters, showing H-bridged water molecules to a carboxylic acid-containing structure in its non-ionic form and in the non-ionic form bound to the Hintern bridge.

Figure 6a illustrates an acid-specific H bridge in a structure that forms an internal acid-specific H bridge only in its nonionic form.

Figure 6b illustrates a specific non-acidic H-point forming structure in both its anionic form and its nonionic form.

Figure 7a shows representative ring structures suitable for advanced pH selectors, including a 4-element ring structure, a 5-element ring structure, and a 6-element ring structure. Figure 7b shows a structure which is unacceptable for use as an advanced pH selector because it has an acyclic structure.

Figure 8a illustrates the isolation of the carboxyl moiety from the appropriate inductive effects for an increasing number of carboxyl-separated carbons from an electron removal group.

Figure 8b illustrates a structure having improper isolation of its carboxyl from inductive effects.

Figure 9a illustrates a structure whose H-tipping site is open to solvent.

Figure 9b illustrates a structure whose H-tipping site is partially hindered by solvent.

Figure 10 shows low H-bridge bridge components.

Figure 10a shows H-bridge donor comprising a carboxylic acid which is suitable for forming H-bridge bridges with H-bridge acceptor moieties having a pKa value, in an independent form, in the range of approximately 3.0 to 6, 5

Figure 10b shows representative H-acceptor moieties suitable for forming low-H-bridging bridges with a carboxylic acid donor.

Figure 10c illustrates representative advanced pH selectors designed to form internal specific acid-low H-bridges. Figure 11 illustrates the statistical bases for increasing specificity of pH-selective oncological instruments.

Figure 11a illustrates nonisolated anionic and nonionic tumor-isolated forms of an oncological instrument with two pH selectors.

Figure 11b illustrates nonisolated anionic and nonionic tumor-isolated forms of an oncological instrument with three pH selectors.

Figure 12 shows calculated specificity and efficacy factors for pH multi selectors of oncological instruments.

Figure 12a shows specificity and effectiveness factors calculated as a function of the pKa value for structures containing a pH selector.

Figure 12b shows specificity and effectiveness factors calculated as a function of the pKa value for oncological instruments containing two pH selectors.

Figure 12c shows specificity and effectiveness factors calculated as a function of the pKa value for oncological instruments containing three pH selectors.

Figure 12d shows specificity and effectiveness factors calculated as a function of the pKa value for oncological instruments containing four pH selector.

Figure 13 shows a composition containing advanced pH selectors.

Figure 13a shows the structure of this composition. Figure 13b shows a graph of the n-Octanol / buffer division of this composition of 2 pH selectors with a pH function.

Figure 14 illustrates representative charge components in both their precursor and final forms.

Figure 15 illustrates two representative paracomponent synthetic routes of charge, including a simple procedure for converting precursor forms to their final forms.

Figure 16 illustrates representative 2-pH selector oncological instruments.

Figure 16a illustrates two representative 2-pH selector instruments containing single pH selectors.

Figure 16b illustrates five representative 2-selector pH instruments containing advanced pH selectors.

Figure 16c illustrates ten representative 2 pH selector oncological instruments containing advanced pH selectors designed to form a low-H-bridge. Structures 1 and 2 of Figure 16c show cancer instruments wherein the H-acceptor portion of pH selectors are N-oxide moieties, and where each N-oxide moiety serves as the H-bridge acceptor for two carboxylic acid moieties. H. donor donors

Figure 17 illustrates two representative pH selector oncological instruments. Figure 17a illustrates two representative 3 selector pH oncological instruments containing advanced pH selectors.

Figure 17b are two representative 3-selector pH-oncological instruments containing advanced pH selectors designed to form a low barrier H-bridge.

Figure 18 illustrates representative 4-selector oncological instruments.

Figure 18a illustrates 4 pH selector oncological instruments where the H-bridge acceptor moieties of pH selectors are alkoxy amine moieties.

Figure 18b illustrates 4 pH selector oncological instruments, where the H-point acceptor moieties of pH selectors are N-oxide moieties, and in each portion of N-oxide serves as H bridge acceptor for two moieties of carboxylic acid. H bridge donors.

Figure 19 illustrates the synthesis of oncological instruments containing simple pH selectors, where allipophilicity can be adjusted by varying the R group.

Figure 20 illustrates a synthetic advanced pH deselectors scheme in which lipophilicity can be adjusted by varying the R group.

Figure 21 illustrates useful amine ester and ketone ester intermediates for instrumentation synthesis. Figure 22 illustrates syntheses of representative pH selectors designed to form an internal specific low acid H-bridge.

Figure 22a shows the syntheses of a depH selector wherein the H-bridge acceptor is a decyanomethyl amine moiety.

Figure 22b shows the syntheses of a depH selector wherein the H-bridge acceptor is an N-oxide moiety.

Figure 22c shows the syntheses of a depH selector wherein the H-bridge acceptor is a detrifluoroethyl amine moiety.

Figure 23 shows titration results for a variety of pH selector structures.

Figure 23a shows a conventional titration curve, where titration was performed in methanol / water, 1: 1 to volume, for a simple carboxylic acid (butyric acid) and an advanced pH selector (an acid starch derivative of camphoric acid), plus that same data plotted in the form derived from the first information.

Figure 23b shows a conventional titration curve, where titration was performed in water and each species was present at a concentration of 33 milliMolar, for the two pH selector oncological instruments (but with stable iodine rather than radioactive iodine) varying in their group. R is a structure of three pH selectors.

Figure 23c shows a titration curve plotted as the first derivative for an advanced pH selector designed to form a low barrier H bridge. This titration was performed in water and the depH selector, comprising an N-oxide / camphoric acid-derived acid structure, was present at a 5milliMolar concentration.

Figure 24 shows experimentally determined pKa values for three pH-deselecting structures, including: the acidic acid-derived camphoric acid-derived starch / selector shown in Figure 23a; an N-oxide / acid-enhanced pH selector designed to form an H bridge low barrier, derived from the isipipotic acid; and the advanced acid pH N-oxide / pH selector designed to form a low barrier H-bridge derived from camphoric acid, shown in Figure 23c.

Figure 25 illustrates synthetic schemes for representative oncological instruments containing advanced pH selectors.

Figure 25a shows an oncological instrument in which its two pH selectors are joined by a di-acylhydrazide structure.

Figure 25b shows an oncological instrument in which its two pH selectors are joined by a di-amide structure.

Figure 26 illustrates synthetic schemes for representative oncological instruments containing advanced pH selectors designed to form a low-flow H-bridge.

Figure 26a shows a synthetic scheme for a 2-pH selector oncological instrument wherein the single N-oxide moiety serves as the H-bridging receptor moiety for two carboxylic acid H-bridging donor moieties.

Figure 26b shows a synthetic scheme for a 2-pH selector oncological instrument wherein both nitrogens from one hydrazine moiety serve as the H-bridging moiety for two H-bridge donor moieties of carboxylic acid.

Figure 26c shows a synthetic scheme for a 2-pH selector oncological instrument wherein the cyanomethyl amine moiety serves as the H-bridging receptor moiety for two carboxylic acid H-bridging donor moieties.

Figure 26d shows a synthetic scheme for a 2-pH selector oncological instrument wherein N-oxide co-evaporation serves as the H-bridging receptor moiety for two H-bridging carboxylic acid donor moieties.

DEFINITIONS OF TERMS

Terms used in this context have the following specific meanings, unless otherwise noted.

The words "understand," "have," "contain," and "include," and other forms thereof are equivalent in meaning and not exclusive to an item or items referring to any of these words, that is, they are not part of an exhaustive list of such item or items or limited solely to the item or items listed. The words "consistent" "consist of" and other forms thereof are equivalent in meaning and limited in the sense that an item or items referring to any of these words are part of an exhaustive list. of talitem or items and are limited only to the item or items listed.

PH Selector Component - A structural component of an oncology instrument that is able to subtract to a pH-mediated transition between an anionic hydrophilic at a higher pH and a nonionic formaliphilic at a lower pH.

Advanced pH Selector Component - A pH change component designed to form an internal specific acid hydrogen bridge. An advanced pH selector has the following properties:

(a) contains an aliphatic ring structure selected from a group consisting of: 4-element rings, 5-element rings, and 6-element rings;

b) contains a carboxylic acid moiety directly linked to an aliphatic ring structure;

c) a carboxylic acid moiety is separated from an electron withdrawing group by at least two carbons;

d) contains an Hselected bridge acceptor moiety within a group consisting of:

(i) part of the aliphatic structure of the ring;

ii) direct bonding to the aliphatic structure of a ring; eiii) bonding through an atom to the aliphatic ring structure;

e) the acceptor moiety of H has a structure which in its nonionic form does not serve as the H-spike donor moiety; and

f) the carboxylic acid moiety and said H-bridge acceptor moiety are positioned in close proximity to and are correctly positioned and oriented such that they are compatible with the formation of an internal specific acid H-bridge.

Charge component - A structural component of an oncological instrument that serves to connect an isotope radioactive that is effective in reporting the presence of the cancer instrument, that is, effective in destroying cells. The charge component may exist in either a precursor form ready to bind a radioactive isotope or a final form containing a radioactive isotope.

Precursor Charge Component - A charge component that has a structure that is capable of readily deleting to a selected radioactive isotope, but does not yet determine a radioactive isotope. An oncological instrument with this charge decomposing precursor form is suitable for long term storage.

Charge component in final form - A charge component that contains at least one determined radioactive isotope. An oncological instrument with this final charge component form is suitable for distribution within an individual for the purpose of detecting and / or destroying tumors containing acidic areas.

Cancer instrument - a non-peptide composition that includes at least two select pH components and at least one charge component.

Diagnostic Cancer Instrument - An oncological instrument containing a radioactive isotope that is effective in reporting the presence of the cancer instrument in a tumor to a detector outside the individual undergoing the diagnostic procedure.

Cancer Instrument for Therapy - An oncological instrument containing a radioactive isotope that is effective in destroying cells.

Two-way radioactive isotope cancer therapy therapy strategy - a treatment strategy where at least two different cancer instruments are used to treat tumors, where one cancer instrument contains a radioactive isotope that emits high linear transfer energy, and another cancer instrument contains a radioactive isotope. issuer.

Interstitial space - The area of a tissue or tumor that is outside the vascular network and outside the cells.

Acid Tumor Area - An area of a tumor where the interstitial space has a pH less than 7.0.

Physiological conditions - an aqueous solution having a temperature in the range of 20 ° C to 40 ° C and which has a concentration of sodium chloride between 0.13 M and 0.17 Ma.

Significant Fraction - greater than approximately 1%.

Anionic hydrophilic form - a form that carries at least one negative charge and has an octanol / water partition coefficient of less than 1.

Nonionic lipophilic form - a form that does not carry an ionic charge and has an octanol / water partition coefficient greater than 1.

Efficacy factor - A percentage of oncological instrument molecules that are in their nonionic form with pH 6.4.

Specificity factor - the ratio: (percentage of oncological instrument molecules that are in pH 6.4 ion ion) divided by (percentage of oncological instrument molecules that are in pH 7.4 nonionic form).

Effective for reporting presence - an radioactive isotope whose emission, such as a gamma ray or a positron, generates a signal that is readily detectable outside the individual.

pT - the pH value at the midpoint of the transition between the hydrophilic form of a substance and the formalipophilic form of that substance, where this transition between the two forms is measured with 5 milliMolar of the substance present in physiological saline (or a near equivalent containing some buffer). When the pT value matches the pKa value for a substance, the depT value differs in that it is influenced by both the substance pKa and the lipophilicity of the substance. The pT value is useful for describing carboxyl containing substances whose non-ionic form is strongly lipophilic and may then detach from the aqueous solution during titrations, or may divide within a nonpolar phase during partition assays. Additionally, the pT value is measured in the approximate presence of a physiological saline concentration and thus better reflects the properties of the detestant substance in vivo in an environment.

Radioactive isotope emitting high linear energy transfer radiation - an isotope radioactive that emits an alpha particle or emits an Augar and / or Coster-Kronig electron.

Inventor - Ph.D James E. Summerton

DISCLOSURE OUTLINE:

A. Molecular Design of Oncological Instruments

1. pH Selector Components

a) pH selector lipophilicity adjustment

b) Incorporation of a new specific acidic H-bridge into the pH selector

c) Incorporation of multiple pH deselector components in oncological instrument

2. Load component

a) Structural requirements

b) Precursor and final forms

c) Radioactive isotope charge selection

i) for tumor detection

ii) for treating tumors3. Structure of the Oncological Instrument

a) Structural requirements

b) Cancer instruments with 2 pH selectors

c) Cancer instruments with 3 pH selectors

d) Cancer instruments with 4 pH selectors

B. Synthesis, Testing, and Optimization of Oncological Components and Instruments

1. Preparation of structures containing representative pH selectors

a) Simple pH selectors

b) Advanced pH Selectors

c) Advanced pH selectors designed to form a low barrier H bridge

2. Testing pH Selectors

3. Preparation of representative oncological and final form conversion instruments

a) Cancer instruments with simple pH selectors

b) Cancer instruments with advanced pH selectors

c) Oncological instruments with advanced pH selectors designed to form a low barrier H bridge

4. Testing and optimization of cancer instrumentsa) Iterative optimization process: synthesis and

cell culture test

i) Joint preparation of various oncological instruments in lipophilicity iii) Evaluation of cell culture input at pH 6.4 and pH 7.4

iii) Repeat steps as required

(b) Assessment of disposition in normal mice

c) Evaluation of disposition in tumor-bearing mice

d) Preclinical and clinical tests

C. Methods for Using Oncological Instruments

1. Diagnostic Method

2. Therapeutic Method

a) Simple radioactive isotope method

b) Double radioactive isotope method

3. Detailed method for detecting and treating tumors

4. Strategy for dealing with micro-metastasis

5. Treat to lower pH in tumors for increased effectiveness and specificity.

6. Using multiple oncological instruments that vary in pKa to increase efficacy and specificity

7. Treat to increase urine pH to protect kidney from damage

8. Monitor and wash bladder for increased safetyMETHOD OF THE INVENTION:

A. Molecular Design of Oncological Instruments

Cancer instruments comprising the present invention are a novel class of molecules designed for the detection and treatment of tumors. Each cancer instrument contains two or more pH-deselecting components which readily undergo a pH-mediated transition between a higher pH anionic hydrophilic form and a lower pH nonionic lipophilic form. Each cancer instrument also contains a charge component that is effective for binding a selected radioactive isotope that is suitable for performing the diagnostic or therapeutic role of the cancer instrument.

1. pH Selector Components

If an oncological instrument is to achieve adequate specificity for acidic areas of tumors, it must have a structure such that in aqueous pH 7.4 solution that almost all molecules of the oncological instrument occur in a negatively charged (anionic) form which has poor affinity for negatively charged surfaces. Conversely, in order to achieve adequate specificity, at the pH present in acidic areas of tumors a significant fraction (greater than 1%) of the instrument's molecules must change to the nonionic lipophilic form that readily inserts cell membranes. A major challenge in designing cancer instruments is to design a structure in which a sufficient fraction of the cancer instrument molecules undergo the transition between the anionic hydrophilic form and the nonionic lipophilic form as an assessment of various normal woven pH difference limits and acidic areas of tumors. When this pH difference is usually only around 0.5 to 0.7 pH units, this can be appreciated by simple steps (described later in Section C in this context) can easily be performed to substantially increase this pH difference between normal tissues and acidic areas. tumors. With such steps the pH difference between normal and acidic tumor areas can predictably be increased to 1.0 or more pH units.

To meet the many demands of the inherent challenge of molecular design by exploiting acidity in others, pH selector structures have been available which each contain a weak acid moiety (usually a carboxylic acid) that leads to a negative charge at pH 7.4. Therefore, because of this essential weak acid moiety exhibiting substantial hydrophilic condition in its nonionic free acid form, in order to achieve adequate efficacy, two molecular design strategies have been used to provide sufficient lipophilicity for rapid membrane insertion when the oncological instrument enters. in the acidic area in a tumor. These two design strategies, used separately or combined, when combined with a third design strategy, can also play the key role dramatically increasing the specificity of the cancer instrument for acidic tumor areas. The physical and mathematical bases for these three molecular design strategies, two of which are believed to be unique to oncological instruments, are described below.

a) Adjustment of pH selector lipophilicity by incorporation of multiple lipophilic portions to counteract the inherent hydrophilicity of the free acid moiety. In this regard it is primarily useful to obtain a quantitative assessment of how the increase in the non-ionic free acid form of the pH selector affects its partitioning properties (ie, as the division between an aqueous phase and a lipophilic phase such as octanol or a cell membrane). ). To this end, mathematical modeling was performed to calculate the pH-dependent transition between the forms of a weak acid substance, which exists at high pH in a water-soluble anionic form, [A-], and which converts lower pH to one. nonionic lipophilic form which has some aqueous solubility, [HAsol], and which may also become water-insoluble / lipid-soluble, designated [HAinsol], by virtue of the precipitation or oleation of the aqueous solution, or by virtue of partitioning into a lipophilic phase, such as octanol or a cell membrane. The interconversion of these three forms, together with the key equations used in mathematical modeling, are illustrated in Figure 3.

The results from the modeling, illustrated in Figure 4, show that the midpoint pH of the transition between the anionic form, [A-], and the nonionic forms, [HAsol plus HAinsol], progressively increases as the octanol partition coefficient. / water, P, nonionic acid free form is increased. However, the transition pH increase will only apply after the free acid form reaches the effective lipophilicity threshold for initiating precipitation or oleation, or if a non-polar phase is present to allow for partitioning. Example 1 describes titration experiments which illustrate this mathematically predicted impact of lypophilicity. of the acid form in the pH-dependent transition between the anionic and nonionic forms.

However, it should be noted that excessive incorporation of lipophilic moieties as an exclusive means to increase non-ionic lipophilicity may be ineffective. This is because when very high lipophilicity is generated only by incorporation of too many lipophilic portions then even the pH-selector shape can begin to have an affinity for cell surfaces - resulting in isolation of the cancer instrument in normal tissues as well as in tumors. This can be a particular problem when lipophilic portions form a large surface and hydrophilic portions are well separated from this lipophilic surface. When the oncological instrument is also isolated in normal tissues, it will cause a high signal depth in diagnostic application and significant toxicity in therapeutic application.

(b) Incorporate a new specific acid internal H-bridge into the pH selector to amplify the solubility differential between hydrophilic and lipophilic forms.

In the foregoing section a somewhat unconventional strategy has been described to improve the entry of the cancer instrument into cells in the acid-depleting areas, and thereby increasing the effectiveness, this strategy is to incorporate lipophilic groups within pH selector components. This simple strategy is commonly used in what is referred to as the "rational drug project" - which has been widely applied to improve the effectiveness and specificity of cancer chemotherapy. However, because there is only a slightly small difference in pH values between normal tissues and acidic areas of tumors, the inventor set out to devise an additional strategy to intensify the entry of cancer instruments into acidic areas, which was also expected to increase the specificity of cancer instrumentation. tumors. To this end, the inventor postulated that both anionic form hydrophilicity and non-ionic pH-selector form lipophilicity could be substantially increased by effective structure design for the pH-dependent acid-specific internal hydrogen bridge. The inventor further postulated that such a Hinterna bridge would raise the pH at which the pH selector transition between its anionic form and its nonionic form, thereby making the most available non-ionic oncological instrument for cell entry into the acidic areas of tumors. .

This predicted increase in the H-bridge mediating non-ionic form lalipophilicity and concomitantly increasing anionic form hydrophilicity is based on the expectation that in an aqueous environment such an internal H-wall will displace several (probably two) free acid bound waters. To illustrate, Figure 5a shows the expected hydration waters directly bound to H for a carboxyl standard in its anionic and nonionic free acid form. Figure 5b shows that the corresponding expected hydration waters directly bound to H for anionic and nonionic acid free form of an effective structure to form the desired specific acid Hinternal bridge. Clearly, both the standard carboxyl and the effective carboxyl to form the Hinterna bridge lose their ion counter going from anionic to nonionic free acid form. However, while the standard carboxyl is expected to have the same number (probably 4) of waters directly bound to H for both anionic and nonionic free acid forms, the inventor believes that for carboxyl capable of forming an internal H bridge will be a net loss from several (probably two) H-bound waters where it will come from anionic form (probably 5 H-bound waters) to nonionic free acid form (probably 3 H-bound waters) - and this loss from several H-bound waters H will substantially amplify the hydrophilicity / lipophilicity difference between the two forms. The inventor predicted that if such a specific acid internal bridge could be formed in an aqueous environment, then the resulting loss of various hydration waters would significantly increase the ability of cancer instruments to rapidly insert cells into acidic areas of tumors, while also significantly increasing repulsion of the cancer instrument. of cells in normal tissues. In summary, the incorporation of such a specific acid internal H-bridge has been envisioned to increase the specificity and effectiveness of the cancer instrument by differentially altering the number of water binding molecules for each of its two forms of pH selector components.

The inventor also predicted (and has now shown) that the formation of the specific internal acid H-bridge in the pH selector component should favor the transition of the carboxyl portion of the depH selector component to its free acid form, resulting in a significant increase in the value of pKa for that carboxylic acid moiety. As will be detailed in the following section, such an increase in pKa of the carboxyl moiety is desirable in the context of a strategy for dramatically increasing tumor cancer instrument specificity by incorporating two or more pH selectors into this cancer instrument.

With respect to designing pH selector structures capable of forming an internal H bridge in an aqueous environment, conventional wisdom among hydrogen bridge experts typically supports that a solitary hydrogen bridge will not be stable in an aqueous environment because of the competition with the vast concentration of water in the water. H-bridge donor sites (110 Molar) and H-bridge donor sites (110 Molar). Instead, this is generally supported by a stable noncovalent interaction in aqueous solution that requires a multiplicity of interactions, selected from H bridges, hydrophobic interactions, and electrostatic interactions. While this requirement for a multitude of noncovalent bonds appears to be well known. For intermolecular interactions, the inventor has postulated that, contrary to conventional wisdom, it may be possible to design compact structures that will form a single relatively stable pH-dependent intramolecular H-bridge in aqueous solution, where that H-bridge increases the hydrophilicity / lipophilicity difference between the two forms and serves to significantly favor the free acid form over the anionic form (ie, elevates opKa). The crucial question then was: could the practical pH selector structures be designed which would form such an internal H bridge in aqueous solution?

After considerable experimentation, new structures have now been devised which have been shown to form the only desired pH-dependent internal H bridge, and which are suitable for incorporation within the oncological instruments of the present invention. As originally envisioned by the inventor, such Hinternal bridge appears to be significantly enhanced. The hydrophilicity / lipophilicity difference when the pH selector goes from its anionic to its nonionic form. Several acid-specific internal H-bridges have also been shown to significantly increase the pH at which the structure changes from its hydrophilic anionic form to its nonionic lipophilic form relative to opH in which a similar portion of simple carboxyl undergoes this transition. which has a structure designed to form a specific acid internal bridge is called an "advanced pH selector". Results from molecular modeling and extensive experimental work suggest that the following three properties are essential for an advanced pH selector to form an acceptable and stable internal acid H-bridge in aqueous solution.

i) Specific acid H bridge

The structure should contain a carboxyl moiety which is positioned in the appropriate vicinity of an H-bridge acceptor moiety for formation of the H-bridge. When that carboxyl moiety is in its free acid form it can serve as an H-bridge donor, and The proximal H-bridge acceptor should be such that its nonionic form can only serve as an H-bridge acceptor, and cannot serve as a ponte-bridge donor. The inventor refers to an H bridge formed by a structure as a "specific acidic internal H bridge". Figure 6a shows a representative structure of the pH selector which can form only one specific acid internal H bridge. Conversely, Figure 6b illustrates a similar but unacceptable structure which can form both an internal bridge when carboxyl is in its anionic form and an Internal bridge when carboxyl is in its free acid form. The inventor calls this dual H-bridge capability "specific non-acidic internal H-bridge", and his experimental results indicate that such specific non-acidic H-bonding is unacceptable because it does not provide the desired lipophilicity increase of the acid form, and does not raise the pH at which the structure changes from its anionic hydrophilic form to its nonionic lipophilic form. Example 2 describe experimental results in which a 1 to 1 mixture of water and methanol the structure in Figure 6a specifies acidic internal acid bridge form under mildly acidic conditions - evidenced by a significant increase in pH during the transition between the anionic and free acid forms (ie a Conversely, this same example also shows that the very similar structure in Figure 6b, designed to form specific non-acidic H, does not increase the pH of the transition between anionic and nonionic forms under these conditions.

ii) Minimum freedom of conformation between the supporting portions of H

The H-bridging acceptor moiety and the carboxyl serving as the H-bridging donor moiety should be realized in close proximity to each other by a structure which has the minimum conformational freedom. This limited conformational freedom can be achieved by using an appropriate ring structure. Molecular modeling and experimental work suggest that 4-element, 5-element and 6-element aliphatic rings are preferred for this purpose. Figure 7a shows representative 4-membered, 5-membered, and 6-membered aliphatic ring structures which allow only limited conformational freedom between the H-bridging moiety (OH of carboxylic acid) and the appropriately positioned H-bridging acceptor. Conversely, Figure 7b shows that a somewhat similar structure is unacceptable because its acyclic structure allows for excessive conformational freedom between H-bridging donor and Δ-bridging acceptor moieties. Example 3 describes experimental results showing that in a 1: 1 mixture of methanol and water, the structure with minimal conformational freedom between H-bridge moieties may provide significant increase in pKa value of the decarboxyl moiety, while similar structure with substantially larger rotational freedom between bridge moieties. deH does not provide a corresponding increase in the pKa value of the carboxyl portion - and thus is presumably not forming stable acid-specific internal H-bridge under these conditions.

iii) Carboxyl isolated from inductive effects of electron removal groups

The carboxylic acid which serves as the H-bridging moiety should be separated from any electron removal group by at least two, preferably three or more carbons. This avoids any excessive reduction in the carboxyl pKa value due to inductive effects from the electron removal groups. The molecular structures and their corresponding pKa values shown in Figure 8a illustrate how an increasing number of aliphatic carbons can progressively isolate a carboxyl from the pKa lowering effects of a phenyl group. Figure 8b shows a structure in which the inductive effects from the proximal amide fraction apparently cause an undue reduction in a pKa of the carboxylic acid moiety. Example 4 describes the experimental results which suggest that even with minimal conformational freedom between H-bridge donor and acceptor moieties, if there is inadequate isolation of carboxyl from inductive effects then the transition pH between anionic and free acid forms will be too low to be useful in pH selector. .

In addition to the above 3 essential properties, but at least one additional property selected from the following two properties, it is desirable to achieve the formation of an internal H-bridge in aqueous solution.iv) Lipophilic groups that partially protect the H-deposing site.

A major challenge in forming a solitary H-bridge in an aqueous environment is to preferably form this H-bridge in the presence of the large concentration of H-bridge donor and H-bridge acceptors comprising the surrounding water. Based on biochemical studies of enzyme catalytic sites and the inventor's extensive molecular modeling, he postulated that the desired intramolecular H-wall could be most favored if the H-bridge site was partially protected from water particles by the presence of lipophilic groups. In the context of the partial impediment from the solvent, Figure 9 shows two relevant structures. In Figure 9a the H-bridge location is completely open to the solvent. Conversely, in Figure 9b the corresponding H-point site is partially hindered by the solvent by an adjacent methylene on one side and an adjacent methylene on the other. Synthesis and titration studies, detailed in Example 5, give results suggesting that such partial impedance of the H-bridge site by the solvent favors the desired intramolecular H-bridge. V) Low-barrier H-bridge

The term "low barrier H bridge" is used in this context to mean noncovalent bond formed between the H bridge donor portion and the H bridge acceptor portion, where the pKa values of the two isolated portions are within approximately 2 pH units of each. one. It may be noted that this definition includes what may also be interpreted as an internal salt wherein hydrogen is closer to the acceptor portion than to the donor portion. Such low-stranded H-bonds are generally found to be exceptionally strong and thus may appreciably favor the desired intramolecular H-bridge in our pH selectors. Figures 10a and 10b show a variety of H-bridged moieties which are suitable for forming low barrier H-bridges into pH selectors. Figure 10c illustrates a number of representative pH deselector structures designed to form low-bridged H-bridges. Example 6 describes the synthesis and representative pH selector test (structures ie iii in Figure 10b), together with experimental evidence for the formation of the desired low barrier H-bridge in aqueous solution. Below is a sum of the properties which can be found for be suitable for an advanced pH selector component which is designed to form an acidic internal hydrogen bridge specifies:

a) contains an aliphatic ring structure selected from the group consisting of: 4-element rings, 5-element rings and 6-element rings;

b) contains a portion of carboxylic acid directly attached to the aliphatic ring structure;

c) the carboxylic acid moiety is separated from any electron removal group by at least two carbons;

d) contains H-bridge acceptor moiety selected from the group consisting of:

i) aliphatic ring structure part;

ii) bonding directly to the aliphatic ring structure; and

iii) bonding through an atom to the aliphatic forward structure;

e) the bridging acceptor portion of H has a structure which in its nonionic form does not serve as the H-bridging donor portion; and

f) the carboxylic acid portion and H-acceptor moiety are positioned in close proximity to each other and are correctly positioned and oriented such that they are compatible with specific acidic H-bridge formation.

c) Incorporate multiple pH selector components within each instrument as a new strategy to dramatically increase the specificity of the cancer instrument for tumors - without causing undue reduction in the effectiveness of the cancer instrument.

The two design strategies described in the preceding two sections, used alone or in combination, are suitable for generating pH selector structures which are effective for inserting into cell membranes at pH present in acidic areas of tumors, and thus providing good efficacy. However, such project strategies may provide only a modest advantage of the specificities of conventional cancer therapies, such as chlorambucil. In this regard, it may be appreciated that as the most frequent use of cancer therapies this has its precarious specificity which is generally low due to its many failures to cure patients. That is, current cancer asterapies are estimated to have specificities in the order of only about 2 to 8. This means that they are typically on the order of 2 to 8 times more harmful to cancers than to normal tissues. The inventor was keenly aware that a similar limited level of specificity and a low success-matching tax would probably contaminate cancer instrument therapy unless some effective strategy could be devised to substantially increase its tumor specificity. Thus, several years ago the inventor became involved in the search to devise some way to substantially increase the specificity of oncological instruments. This search led to the third and most essential strategy of the oncological instrument project, 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. The pKa of an acid, and opH of its aqueous solution are known, so the Henderson-Hasselbalch equation can be used to calculate the anionic, [H-], nonionic, [HA] ratio of the present acid in that solution. : (pH = pKa + Yog [A -] / [HA]).

A simple application of the Henderson-Hasselbalch equation suggests that if the pH in normal tissue interstitial space and the pH in acidic tumor areas differ by only 1.0 pH unit (ie, about pH 6.4 results in tumors and pH 7, 4 present in normal tissues) so the maximum theoretical specificity of a drug containing carboxyl could show for acidic areas of a tumor, relative to normal tissues, seems to be no more than 10. Differently stated, with only 1.0 depH differential between normal and tumor A simple application of the Henderson-Hasselbalch equation seems to suggest that the rate of entry of a conventional weak acid-containing drug into a cell in an acidic area of a tumor cannot be more than approximately 10 times higher than the rate of entry of that drug into a cell in a given cell. a normal tissue.

If this upper limit of approximately 10 were to apply to the specificity of the cancer instruments, then it is likely that in a precarious diagnostic diagnostic application of specificity it would generate a large fundoton signal from normal tissues that could drop much or all of the signal from any tumors. which was trying to detect - particularly for the case very small early stage detectors whose detection would provide the best prognosis for the patient. Likewise, in such a precarious level of therapeutic application of specificity would likely cause considerable patient toxicity, possibly to the extent of destroying the patient - which usually occurs with current chemotherapy which also has similar levels of poor specificity.

In order to overcome the problem of limited specificity, the inventor devised a new design strategy to dramatically increase the specificity of cancer instruments over the apparent theoretical maximum of 10 which seemed to be imposed by the small differential between normal and tumor tissue. Essentially, the strategy is to incorporate two or more pH selectors into a single oncological instrument, where the carboxyl decay pH selector is sufficiently distant (probably greater than approximately 5 angstroms) from the carboxyl of its neighboring pH selector that a carboxyl deionization does not affect significantly anionization of its neighboring carboxyl. Conversely, all pH selectors of the multi-selector oncology instrument must be sufficiently close to each other on the oncology instrument molecule that all pH selector components must be in their ionic form before the oncology instrument can come close and then break the surface. negatively charged exterior of the cell membrane in order to insert into the lipophilic interior, as required for this oncological instrument to be isolated in acidic areas. It is estimated that all carboxyls on the multi-selector pH instrument should no longer have approximately 15 to 20 angstroms of each other to adequately meet this latter requirement.

Figure 11a briefly illustrates the various anionic and nonionic forms of an oncological instrument containing two pH selector components, and Figure 11b similarly illustrates the various anionic and nonionic forms of an oncological instrument that contain three selector components. pH It should be appreciated that it is the concentration of the nonionic form which is of particular interest because, when designed correctly, it should be only this nonionic form of the oncological instrument which is able to efficiently contact and insert itself into cell membranes - with all other forms. which contains at least one anionic portion that is being repelled from the anionic surfaces of the cell by electrostatic forces.

When employing this design strategy of a pH multi selector, the predicted efficacy and specificity factors for a given instrument can be calculated using the pKa value for the carboxy portion of the pH selector used in this instrument and several of these pH selectors in this oncological instrument. In these calculations, the efficiency factor is the percentage of oncological instrument molecules which are in their nonionic form at pH6.4. This factor provides a measure of how efficiently the cancer instrument will insert cells into acidic areas of tumors. The specificity factor is the ratio: (percentage in nonionic form pH 6.4) divided by (percentage in nonionic form nopH 7.4). This factor provides a measure of the rate of cancer instrument entry rate in cells in acidic tumor areas (at pH 6.4) relative to the rate of cancer instrument entry in cells in normal tissues (at pH 7.4). For these calculations, the Henderson-Hasselbalch equation is first used to calculate the selected pH selector, which percentage will be in the non-ionic form at pH 6.4 (obtained in acidic areas) and at pH 7.4 (present in normal tissues). Then a binomial expansion is used to calculate the percentage of oncological instrument molecules that have all their pH selector components in the ion-form.

Figure 12 shows sketches of calculated efficacy and specificity factors for a) 1-pH selector compositions, b) 2-pH selector oncological instruments, c) 3-pH selector oncological instruments, and d) 4-pH selector oncological instruments - as well as a function of the pKa values of its pH deselector components. These calculated efficacy and specificity factors demonstrate that compositions containing a single carboxyl moiety, such as chlorambucil, inherently have somewhat poor specificity for elective entry into cells in acidic areas of tumors (the specificity factor is always less than 10). However by incorporating two or more pH selectors into a single cancer instrument, the calculations suggest that with practical efficacy factors (about 5% of the non-ionic cancer instrument at pH 6.4) and appropriate pKa value adjustment (see section above specific internal acid H point), the specificity factors of cancer instruments can be increased many times as much as possible for single carboxyl compounds. In particular, with pKa appropriate the specificity factor may be more than 50 for cancer instruments containing 2 pH selectors, and more than 200 for cancer instruments containing 3 pH selectors, and more than 900 for cancer instruments containing 4 pH selectors. .

As can be seen from Figure 12, there is an inverse relationship between the effectiveness factor and the specificity factor. Thus, with increasing pH selector pKa the efficacy factor increases while the specificity factor decreases. Since reasonable efficacy and high specificity are both desired in cancer therapy, it is useful to consider that pK value probably provides a reasonably ideal balance between efficacy and specificity. Based on the values calculated in Figure 12b, it appears that a pH selector with a pKa of 5.9 should provide a desirable balance between efficacy (6%) and specificity (60) for oncological instruments containing 2 pH selectors. Additionally, values calculated in Figure 12c suggest that a depH selector with a pKa of 6.2 should provide a desirable balance between efficacy (5.8%) and specificity (277) for cancer instruments containing 3 pH selectors.

Further, values calculated in Figure 12d suggest that a pH selector with a pKa of 6.4 should provide a desirable balance between efficacy (6.3%) and specificity (915) for cancer instruments containing 4 pH selectors.

In this context, it should be appreciated that incorporation of a specific acid internal H-bridge into a pH selector, as described in the preceding section, provides appropriate means for adjusting the pH selector pKa value to an appropriate scale to obtain such dramatically improved specificities in these instruments. multi-selector oncologic studies.

The discussion and background calculations focused on optimizing the pH selector pKa value (via incorporation of a specific acid internal H-bridge) from an oncological instrument as a means of achieving a desirable balance between efficacy and specificity (Figures 11 and 12). However, it should also be appreciated that increasing the partition coefficient (i.e., alipophilicity) of the oncological instrument should have a similar effect on balancing the efficacy and specificity of depH multiselector oncological instruments. This is because, as shown in Figure 4, such increase in lipophilicity is also desirable to increase the pH at which the oncological instrument can fit into the phaselipophilic, such as the cell membrane. Thus, in this strategy of pH multi-selector to increase tumor cancer instrument specificity, the balance between efficacy and specificity can be adjusted by adjusting the lipophilicity of the cancer instrument, or by adjusting the pH selector pKa (via incorporation of a specific internal acid H bridge ), or by a combination to adjust the lipophilicity of an oncological instrument and adjust the pKa of the pH selector components.

At this point it is appropriate to ask: do these calculated values of specificity for multi-selector pH instruments truly reflect the oncology instrument's ability to selectively enter the lipophilic phase (such as octanol or cell membrane) at the pH obtained in acidic areas of a tumor (pH 6.4), while largely preventing entry into a lipophilic phase in the pH of normal tissues (pH 7.4)?

To address this question, a representative composition containing two depH selector components shown in Figure 13a has been synthesized. After synthesis, this composition was partitioned between n-octanol and aqueous buffers ranging from pH 5.6 to pH 8.0. The results of this partition experiment are outlined in Figure 13b. These results show that at pH 6.4 (obtained from the tumors) approximately half of this composition partitioned into the octanol phase, while at pH 7.0 only about 2% partitioned into the octanol phase, and at pH 7.2 none of the composition was detected at the octanol phase. octanol.

As suggested by the calculated values outlined in Figure 12, these partition results also suggest that most of the composition containing two pH selector components may exist in their nonionic form (ie, octanol soluble form) at pH which is present in acidic areas of tumors, and so that the composition may change almost completely to its kanionic form (ie, soluble in buffer) at the pH present in normal tissues. This provides an experimental basis for the new strategy of incorporating multiple pH selector components into an oncology instrument as a means to dramatically increase the specificity of an oncological instrument for acidic tumor areas.

A variety of representative structures of the multi-selector pH instrument will be illustrated later in section A.3 in this context.

2. Load component

a) Structural Requirements

The charge component is a structural component of an oncological instrument that serves to incorporate a radioactive isotope whose emission is effective to enhance the presence of the oncological instrument or to destroy cells. The loading component must meet the following three design requirements.

(i) The charge component in its precursor form shall be effective for immediately and efficiently incorporating, with minimal manipulation, its selected radioactive isotope.

ii) The radioactive isotope that is bound to the charge component in its final form must remain highly bound during the course of the diagnostic procedure or through the course of the therapeutic process where emissions from the radioactive isotopes are tumor-destroying cells.

(iii) The charge component in its final form, including a bound radioactive isotope, must be sufficiently small and of such a composition as to have no undue impact on the pH-dependent hydrophilicity / lipophilicity properties of the cancer instrument. It is determined differently if the final shape of the charge component contributes to excessive hydrophilicity, it may suppress the entry of the nonionic shape of the cancer instrument into cells in acidic areas of tumors - thereby reducing efficacy. Conversely, if the final form of the charge component contributes to excessive lipophilicity, it may cause improper isolation in normal - desemode tissues, reducing specificity.

Based on these requirements for the charge component, it appears that radiohalogens selected from F, Br, I and At constitute the best types of radioactive isotopes, and that a vinyl group or simple unsaturated aromatic ring or simple aromatic ring will best bind radiohalogen. . Figure 14 illustrates a number of perspective components of the charge component in both selected precursor forms and their final forms containing radioactive isotope ready for diagnostic or therapeutic use.

b) Precursor and Final FormsWhen the selected radioactive isotope which is to be linked to a short-lived oncological instrument, it is often desirable to make, ship, and store the oncological instrument in its precursor form, and then to add the isotopic radioactive charge. a well before the transfer of the instrument within the individual to be diagnosed later. While a large number of different structures have been restated in the field of nuclear medicine suitable for linking a wide variety of radioactive isotopes, many of these structures are unsuitable for use in oncological instruments. Figure 14 shows some selected charge components, both in their precursor forms and in their final forms, which meet the particular requirements for use in oncological instruments. Figure 15 shows synthetic schemes for preparing two of such charge components in their precursor forms.

These syntheses use key reactions to add a group of trialkyl tin which have been described by: Thibonnet, et al., Tetrahedron Letters, Vol.39, page 4277 (1998); Miyake & Yamamura, Chemistry Letters, pages 981-984 (1989); Marshall & Bourbeau, Tetrahedron Letters, Vol.44, pages 1087-1089 (2003); and Corriu, Geng, & Moreau, J. Org. Chem. Vol. 58, page 1443 (1993). Figure 15 also illustrates a simple procedure for converting the precursor forms to their final forms containing radioactive isotope, previously described by Kalutsky, page 96 of chapter 4 titled: Radiohalogensfor Radioimmunotherapy, in the book: Radioimmunotherapy of Cancer, Ed. By Abrams and Fritzberg, Pub. By Mareei Dekker, Inc. (2000). Procedures for preparing other suitable charge components and incorporating the isotope radioactive to the final form are described in: Zalutsky et al., Proc. Nat. Acad. Know. USA, Vol. 86, pages 7149-7153 (1989); and Vaidyanathan & Zalutsky, Nature Protocols, Vol. 1, pages 1655-1661 (2006).

c) Radioactive Isotope Charge Selection

It should be noted that it is the radioactive isotope attached to the cancer instrument that determines the application of this cancer instrument. If the bound radioactive isotope emits a signal that is immediately detectable outside the body then this cancer instrument may be suitable for detecting tumors containing acidic areas. Conversely, if the radioactive isotope has an emission that is effective in destroying cells then this instrument may be suitable for the treatment of tumors that contain acidic areas. In addition, if the instrument contains a radioactive isotope, such as Iodine-131, which emits a signal that is immediately detectable outside the body (eg, gamma ray), it has an emission that is effective in destroying cells (eg, beta particle) so that this instrument can be suitable both for the diagnosis and treatment of tumors containing acidic areas,

i) for tumor detection

In the oncological instruments used for tumor detection one has considerable latitude in the selection of two radioactive optics which is to generate the appropriate signal for detection outside the individual. Several radioactive isotopes with favorable properties for diagnostic application include:

Radioactive Isotope Half LifeFlower-18 1.8 hourBromo-75 1.7 hourBromo-7 6 16 hoursBromo-77 2.4 daysIodine-123 13 hoursIodine-124 4 daysIodine-125 60 daysIodine-126 13 daysIodine-131 8 days

ii) for treatment of tumors

Successful treatment of a tumor faces two challenges. One challenge is to completely destroy all treatment-sensitive rapidly dividing tumor cells with near-normal pH near tumor vessel capillaries. The other severe challenge is to completely destroy all treatment-resistant inactive tumor cells in acidic areas of the tumor. Therapeutic oncological instruments were initially developed solely as a means to destroy inactivating resistant cells in acidic areas of tumors. This was because cancer instruments were designed to be isolated only in acidic areas of tumors, and it was assumed that such cancer instruments would have to be used in conjunction with more conventional cancer therapies, such as radiation or chemotherapy, where conventional therapies would work by destroying tumor cells. treatment-sensitive rapid-division cells in more neutral areas of tumors closer to the vasoscapillary - because such areas could be more devoid of cancer instruments.

More recently, the inventor has developed a dual radioactive isotope strategy in which single cancer instruments can be used to exterminate the entire tumor, thereby preventing the need for co-treatment with conventional maistoxic and less specific cancer therapies. This dual radioactive isotope strategy involves using at least two effective cancer instrument formulations to destroy cells.

The oncological instrument formulation used to destroy inactivation resistant resistant tumor cells must contain a radioactive isotope that emits high linear energy transfer in order to destroy all inactive tumor resistant tumor cells that is positioned within or near such a therapeutic conductive cancer conduction instrument. radioactive isotope.

One option for meeting this demand requirement is to use a radioactive isotope that emits an alpha particle that releases a vast amount of energy over a very short distance (some cell diameters), such that the energy released is highly effective in destroying even cells that are highly resistant to most of the radiation. The radioactive isotope for this purpose appears to be oradiohalogen, Astatine 211. This radioactive isotope is generated from natural Bismuth 209 in a medium energy cyclotron equipped with an alpha particle beam. Astatine-211 has a half life of 7.2 hours and Emits two alpha particles of 5.87 and 7.45 million electron volts, which have been shown to devastate cells within approximately 50 to 80 microns in length of the emitted alpha particles (some cell diameters). Only a few such alpha emissions can destroy even the most radiation-resistant inactive tumor cells.

Another class of radioactive isotopes that emit high linear energy transfer radiation is the one that emits Augar and / or Coster-Kronig electrons. Such radioactive isotopes, if placed within the cell to be destroyed, can cause devastating damage to the DNA cells and this. form are extremely cytotoxic when located in close proximity to the cell nucleus. Radioactive isotopes of this type which are particularly suitable for oncological therapeutic instruments include: Bromo-77 (57 hour half life) and Iodo-123 (13 hour half life).

The oncological instrument formulation used to destroy sensitive rapid-dividing tumor cells closest to capillaries should contain one or more radioactive isotopes that emit particles that have sufficient energy along their pathway to destroy sensitive rapid-dividing tumor cells. Most importantly, those particles emitted must have a sufficiently long path length to be effective against both cells and several hundred microns from where the cancer instrument is isolated - allowing them to destroy rapidly dividing tumor cells sensitive to treatment in areas. Neutral tumors, which can have up to several hundred microns from acidic areas where cancer instruments are isolated. Beta-emitting radioactive isotopes best meet this requirement. Where there are a number of beta-emitting radioactive isotopes that can be used in this dual isotope therapy strategy, the following radiohalogens have favorable properties for this application, including average path lengths greater than 600 microns.

<table> table see original document page 58 </column> </row> <table>

Compared to treatment where an oncological instrument is used only to destroy inactive cells in acidic areas of tumors, and is combined with conventional cancer therapies effective to destroy rapidly dividing tumor cells in other areas of tumors that have higher HP, This new strategy of radioactive isotope cancer instrument therapy in which cancer instruments are used alone to destroy the entire tumor will afford a simpler, less expensive, much less toxic, and much more effective treatment.

3. Cancer instrument structures a) structural requirements

Each cancer instrument must contain two or more pH selector components. These "two or more" requirements are essential to achieve a substantially higher specificity (at least about 20, and preferably over 40) than is typically provided by current cancer therapies (specificities estimated to be typically in the range of about 2 to 8). ) In addition, each cancer instrument must have a structure such as at pH 7.4 this is almost completely an anionic hydrophilic form, but at pH 6.4 a significant portion (preferably 1% or more) transfers an effective non-ionic lipophilic form. to be isolated in acidic areas of tumors. Each cancer instrument must also contain a charge component that is effective in binding a radioactive isotope, or which contains an appropriate radioactive isotope to report the presence of the oncological instrument and / or appropriate for destructive cells.

The following three sections describe the instrument with 2, 3, and 4 pH selectors. With regard to such oncological instrument structures, it should be valued by any selected component combination that will generally require the optimization of the R groups in order to achieve adequate efficacy and a desirable balance between efficacy and specificity. Procedures for such R group optimizations are described. and illustrated later in this Section B and Figures and Examples in relation to this section.

b) Cancer instruments with 2 pH selectors

These cancer instruments have the simplest structures and are generally easier to synthesize. They typically have potentially variable specificity factors ranging from about 20 to about 80. Figure 16 illustrates a variety of two representative pH-selective oncological instruments that meet key structural requirements for oncological instruments. Figure 16a illustrates oncological instruments containing two simple pH selectors. Figure 16 illustrates cancer instruments containing two advanced pH selectors. Figure 16c illustrates oncological instruments containing two advanced pH selectors designed to form low barrier H bonds.

c) Oncological instrument with 3 pH selectors

Cancer instruments containing 3 depH selectors are more complex than the 2 pH selector cancer instruments described above, and are generally more challenging to synthesize. However, the 3-pH selector oncological instruments have the merit of high-specificity-valued terracotta potentials, potentially ranging from approximately 100 to approximately 500. Figure 17 illustrates a variety of oncological instruments from three representative pH-selectors that meet the key structural requirements for oncological instruments. Figure 17a illustrates oncological instruments containing three advanced pH selectors and Figure 17b illustrates oncological instruments containing three advanced pH selectors designed to form low barrier H bonds.

d) Cancer instruments with 4 pH selectors

Cancer instruments that contain 4 pH selectors are even more complex than the 3 pH selector oncological instruments described above, and are generally even more challenging to synthesize. However, depH 4-selector cancer instruments have the potential for even higher specificity factors. , varying potential from approximately 400 to approximately 2500. Figure 18 illustrates two representative oncological instruments that meet the key structural requirements for oncological instruments and contain four advanced pH selectors designed to form a low-bar H-bond.

B. Synthesis, Testing and Optimization of Components and Oncological Instruments

1. Preparation of structures containing representative pH selectors

Before beginning the development of any new type of cancer instrument, it is generally desirable first to prepare and evaluate the properties of a variety of pH selector types, as well as variations within a type in which variations of the R group may share a wide range of lipophilic substances with the structures. .

a) Simple pH selectors

Figure 19 illustrates a representative synthetic route for oncological instrument structures containing simple pH selectors, with lipophilicity adjusted by varying from the R group.

b) Advanced pH Selectors

Figure 20 illustrates a synthetic route for structures containing an advanced pH selector type, with lipophilicity adjusted by varying from the R1 group.

c) Advanced pH selectors designed to form

a low barrier bridge H

Figures 21 and 22 illustrate synthetic routes to structures containing a number of types of advanced pH selectors designed to form a low barrier H bridge.

Figure 21 shows amine ester and ketone ester intermediates useful in the preparation of pH selectors and in the preparation of oncological instruments. Synthetic procedures for making intermediate key detal core components have been reported from the following sources: 1) Goldman, Jacobsen, and Torssell, Synthesis in the CamphorSeries. Alkylation of Quinones with Cycloalkyl Radicals.Attempted Synthesis of Lagopodin A and Desoxyhelicobasidin.Acta Chemica Scandinavica B 28 (1974) 4 92 -500; and, 2) Brian Thomas Connell, Synthesis and Evaluation of a NewCamphor-Derived Lactate as a General Chiral Auxiliary forthe Asymmetric Diels-Alder and Aldol Reactions. The paper was submitted to the Department of Chemistry, University of Rochester, Rochester, New York (1995).

Figure 22 below illustrates representative synthetic routes for converting several such intermediates to the various pH selectors designed to form a specific low acid barrier H bridge.

2. Testing pH Selectors Simple titration assays provide useful information on the pH solubility properties dependent on pH selector structures, as well as their pKa values. Example 7 describes the titration procedures that were used to study pH selector components. Figure 23 shows representative results from a variety of previous titrations.

Figure 23a shows a conventional titration curve, where titration was carried out in methanol / water, 1: 1 by volume, for a single carboxylic acid (butyric acid) and an advanced pH selector (a camphoric acid amide derivative). . At the same time the same titration results are outlined in the most informative form of the first derivative form.

Figure 23b shows the conventional titration curves where the titration was carried in water and each species was present at the concentration of 33 milliMolar. Titration Results are shown by three reported 2-selector (but with iodine rather than radioactive iodine) instruments. which vary only in their R group.

Titration results are also shown for a structure of three reported pH selectors where group R is methyl.

Figure 23c shows a titration curve, sketched as the first derivative, for an advanced pH selector designed to form a low barrier H bridge. This titration was carried in water and the depH selector, comprising an N-oxide / acid structure derived from camphoric acid, was present at a concentration of 5milliMolar.

Figure 24 shows experimentally determined pKa values for structures of three pH selectors, including: an amide / advanced acid pH selector derived from camphoric acid; an advanced N-oxide / acid pH selector designed to form a low barrier H-bridge derived from lsonipocotic acid; and, an advanced pHN-oxide / acid selector designed to form a low barrier H-bridge derived from camphoric acid (also shown as structure i of figure 10c, and the structure shown in figure 23c).

3. Preparation of representative oncological and conversion instruments to final form

a) Simple pH selector oncological instruments Figure 19 illustrates a synthetic route for preparing a representative oncological instrument that contains a simple pH selector.

b) Cancer instruments with advanced pH selectors

Figure 25 illustrates synthetic schemes for representative oncological instruments containing advanced pH selectors. Figure 25a shows an oncological instrument where its two pH selectors are joined by a di-acyl hydrazide structure. Figure 25b shows an oncological instrument where its two pH selectors are joined by a dimide structure.

c) Oncological instruments with advanced pH selectors designed to form a low barrier H bore.

Figure 26 illustrates synthetic schemes for representative oncological instruments containing advanced pH selectors designed to form a low barrier H-wall.

Figure 26a shows a synthetic scheme for a 2-selector 2-pH selector oncological instrument in which a single N-oxide group such as the H-bridge acceptor group for two carboxylic acid H-donor donor clusters. Figure 26b shows a synthetic scheme for a 2-pH selector oncological instrument, in which both the hydrazine group nitrogen is suitable and the H-bridge acceptor group for two carboxylic acid H-donor donor clusters. Figure 26 shows a synthetic scheme for a 2-pH selector oncological instrument, in which cyanomethylamine grouping is accepted as the H-bridge acceptor groups for the carboxylic acid H-bridge donor groups. Figure 26d shows a synthetic scheme for an instrument of 4 pH selectors, where N-oxide coupling is suitable as the H-acceptor acceptor group for two carboxylic acid H-bridge donor clusters.

It should be noted that in the foregoing syntheses oncological instruments containing advanced pH selectors designed to form a low barrier H bridge when a ketone / intermediate ester such as b, d, ef or Figure 21 structures is used to reduce the alkylation of a where the amine / ester group is useful for ester grouping, such as the structures a, c, and e of Figure 21, generally the 2 pH selector producer comprises 2 isomers, where both pH selector components are useful in the it concerns amine and ester, and the other product wherein one of the pH selector components is useful and one of the pH selector components is trans with respect to the amine and ester groups. In such cases it is desirable to purify the desired cyclic product, and reject the useful / trans product. One way of doing this is to explore the fact that the trans pH selector component cannot form a specific low acid barrier H-wall. Accordingly, pKa thereof typically approaches 5.0 in an aqueous solution. In contrast, the useful pH selector component can generally form the desired internal specific acid low-barrier H-bridge, which typically feeds the contained carboxyl group pKado to approximately 6 µM. , 0 or higher. As a consequence, after ester groups have been cleaved to give carboxy groups, the desired form should generally be immediately separable from the form separated by an organic phase such as Tetrahydrofuran, t-Butylmethyl, or Dichloromethane. , and a buffered 6.0 round aqueous phase. In such a dividing system the desired useful form should generally preferably be divided within the organic phase, while the undesirable formable / trans form should preferably be divided within the aqueous buffer phase.

The same basic separation strategy can also be used to separate the desired all-cis-form 4-pH selector oncological instrument from Figure 26 from multiple forms containing at least one trans-pH selector.

4. Test and optimization of oncological instruments.

The following sources of information are appropriate as a starting point for designing and developing an effective and specific cancer instrument. The efficacy and specificity graphs in Figure 12 provide the orientation and desirable values of pKa for the pH selector components as a function of how many pH selector components the selected oncological instrument is to contain. Figure 4 provides guidance on the impact of the division coefficient on the nonionic form of the oncological instrument - where the division coefficient is typically adjusted by altering the lipophilicity of the R groups of the pH selector components. In addition, synthesis of various types of prospective pH selectors and R group variants thereof, as described in Section B above, and in subsequent titration studies of those pH selectors, as described in Section B.2 above, valuable information. provided for proper selection of pH selector components for a prospective oncological instrument. Finally, Figure 14 illustrates a number of charge components suitable for incorporation within oncological instruments.

While background information on the decomposing parts provides a valuable basis for the design and development of an effective and specific cancer instrument, however, since those parts are assembled within a prospective cancer instrument in its radioisotope-containing form, in order to achieve adequate and A desirable balance between efficacy and specificity is generally necessary to optimize this structure of oncological instrument.

a) Iterative process of optimization: synthesis and test in cultured cells

A preferred optimization process involves the following:

i) Prepare the set of oncological instruments that vary in lipophilicity The optimization process is started by preparing a set of prospective oncological instrument structures incorporating the selected components, in which oncological instruments of the set exhibit a wide range of lipophilicities due to their R1 and R2 groups. Each joint cancer instrument must also contain a suitable radioactive isotope, such as Iodine-131, which has easy detection and quantification capabilities in biological systems.

ii) Evaluate entry into cultured cells at pH 6.4 and pH 7.4

Each of those prospective instrument structures must be tested in a relatively simple biological system in which the prospective instrument is exposed to the main biological environments and structures that will be found in a living individual. As described in example 8, a preferred biological system for such an initial test comprises cultured mammalian cells in pH 7.4 buffered medium to emulate normal tissues, and pH 6.4 to emulate acidic areas of tumors. Different cells that have been cultured are exposed for one hour to a given cancer instrument. In a culture cavity the oncological instrument is in the midst of pH 6.4. In another culture cavity the oncological instrument is in the midst of pH 7,4. After being incubated for 1 hour at 37 degrees Celsius, the medium containing the cancer instrument is removed and the cells are washed thoroughly at the same pH, and then the cell-retained radioactive isotope is counted to provide a measure of the relative amount of cancer instrument that was isolated under each. one of two pH conditions.

The preferred oncological instrument structures are those that are maximally isolated by cells at pH 6.4, but minimally isolated by cells at pH 7.4.

The first application of the above two optimization steps should generally be appropriate to identify a reasonably narrow range of oncological instrument lipophilic substances that provide acceptable efficacy (relative to pH 6 isolated) and specificity (relative to pH 6 isolated) , 4 / amount isolated at pH 7.4) values. Alternatively, such an initial test may instead be appropriate to indicate that the selected cancer instrument components are inadequate and different pH selector components and / or a different charge component should be used in cancer instrument structures in one perspective.

iii) Repeat above steps as needed

Additional optimization cycles within progressively narrower structural rates may lead to the further improvement of oncological instrument activity.

b) Evaluate disposition in normal mice

The above relatively fast, simple cell culture assays provide quantitative results, and are favorable for initial testing of a substantial number of oncological instrument structures in perspective. However, this system of initial projection of cell culture should not be appreciated, but not perfectly true to the true complexity of a living individual. Accordingly, it is desirable to then take a reasonable number of the most promising forward-looking cancer instrument frameworks identified in the iterative optimization process above and test the same live mammals. As a first step in such an animal test, it is useful to test these promising instrument structures and normal mice (pretreated to ensure that their urine is slightly basic; see Section C.7 below) as described in Example 9. Taistens in Normal Mice allow a discard any of the oncological instruments that have been found to exhibit an excessive affinity for normal ones.

c) Evaluate disposition in tumor-bearing mice

When the above relatively simple tests in normal tumor-free mice allow those cancer instruments that have excessive affinity for normal tissues to be discarded, the most decisive test for a prospective cancer instrument structure is to test this in tumor-bearing mice, as described in Example 10. Momentarily , this requires injection (preferably intravenously) of each of the Iodine-131-containing cancer instruments into a variety of tumor-bearing mice, and then for an appropriate period of time (about 5 to 24 hours) to allow normal excretion by the kidneys to defrost. of the administered dose that was not isolated in tissue / or mouse tumors. Someone then executes the mice and extirpates the major organs and any of the obvious tumors, then is dosed by the radiation emissions from the extirpated organs, tumors, and the remaining carcass.

Cancer instruments will work best when two auxiliary methods are used. Such a method involves pretreatment of mice to prevent the absorption of oncological instrument within the cell lines of the proximal ducts of the kidneys. This reabsorption is blocked by making the urine slightly basic, as described in section C.7 below. The other method involves pretreatment of the mice to further increase the acidity (reduce the pH) in hypoxic / acidic areas of the same. Three pretreatments are described for this purpose later in this section C.5. At least one, and preferably a combination of two or three such pre-treatments should be employed in order to adjust the tumor microenvironment to be better adapted to the effective and specific oncological instrument activity.

d) Preclinical and clinical test

Test procedures containing radioactive substances in living animals, including humans, are known in the field of nuclear medicine, and particularly in the field of radioimmunotherapy. Such known methods can readily be adapted to test oncological instruments by incorporating the methods of using oncological instruments described in section C. below.

C. Methods of Using Cancer Instrument

1. Diagnostic Method

Due to the fact that oncological instruments benefit from the acidity that is an almost universal feature of oncology, oncological instruments should be effective in detecting most or all types of oncology with sizes ranging from almost microscopic to very large. The next method of using cancer instruments to detect tumors is suitable for many research applications, as well as for veterinary medicine and human medicine. The diagnostic method usually includes, but is not limited to, the following steps:

Step 1. Provide a diagnostic oncological instrument in its final form - either by contacting the precursor form of the cancer instrument with a suitable radioactive isotope that is effective in reporting its presence within a tumor to a detector outside the living individual, or by obtaining directly from of a supplier of the final form of a diagnostic oncological instrument already containing such a radioactive isotope.

Step 2. Transfer this diagnostic oncological instrument within the individual - typically by intravenous injection.

Step 3. Allow adequate time for the cancer instrument to be isolated in acidic areas from any tumor that may be present (for example, from approximately 10 minutes to approximately 50 minutes). Considerably increased sensitivity can be obtained by waiting for additional time (hours) for excretion through the kidneys of most of that fraction of cancer instrument data that has not been isolated in acidic areas of tumors. Waiting for this additional time will suit the extremely lower background from normal tissues and allow even very small tumors to be detected. In this respect, in the course of a few hours most of the injected dose of cancer instrument should be excreted by the kidneys, with significant retention of the cancer instrument occurring only if one or more tumors are present. It is anticipated that typically in individuals who do not have tumors most of the cancer instrument will be excreted in less than 24 hours, and probably in less than approximately 4 hours. The uninsulated oncological instrument excretion rate may be increased by increasing the fluid intake of the individual, particularly if such fluid contains a diuretic.

Step 4. The final step in the diagnostic method is to scan the individual with appropriate equipment to detect emission from the radioactive isotope decomposing component of the oncology instrument to assess whether the significant oncological instrument has been isolated in one or more tumors. With modern imaging equipment, such as gamma ray scanners and PET scanners, tumors should appear as an evident isotopic radius spot at the site of each tumor.2. Therapeutic method

Due to the fact that cancer instruments derive the acidity that is an almost universal feature of cancer, cancer instruments should be effective in treating most or all types of tumors with sizes ranging from close to microscopic to very large. The following methods of using cancer instruments to treat tumors are suitable for many research applications as well as for veterinary medicine and human medicine. Therapeutic methods generally include, but are not limited to the following.

a) Simple radioisotope method

If an individual is found to have an ear tumors, those tumors may be treated with a therapeutic cancer instrument that contains an isotope radioactive effective in destroying cells. The therapeutic method generally includes, but is not limited to, the following two steps:

Step 1. Provide a final therapeutic oncological instrument that contains an effective isotope radioactive to destroy cells. This can be accomplished by contacting the precursor form of the oncological instrument with a suitable radioactive isotope, or by direct obtaining from a supplier of the final form of the therapeutic cancer instrument that already contains radioactive talisotope. Step 2. Deliver this therapeutic oncological instrument within the individual - typically by intravenous injection.

In this therapeutic method the radioactive isotope effective for destroying cells can be anyone whose emission has high linear energy transfer, such as an alpha particle, or one that emits a particle. The particular virtue of using a radioactive isotope that has a linear energy transfer emission, such as alpha emitting the Astatine-211 isotope radioactive, which is considerably more effective than beta-emission radioactive isotopes to destroy inactive cells in acidic areas of tumors. With respect to the use of oncological instruments containing such radioactive isotopes, their emissions generally have a very short path length (for example, 80 microns or less). Thus, it may be necessary to use such cancer instruments in combination with conventional cancer therapies to also ensure the destruction of rapidly dividing tumor cells in areas of a more normal pH near capillaries. In addition, it should be estimated that the preferred alpha emitter, 211, have a short half life (7 hours) and so emitting this radioactive isotope, or an oncological instrument containing this radioactive isotope, a substantial distance from where it is generated may be a problem. Still further, At-211 must be generated in a high capacity cyclotron with an alpha beam potential, and at present there are only relatively few places where such equipment is available.

An alternative simple radioactive isotope method involves providing a therapeutic oncological instrument containing a radioactive isotope of beta emission. While beta emissions are significantly less effective than alpha emissions in destroying native cells in acidic areas of tumors, this poor efficacy of beta emissions can be offset by the use of a much higher dose. While this much larger dose will compromise specificity in some way, however, it has the potential to provide acceptable therapeutic results because of the exceptional high level of specificity for tumors achievable by cancer instruments. In this respect, the beta emitter, Iodine-131, has several useful properties: it is readily available in large quantities at moderate price; Because of its relatively long half life (8 days) it can be shipped and stored for reasonable time periods before use; and while this has only a moderate mean path length (approximately 900 microns), which is nevertheless sufficient to destroy cells throughout the tumor, including rapidly dividing tumor cells at the nearest pH neutral regions near the capillaries - which will be relatively devoid of isolated oncological instruments.

b) Double radioactive isotope method As noted earlier in this article, radioactive isotopes that have high linear energy transfer emissions are optimal for the destruction of inactive tumor resistant cells in detrimental acid areas, but the short path length of such emissions makes an oncological instrument which contain such especially ineffective radioactive isotopes to destroy rapidly dividing cells in areas distant from the point of origin of tumors near capillary vessels where the pH is closer to neutral - because such areas will be relatively devoid of an isolated oncological instrument.

Conversely, an oncological instrument containing a beta-emitting radioactive isotope is relatively ineffective against tumor radiation-resistant inactive cells, but that oncological instrument containing the beta-emitting radioactive isotope may be quite effective in destroying radiation-sensitive fast-dividing cells in Tumor areas where pH is closer to neutrality - even when this cancer instrument is only present in the acidic areas of the tumor. This is because the longer course length of beta particles allows them to reach and destroy those most sensitive tumor cells.

Accordingly, a preferred method for treating tumors is used as a combination of two therapeutic cancer instruments, where an instrument contains a radioactive isotope which has a high linear energy transfer emission (preferably selected from Astatine-211, Bromo-77 and Iodo-123). ) to destroy inactive cells resistant to proximal radiation in acidic tumor areas, and the other 5 cancer instrument contains a beta-emitting radioactive isotope to destroy radiation-sensitive rapidly dividing cells at high pH regions near tumor capillaries, which are relatively devoid of cancer instrument isolated.

3. Comprehensive Method for Tumor Detection and Treatment

Cancer instruments offer the highly desirable properties because they are capable of both detecting and treating most or all of the types and sizes, ranging from almost microscopic to very large. For this reason, therapeutic and oncological diagnostic instruments may be virtually identical - but typically only in the contained radioactive isotope - usually if a provided oncological instrument structure is effective in detecting a tumor, then the same oncological instrument structure, but typically with a radioactive isotope. differently, it should also be effective in treating this same tumor. These special properties of cancer instruments facilitate a comprehensive method for detecting tumors in living individuals, followed by treating any of the tumors thus detected. This comprehensive method is suitable for both veterinary medicine and human medicine. This includes, but is not linked to, the following steps. A) Detecting tumors

Step 1 The first step provides a diagnostic diagnostic instrument in its final form that contains a radioactive isotope that is effective for reporting its presence within a tumor to a detector outside the living individual.

Step 2. The next step is to deliver this diagnostic cancer instrument within the individual - typically by intravenous injection.

Step 3. The subsequent step is to wait for a suitable period of time for the oncological instrument to be isolated in acidic areas of any of the tumors that may be present. This step may also include safeguarding the additional time for excretion through the kidneys of most of that fraction of the cancer instrument dose that have not been isolated in acidic areas of tumors. During this time the individual may also be provided with a particular fluid, a diuretic-containing fluid, to increase the excretion of that fraction of the oncological instrument dose that has not been isolated in acidic areas of tumors.

Step 4 The last step in the detection process is to scan the individual with appropriate equipment to detect the emission of the radioactive isotope from the cancer instrument in order to assess whether the significant cancer instrument has been isolated in one or more tumors. With modern imaging equipment such as gamma scan and PET scan, tumors should appear as a clear spot of radioisotopic light on each tumor. When one or more tumors are detected in step 4, one then proceeds to treat detected tumors. .

b) Treating detected tumors

Step 5. To treat the detected tumor, one or

Several therapeutic cancer instruments have been provided. While this may be a single oncological instrument containing a radioactive isotope of beta emission, it is generally preferred to provide two or more oncological instruments, where one contains an isotope radioactive which emits high energy transfer radiation, and the other contains a beta-emitting radioactive isotope.

Step 6. One or more provided oncological instruments are transferred into the individual usually by intravenous injection.

4. Strategy for Coping with Micro Metastases When a tumor reaches a substantial size (such as one centimeter or larger) it usually begins to be metastasized, where single tumor cells or very small aggregates of tumor cells are released from the original tumor, and those released cells can form colonies at distant locations in the body. These cell colonies, called micro-metastases, can then grow into original novel tumors. The difficulty of these presented for the therapeutic method of cancer instrument refers to the time period between the formation of the micro-metastases and the time taken by such metastases to grow to a size (approximately 1 millimeter in diameter) sufficient to generate their own acidic areas. Submillimeter originals cannot typically be detected or destroyed by oncological instruments. Thus, while the original tumor containing acidic areas can be detected and extinguished by cancer instruments, it is expected that any original micro-metastases less than approximately 1 millimeter in diameter will survive treatment of cancer instruments and eventually lead to a return of symptoms - although such a return symptoms may not occur for several years after the initial treatment of the cancer instrument.

A strategy for solving these demicro-metastasis problems is defined as waiting for a long time after initial oncological instrument therapy (steps 5 and 6 in Section 3 above) sufficient for any micro-metastases that might be present and have survived the initial treatment, which grew to a size where they developed acidic areas (typically approximately 1 millimeter in diameter). Conversely, those original micro-metastases should not be allowed to grow to a much larger size (probably around 1 centimeter in diameter) where they too start to reproduce by metastasis. Since the micro-metastases that could escape the first instrument treatment are in this appropriate range of size (large enough to contain acidic areas, but not too large to have started metastatic reproduction), again perform the detection of the cancer instrument and the treatment process (repeat steps 1 through 6 in section 3 above).

A complication of the above strategy is defined by the display of tumors over a wide range of rate of decrease, and thus it is difficult to predict how long it will take for any of the micro-metastases to escape the first treatment to reach a size containing acidic regions. Consequently, the prudent course is defined by repeating diagnostic steps 1 through 4 above at appropriate intervals (perhaps once a year) that continue for a sufficient duration (perhaps 4 to 6 years) to ensure in practice that the possibility of any micro-metastases is not present. escaping extinction at the initial oncological instrument therapy, then such micro-metastases grew to a size sufficient to disrupt acidic regions and as soon as they were detected in one of the subsequent repeat diagnostic procedures (repeats from steps 1 to 4 above). If and when one of the repeat diagnostic procedures detects an ear tumors, then the patient could be treated again by steps 5 and 6 as above. It seems likely that such a second treatment would have a high probability of completely extinguishing any tumorsoriginis that might have escaped in the form of micro-metastases during the course of the initial instrument treatment - thus, the patient's original tumor and progeny were completely exterminated.5 . Treating Decreasing pH in Tumors to Increasing Effectiveness and Specificity

Both the efficacy and specificity of a cancer instrument for acidic tumor areas are a strong function of the pH differential between the tumoral acid areas and normal tissues. Thus, if one can selectively lower the pH in acidic areas of tumors even if found in the natural condition - without concomitant pH reduction in normal tissues, it will afford greater sensitivity in diagnostic applications and greater efficacy and specificity for therapeutic instruments applications.

During the middle of the last century a number of treatments have been reported to alter the pH in tumors - some causing pH elevation and some causing pH decrease. In the context of diagnostic and therapeutic oncological instruments, that is, treatments that selectively lower the pH in tumors without a concomitant reduction in pH in normal tissues of interest. Following are three such treatments that have been reported to selectively lower the pH in tumors.

a) It has long been known that introducing deglucose into tumor-bearing animals acts to decrease pH in the interstitial space in hypoxic areas, typically for a period of approximately 2 to 3 hours, while having little or no effect on interstitial space pH. in normal tissues (Naeslund & Swenson (1953) Acta Obstet. Gyneocol. Scand. 32, 359 - 367). Additional oral glucose entry may significantly extend the duration of time while opH in tumors thus remains reduced.

b) The pH in acidic areas of tumors can also be further reduced by treatment with mitochondrial inhibitor, meta-iodobenzylguanidine, again apparently without undue effect on pH in normal tissues (Jahde et. al., (1992) Cancer Research 52, 6209 - 6215 )

It has also been reported that the pH in acid-depleting areas can be further reduced by about 0.7 pH units by using a combination of glucose and meta-iodobenzylguanidine (Kuin et al., (1994) Cancer Research 54, 3785 - 3792).

c) Still further, the pH in acid-depleting areas may be further reduced by vasodilatory drugs that are routinely used to treat people with hypertension (Adachi and Tannock (1999) Oncology Research 11, 179 - 185). Such drugs are apparently effective because anomalous vasculature detumors often lack muscle-constricting cells and their nerve fibers. Consequently, when a vasodilator drug is administered to an individual, resistance to blood flow is unchanged in tumors but decreases in normal tissues. These different effects of vasodilator result in significantly greater blood flow through normal tissues and a concomitant reduction in blood flow through the tumor. In turn, this reduced blood flow through the tumor probably reduces the fading of lactic acid produced by tumor cells in hypoxic areas of the tumor - resulting in a vasodilator mediated pH observed in acidic areas of the tumor.

Such treatments, but preferably a combination of two or more such treatments further reducing the pH in acidic tumor areas should increase the isolation of cancer instrument in the most acidic tumor areas now, as well as lead to an increase in tumor areas that are sufficiently acidic to isolated cancer instrument. . Both of these effects served to increase the effectiveness and specificity of the cancer instrument.

Making the tumor more acidic also leaves anyone using a cancer instrument with pH selectors that have a lower pKa - which can result in a significant increase in the specificity of the cancer instruments.

6. Use Multiple Oncological Instruments that vary in pKa to Increase Effectiveness and Specificity

Reported experimental measurements of acidity in tumors have shown a pH gradient ranging from approximately 7.0 close to tumor capillaries and decreasing distance from tumor capillaries, with values as low as approximately 6.0 that are being reported for more distant areas. tumorous capillary tumors. For tumors having a wide range of pH values there is the potential that therapeutic cancer instruments with too much pKa elevation can rapidly insert cells in moderately acidic areas near the tumor capillaries to bring this inadequate cancer instrument to reach the more pH areas. low dotumor farthest from the capillaries.

Alternatively, therapeutic oncological instruments with too low a pKa may be too isolated in moderately acidic areas closer to the tumor's hair capillaries, thereby leading to inadequate treatment of such areas. A strategy to validate a wide pH range within a tumor is defined by using a combination of two or more cancer instruments, where a cancer instrument with a higher pKa is potentially effective in regions of higher tumor pH closer to the vascapillaries, and where another A low pKama cancer instrument is potentially effective in lower pH regions of the tumor that are farther from the capillary vessels. The argument for using such a combination of cancer instruments is defined by the higher pKa cancer instrument and may be more isolated in areas close to vessels. capillaries - leaving little available to reach lower pH areas further away from capillaries. Otherwise, the lower pKa instrument should be poorly isolated in areas of higher pH near capillary vessels and thus remain available for diffusion within lower depH areas closer to capillaries where they can then be effectively isolated. Thus, together, such as a combination of high and low pK oncological instruments, the complete tumor extinction should be better achieved.

7. Treat to Increase Urine pH to Protect Kidney Damage

One of the functions of the kidneys is to keep the pH in the body very close to 7.4. To perform this function the kidneys can excrete urine ranging from moderately basic to reasonably acidic. In this process a substance is filtered from the blood into the kidney glomerulus, after which that substance (dissolved in the urine) passes through the proximal ductum where the critical components excreted in the glomerulus are reabsorbed by cells that line the duetoproximal. If the urine is acidic at this time then an oncological instrument in this acidic environment has the potential for binding and insertion of the cells that line the proximal kidneys, rather than the cancer instrument inserts tumor cells from an acid extracellular environment. Described differently, if the excreted urine is sufficiently acidic the oncological instrument will transfer from its nonionic lipophilic form. If this selector to a lipophilic form occurs in a region where the urine is in direct contact with cell membranes, as is the case for cells that line the proximal kidney ducts, then the instrument can insert such cells. In the case of a diagnostic cancer instrument this entry of the cancer instrument into the cells that line the proximal ducts may lead to the obvious signal that emanated kidneys, which could obscure a nearby tumor or kidneys. In the case of a therapeutic oncologic instrument being an oncological instrument entry into the cells that line the proximal ducts can lead to the destruction of the cells that line the proximal ducts - effectively rendering the bones nonfunctional.

Fortunately, there are well-known methods in the medical field for raising the urine pH of a subject long enough to make diagnosis or therapy with cancer instruments. By using an appropriate drug to maintain urine at slightly basic pH during the period when most of the cancer instrument is not isolated is being excreted by the kidneys, this urine excreted cancer instrument will remain in its anionic hydrophilic form and be safely passed from the body to urine. . A safe and effective substance to make urine basic is the carbonic anhydrase drug inhibitor, Acetazolamide. Thus, a prudent course in any diagnosis or treatment with cancer instruments is approximately one to two hours before transferring the cancer instrument to the individual, first treating the individual with stadroga, or the other effective substance to increase and maintain urine pH at a pH above approximately 7.4.

8. Monitor and Wash Bladder to Increase Safety

Because of the potential damage to the kidneys by therapeutic cancer instruments if the kidneys are excreting acidic urine, it should also be desirable to continuously monitor the pH of urine entering the bladder by using a pH probe at the end of a catheter. The preferred time period for such monitoring is immediately prior to injection of the cancer instrument until such time that most cancer instruments are not isolated in acidic areas of tumors are excreted by the kidneys (typically approximately 3 to 6 hours). Such monitoring will allow emergency intervention (such as injection of an additional Acetazolamide dose) at the time when the newly excreted daurine pH begins to fall below approximately pH 7.4.

During the course of a few hours of the injected dose an oncological instrument will be excreted through the kidneys and stored in the bladder until the urine is cleared. For the example of a diagnostic application of a cancer instrument, this accumulation of radioactive cancer instrument in the bladder may obscure the presence of tumors in close proximity to the bladder. For example of a therapeutic cancer instrument the accumulation of radioactive cancer instrument in the patient's bladder has the potential to damage the bladder. If the accumulation of a radioactive bladder cancer instrument proves to be a significant problem, it can be substantially reduced by using a surging / drainage catheter to irrigate the bladder from the time the oncological instrument is injected until such time as most of the injected dose is released from the bladder. The irrigation of the bladder over the period of time as soon as most of the dose of cancer instrument is being excreted by the kidneys is appropriate as an additional purpose, so that the solution made in the bladder may pass into a storage container where it may be until the radioactive isotope has decomposed to a safe level (typically 10 half-lives of the radioactive isotope). This serves to ensure the safety of medical professionals who serve the individual, largely avoids contamination by negligence with respect to the lavatory and other areas by radioactive chronological instrument eliminated by the individual.

EXAMPLES:

Example 1. Effect of lipophilicity on transition pH between ionic and nonionic forms

The sodium salts of propionic acid (a hydrophilic acid carboxylic acid) and octanic acid (a lipophilic acid carboxylic acid) at a concentration of 33 milliMolar in water were titrated with 5 H HCl. The midpoint between salt and acid (pKa value) was found to be 4.83 predicted for propionic acid, but surprisingly high 5.5 for octanoic acid. The first derivative of the titration curve was symmetrical for propionic acid, but highly asymmetric for octanoic acid. In addition, drops of octanoic acid oleation of the solution during the course of octanoic titration.

In contrast, when salts of these same two acids were titrated in a semi-aqueous medium: methanol / water, 1: 1 by volume, their transition midpoints were seen to be virtually identical, and the first derivative of their titration curves was now symmetrical for both. . Finally, on titration in semi-aqueous medium, methanol / water, no octanoic acid oleation of the solution.

These results suggest that the surprisingly high mean point in titration seen for octanoic acid in aqueous solution was simply due to the solubility effects illustrated in Figures 3 and 4. The results further show that performing titrations in semi-aqueous, methanol / water, 1: 1 by volume. , these solubility effects on the titration curves are avoided.Example 2. Comparison of specific acidic and nonacidic acid H bridges at transition pH between ionic and nonionic forms

Thirty milliMolar solutions of sodium salts of the compounds shown in Figure 6a (designed to form specific acid internal H bridge) and 6b (designed to form specific non-acid H bridge) titrated with 5 MHCl in the aqueous solution. The first derivative of the titration curve for the compound in Figure 6b was symmetrical showing an expected pKa value of 4.82 (typical for simple carboxylic acids), and there was no apparent insoluble material generated during titration. In sharp contrast, the first derivative of the titration curve for the compound in Figure 6a was highly sloped and showed a surprisingly high minimum of pH 5.8, with approximate mean titration point at pH 5.6. Further, massive precipitate had formed with each addition of HCl, beginning when the pH dropped below 5.8. These dramatic differences in titration properties between nearly identical structure compounds support the conclusion that the structure in Figure 6a is forming a specific acid internal H bridge. H bridge is acting to increase the pH of the transition between the ionic and nonionic forms. Conversely, while the structure in Figure 6b may be forming internal H bridges, in this case then because H may form or with the shape. of the carboxylate salt or the carboxylic acid form, such a H-bridge does not significantly favor the nonionic form over the ionic form and thus does not significantly change the transition pH between the two forms relative to what would be for a simple carboxylic acid such as propionic acid. .Example 3. Effect of low conformational freedom of H point on the transition pH between forms ionic and nonionic

Aqueous titration of the acyclicized structure Figure 7b showed an expected transition pH value of 4.95 (typical of simple carboxylic acids) without acid precipitation. In contrast, the 5-element ring structure titration of Figure 7a showed an unexpectedly high value of 5.6 for the transition pH between anionic and nonionic forms. Titration of this ring structure was also accompanied by massive precipitation when the pH dropped below approximately 5.8, and the shape of the titration curve was highly asymmetric.

However, when these two compounds were instead titrated in semi-aqueous medium, methanol / water, 1: 1 by volume, there was no sign of insolubility and the outlines of the titration curves of the first symmetrical outmagora derivative. For the acyclic compound shown in Figure 7b the pH at the midpoint was 5.8, while the pH at the midpoint was substantially higher 6.45 for the ring structure at Figure 7a.

Thus, even in the absence of slope of the titration curve due to precipitation of the acid-lyophilic form, in the semi-aqueous titration experiment a substantial increase of 0.65 pH unit in the midpoint of the titration curve for the cyclic compound is seen in Figure 7a.A The most reasonable explanation for this seems to be that in the semi-aqueous solution a specific acidic internal H-bridge is formed only in the case of cyclic ring structure, and this H-bond is guiding the equilibrium in favor of the acid form.

Example 4. Importance of Carboxyl Isolation of Electron Linker Removal Effects

Molecular modeling of structure 8b suggests that an internal H-bridge should be strongly favored by the near-perfect phageometry of said H-bridge and the very limited conformational freedom between the H-bridge donor fraction and the H-bridge acceptor. Despite these favorable factors for the formation of a H-bridge. specific acid H bridge, when the salt of the structure in Figure 8b was prepared and titrated with HCl in aqueous solution, the titration curve indicated a pKa value below 3.0. It is concluded from this result that the amide inductive effects, where said effects must be readily propagated through double bond intervention, are likely to cause a large reduction in pKa dacarboxyl, with these negative inductive effects can outweigh any positive effect on a specific acidic H-bridge that could elevate carboxyl pKa.Example 5. Advantage partially prevent the H-point location of the aqueous solvent

The two compounds in Figure 9 were prepared and then titrated in semi-aqueous solvent methanol / water 1: 1 by volume. This semi-aqueous solvent was used in these titrations to prevent insolubility effects from impacting titration results. In these experiments the first derivative of the titling sketches was symmetrical and there was no visible sign of precipitation. 'Thus, the goal of avoiding insolubility effects has been met.

What was found is that structure (a) of Figure 9 showed a mean point for the titration curve at pH 5.6, while structure (b) of Figure 9 showed a significantly higher mean point for titration at pH 6.14. .

Again, the likely explanation for the significantly higher transition depH value for structure (b) in these titrations is that in this semi-aqueous solution structure (b) is forming a specific acidic H-bridge that is acting to alter equilibrium in favor of forms acid, while the closely related structure (a) does not form such an acid-specific internal H bridge. It seems likely that the moderate impediment at the H-bridge site in structure (b) is the factor that favors the formation of the H-bridge at structure (b), while the lack of such an impediment at the H-bridge site in structure (a) is probably responsible for the apparent absence of an internal H-bridge in structure (a). Example 6. Preparation and titration of two pH-depleting structures designed to form a low-barrier acid-specific Hinternal bridge.

The isonipocotic acid methyl ester was reduced alkylated with 3-pentanone and then the resulting tertiary amine converted to N-oxide with meta-chlorobenzoylperoxide. Finally, the ester was partitioned with aqueous sodium hydroxide to give structure iii in Figure 10b. This product, in its sodium carboxylate form, was then titrated in water. The first derivative of its titration sketch shows two minima, one at pH 3.8 (which is approximately that expected for the N-oxide moiety), and another minimum at pH 6.0, which is substantially higher than would be expected for the N-oxide moiety. N-oxide or the carboxylic acid moiety. However, in light of the results in the previous examples in this context, the most likely explanation is that this pKa 6.0 value is due to an internal low-barrier H-bridge formed between the carboxyl-H-bridge donor and the H-N-deponder acceptor. oxide, and which internal H-bridge acts to balance the equilibrium to the acid form - ie, raise the pKa. The useful (3-amino-1-acid) derivative of the camphoric acid was by reduction of excess alkylated deacetaldehyde and resulting tertiary amine converted product. N-oxide with meta-chlorobenzoylperoxide to give the structure in Figure 10b. This product, in its sodium decarboxylate form, was then titrated in water. The first derived from his titration sketch shows two minima, one at pH 4.2 (which is approximately that expected for the N-oxide group), and another minimum empH 6.5, which is substantially higher than would be expected for the portion. N-oxide or the carboxylic acid moiety. Again in light of the results from previous examples in this context, the most likely explanation for this exceptionally high pKa value of 6.5 is that a low barrier inner H bridge is forming between the carboxyl H bridge donor and the bridge acceptor. H-N-oxide, and which H-bridge acts strongly to guide equilibrium to the acid form, thereby raising the pKa value.

It is noteworthy that the above derivative of internally linked H-isotropic acid has a considerably lower value of pKa (6.0) than the similar derivative of H-bound camphoric acid despite the H-bridging moiety and the bridging-accepting moiety. H are virtually identical in both structures. The most likely explanation for a significant difference between pKa values is that the lower energy conformation for 6-element aliphatic rings, such as the isonipocotic structure above, is typically the conformation in chair and usually takes as 5 Kcal to convert this conformation into chair for following more stable conformation, the form of barcotchloride. Because the molecular modeling of the inverter has shown that only the twisted boat conformation of the isipocytic derivative can form the specific acidic inner H bridge, it is likely that the strength of this H bridge will be reduced by the energy cost of converting the chair-to-twisted boat conformation.

In contrast, the camphor-doped 5-membered aliphatic ring can readily and cost-effectively change between different shaping folds, and thus there is little energy cost to adopt the appropriate conformation to form the specific acidic H-bridge. As a consequence, essentially the complete energy of this H bridge is available to favor the acid form over the salt form - thus raising pKa to the exceptionally high value of 6.5.

The above meaning is that it demonstrates that subtle structural properties can be exploited to quite precisely adjust the pKa value of a pH selector to achieve a desirable balance of efficacy and specificity, the importance of which is illustrated in Figure 12.

Example 7. Titration Procedures for Studying pH Selector Structures

a) Aqueous titrationsTypically new structures that may be appropriated as advanced pH selector components are primarily evaluated for their pT value, ie, the pH value at the midpoint of their transition between ionic and nonionic forms. This requires preparing the sodium salt form of the structure and dissolving it in a demiliMolar concentration in 50 mL of 0.15 Molar NaCl (used to minimize carbonic acid content). A small stirring bar is added and the pH of the solution is adjusted to approximately 9 with a small amount of 1 M NaOH. When stirring, 5 microliters of 1 M HCl are added and the pH is recorded 1 minute after the addition of each HCl fraction. . If precipitation or oleation of the pH selector does not occur during titration, a pH sketch versus the volume of added HCl provides the conventional titration curve. However, a much more useful presentation of the titration results, which allows a very accurate measurement of the pKa value, is obtained for the first derivative of this titration curve. This requires plotting on the X axis the values for: (pHn + pHn + 1) / 2, and plotting on the Y axis the values for: (pHn - pHn + 1).

b) Semi-aqueous titrations

If the pH selector precipitates or olies during the course of titration then the titration curve will generally be steeply inclined. In such cases it is difficult to differentiate between solubility effects and depKa effects at the midpoint of transition between anionic and nonionic form of the pH selector. In such cases the desolubility effect may be prevented by titration in semi-aqueous solvents where the depH selector salt and acid forms are completely soluble. A 1: 1 mixture of water methanole is generally suitable for this purpose. In this solvent the first derivative of the smooth titration curves becomes symmetrical. In the case of a substance which cannot form a stable internal H-bridge, such as octanoic acid, the titration outline now shows a classic pKa value very similar to that seen for plain water-soluble simple carboxylic acid such as propionic acid. However, in the case of a substance designed to form a specific acid stable internal H-bridge, such as the advanced pH selector structures described in the examples above, while the titration sketch becomes symmetrical in methanol / water, the titration sketch may also show a substantially increased value of pKa relative to a very similar structure which cannot form a specific acid internal H-bridge - and this increased value of pKa is indicative of specific internal H-bridge formation under the titration conditions.

Thus, in physiological saline titrations the total increase in apparent transition pH, accompanied by asymmetry in the titration curve, may be due to a combination of the effect of an acidic internal H-bridge plus a solubility effect. However, when titration is preferably carried out in a semi-aqueous solvent such as methanol / water, a substantial increase in pKa value for a substance designed to form a specific acid internal H-bridge relative to pKa for a many-like substance which cannot be forming such an H-bridge is mainly attributable only to the inner H-bridge, and not to any lipophilicity increase in the acidic portion which could be a consequence of that inner H-bridge. However, it should also be appreciated that to some extent the low polarity solvent methanol / water preferentially favors the formation of an internal H-bridge, and as far as possible when testing a new structural type of the pH selector it is desirable to confirm the formation of the specific acidic internal H-wall by titration in a completely aqueous solution.

Example 8. Cell Culture Test System for Cancer Instruments

It is recommended that initially each prospective cancer instrument structure should be tested in a relatively simple biological system in which the cancer instrument is exposed to the major biological environments and structures it will encounter in a living individual - particularly including mammalian cells exposed to serum containing buffered medium. pH 7.4 to limit blood and normal tissues, and buffered to pH 6.4 to mimic acidic areas of tumors.

Such an appropriate test system requires making two preparations of isotonic culture medium. It should contain 10% serum and be strongly buffered to pH 7.4 with 50 ml Molar HEPES buffer (pKa 7.5). The other should contain 10% serum albumin and be strongly buffered to pH 6.4 with 50 milliMolar BisTRIS buffer (pKa6.5). The iodine-131 containing oncological instrument should be added at a concentration equal to each culture medium.

Prior to the experiment, Hela cells should first be grown by confluence of 12-well culture plates. Then the culture medium is removed from four cell wells and replaced with buffered medium containing the oncological instrument at pH7.4. Additionally, culture medium is removed from other four cell wells and replaced with the half-buffered oncological instrument at pH 6.4. The placassas are then incubated at 37 ° C for one hour. After incubation, the medium containing oncological instrument is removed and replaced with free medium of oncological instrument named pH, briefly rotated, and removed. This washing procedure is repeated a total of 4 times. The cells are then lysed with 1 ml of detergent solution and this lysis solution removed and counted in a scintillation or gamma counter to provide a measure of the relative amount of oncological instrument which was isolated under each of the two pH conditions.

The preferred oncological instrument structure is that which is maximally isolated by cells at pH 6.4 but only minimally isolated by cells at pH 7.4.

Example 9. Testing Cancer Instruments in Normal Mice

The above cell culture testing system for oncological instruments allows for a rapid and quantitative initial assessment of the probable efficacy and specificity properties of an important number of prospective oncological instruments. However, this initial assessment should then be continued for the most promising oncological instrument frameworks tested in living mice. It is recommended that mice be first pretreated with a carbon dioxide inhibitor such as acetazolamide to ensure that their urine remains basic for several hours. Thereafter, an appropriate amount of iodine-131 containing instrumentation in phosphate buffered saline should be injected, preferably intravenously, such as into the tail vein. Thereafter the mice should be periodically monitored for up to approximately 24 hours (such as by briefly positioning under an appropriate gamma or camera counter) to determine the excretion rate of the identified instrument.

The main purpose of this test in normal mice is to identify any oncological instruments that are unnecessarily isolated in the normal tissues - presumably due to excessive lipophilicity, or possibly due to some structural element that has an unexpected affinity for normal tissues or any particular organ, etc. Oncological instruments that are rapidly and completely excreted from normal mice, and thus pass this preliminary test, should then be tested on tumor bearing mice, as described in the following examples. Example 10. Testing on cancer instruments on tumor bearing mice

While the above relatively simple tests in normal tumor-free mice have allowed us to rule out these cancer instruments which have an excess affinity for normal tissues, the most decisive test for a prospective cancer instrument structure is to test it in tumor-bearing mice.

In such experiments a first pretreatment in mice is recommended: a) with a carbon dioxide inhibitor, such as acetazolamide, to elevate opH in the urine; and, b) with one or a combination of substances effective to selectively lower the pH in tumors. Substances include: i) glucose (Naeslund & Swenson (1953) Acta Obstet. Gyneocol. Scand. 32, 359-367); ii) mitochondrial inhibitor, meta-iodobenzylguanidine (Jahde etal., (1992) Cancer Research 52, 6209 - 6215); and iii) vasodilatory drugs routinely used to treat people with hypertension (Adachi and Tannock (1999) Oncology Research 11, 179 - 185).

After an appropriate period of time following such pre-treatments, tumor bearing mice are injected (preferably intravenously) with an iodine-131-containing instrument. After an appropriate period of time (on the order of 5 to 24 hours) to allow normal excretion of the kidneys. From this fraction of the dose administered which is not isolated in mouse tissues and / or tumors, the mice are killed and the major organs and any obvious tumors are excised. Remaining organ and tumor and carcass are then counted in gamma counter.

Regarding the pretreatment of mice, it should be emphasized that cancer instruments will work best when both types of pretreatments are used, where one type is appropriate to prevent the absorption of the cancer instrument into cells lining the proximal kidney tubules. This resorption is blocked by making the urine slightly basic. The other type is suitable to further increase acidity (lowers pH) in hypoxic / acidic areas of tumors. Preferably a combination of two or three such pretreatments to selectively lower the pH in tumors should be employed in order to adjust the tumor microenvironment to best suit the effective and specific activity of the cancer instrument.

The above description best describes the compositions and methods of the present invention, and the manner and process of preparing and using such compositions and methods in such complete, clear, concise and exact terms as to enable any person skilled in the art to make and use the same. compositions and methods. These compositions and methods are, however, susceptible to alternative modifications and constructions from the illustrative embodiments discussed above which are entirely equivalent. Accordingly, this is not intended to limit the disclosed compositions and methods for particular embodiments disclosed. Rather, the intention is to cover all modifications and alternative constructions that come within the spirit and scope of the compositions and methods as generally expressed by the following claims, which particularly indicate and claim distinctly the topic of the disclosed compositions and methods.

Claims (35)

1. A composition comprising an advanced pH selector component, said advanced pH selector component, comprising: a) a selected aliphatic ring structure from a group consisting of a 4-element ring, a 5-element ring, and a b) a portion of a carboxylic acid directly bonded to said aliphatic ring structure, said portion of a carboxylic acid is separated from any bond of the electron-removing group to at least two carbons; and c) a selected H-bridge acceptor moiety from a group consisting of a part of the aliphatic ring structure, directly attached to the aliphatic ring structure, and bonded via an atom to the aliphatic ring structure, wherein said bridge acceptor portion H in its nonionic form has a structure that cannot serve as said H-bridge donor portion, and said carboxylic acid portion and said H-bridge acceptor portion are positioned and oriented such that they are compatible with the formation of a H-bridging donor. H bridge specific internal acid. Composition according to Claim 1, characterized in that a form independent of the H-bridge acceptor portion of the advanced pH selector component has a pKa value in the range of from about 3.0 to about 6.5. Composition according to Claim 1, characterized in that a H-accepting moiety of the advanced pH selector component is selected from the group consisting of: (a) a cyanomethyl amine, (b) a trifluoroethyl amine (c) an alkoxy amine, (d) a hydrazine, (e) an N-oxide, (f) an imidazole, (g) an aniline, (h) a starch, (i) a phosphoramide, (j) an thiophosphoramide (k) a urea; and (1) an ether. 4. Composition of an oncological instrument, characterized in that it comprises: (a) at least two pH selector components, each pH selector component being effective for a pH-mediated transition between an anionic hydrophilic form at a lower pH. elevated and a nonionic formalipophilic at a lower pH; and (b) at least one selected component charge from a group consisting of: i) a structure effective for binding an isotope radioactive, and ii) a structure containing an isotopic radioactive. An oncological instrument composition according to claim 4, further comprising a radioactive isotope selected from the group consisting of: (a) an effective radioactive isotope for reporting the presence of the composition, (b) an effective radioactive isotope for matellules; and (c) a radioactive isotope effective for reporting the presence of the composition and effective for killing cells. Composition of an oncological instrument according to claim 4, characterized in that it has a specificity factor greater than 20. A cancer instrument composition according to claim 4, characterized in that the carboxyl portions of pH selectors have pK values ranging from 5.4 to 6.8. An oncological instrument composition according to claim 4, characterized in that two of the pH selector components share a single H-bridge acceptor moiety. An oncological instrument composition according to claim 8, characterized in that the shared H-bridge acceptor moiety is an oxygen atom of an N-oxide. An oncological instrument composition comprising: (a) at least two advanced pH selector components, each comprising: (i) an aliphatic ring structure selected from the group consisting of a 4-element ring, a 5-membered ring and a 6-membered ring ii) a carboxylic acid moiety directly attached to an aliphatic ring structure, the carboxylic acid moiety being separated from any electron moiety attached at least to two carbons; and (iii) an H-bridge acceptor moiety selected from the group consisting of part of the aliphatic ring structure, directly attached to an aliphatic ring structure, and bonded via an atom to the aliphatic ring structure; H in its ionic form has a structure that cannot serve as an H-bridge donor portion, and the carboxylic acid portion and the H-bridge acceptor portion are positioned and oriented such as are compatible with the formation of an H-bridge. internal specific acid; and, (b) at least one charge component selected from a group consisting of: i) a structure effective for binding an isotope radioactive, and ii) a structure containing an isotopic radioactive. 11. Diagnostic composition of an instrument for detecting a tumor containing acidic areas, comprising: (a) at least two effective depH selector components for undergoing a pH-mediated transition between an anionic hydrophilic form at a lower pH elevated non-ionic lipophilic form at a lower pH; and (b) at least one radioactive isotope whose emission from within a tumor in an individual may be detected outside the individual. Diagnosis of an instrument composition according to claim 11, characterized in that the radioactive isotope is a radiohalogen. 13. Therapeutic composition of an instrument for treating an individual who has a tumor that contains acidic areas, which comprises: (a) at least two effective depH selector components to undergo a pH-mediated transition between an anionic hydrophilic form and a Higher pH and a nonionic lipophilic form at a lower pH; and (b) at least one radioactive isotope whose emission is effective for killing cells. Therapeutic instrument composition according to claim 13, characterized in that the radioactive isotope is a radiohalogen. Therapeutic instrument composition according to claim 13, characterized in that the radioactive isotope emits high linear energy transfer radiation. Therapeutic instrument composition according to claim 15, characterized in that the radioactive isotope is selected from a group consisting of Astato-211, Bromo-77, and Iodo-123. Therapeutic composition of an instrument according to claim 13, characterized in that the radioactive isotope emits a particle. A combination of oncological instrument therapeutic compositions according to claim 13, comprising: (a) at least a first composition of an oncological instrument containing a first isotope radioactive that emits high energy transfer radiation; and (b) at least one second oncological instrument composition containing a second isotope radioactive that emits a beta particle. Diagnostic method for detecting a tumor in an individual containing acidic areas, the method characterized by comprising the steps of: (a) providing a diagnosis of oncological instrument composition as described in claim 11. (b) introducing the diagnosis of instrument composition (c) wait approximately 10 minutes to approximately 48 hours; and (d) scan the individual with effective equipment to detect emissions from the diagnostic composition of an oncological instrument in the individual. 20. Diagnostic method according to claim 19, further comprising the step of: pre-treating the individual with at least one selected substance from a group consisting of: (a) a first effective substance for increasing the pH of the urine of (b) a second effective substance to decrease opH in tumors. Therapeutic method for treating tumors in an individual, the method comprising the steps of: (a) providing a therapeutic composition of an oncological instrument as described in claim-13; and (b) introduce the therapeutic composition of the cancer instrument within the subject. Therapeutic method according to claim 21, further comprising the step of: pretreatment of the subject with at least one selected substance from a group consisting of: (a) a first effective substance for increasing the pH of the urine of an individual, and (b) a second effective substance to decrease opH in tumors. 23. Therapeutic method according to claim 21, further comprising the step of: irrigating the individual's bladder for at least one hour and then introducing the therapeutic composition of the cancer instrument within the individual. Therapeutic method for treating tumors in an individual, the method comprising the steps of: (a) providing a combination of the therapeutic compositions according to claim 18; and (b) introducing the therapeutic compositions into the subject. A therapeutic method according to claim 24 further comprising the step of: pretreatment of an individual with at least one selected substance from a group consisting of: (a) a first effective substance for increasing urine pH (b) a second effective substance to decrease opH in tumors. 26. Therapeutic method according to claim 24, further comprising the step of: irrigating the individual's bladder for at least one hour and then introducing the therapeutic composition within the individual. Method for detecting a tumor in an individual containing an acidic area, and treating some of the detected tumors, comprising the steps of: (a) providing an effective diagnostic composition by being isolated in an acidic area of a tumor and containing a radioactive isotope. emitting a signal that can be detected outside the individual (b) introducing a diagnostic composition within the individual (c) waiting approximately 10 minutes to approximately 48 hours (d) scanning the individual with the effective equipment to detect the compositional emitting signal diagnosis; and, for the individual where the scan indicates the presence of the tumor: (e) providing at least one effective oncological instrument therapeutic composition for selectively isolating in the acidic area of the tumor and containing an effective isotope radioactive to kill cells; and (f) introducing at least one of the oncological instrument therapeutic compositions within the individual. A method according to claim 27, further comprising the step of: pre-treating the subject with at least one selected substance from a group consisting of: (a) a first effective substance for increasing the pH of the urine of individual; and (b) a second substance effective for decreasing pH in tumors, wherein the pretreatment step is performed prior to introducing the diagnostic composition into the individual and again prior to introducing oncological instrument therapeutic compositions into the individual. A method according to claim 27, further comprising the step of: irrigating the bladder of the subject for at least one hour and then introducing the therapeutic composition of the oncological instrument within the subject. A method according to claim 27 further comprising a step of: repeating steps (a) through (d) after an estimated time period to be sufficient for demicrometastasis growth within a new tumor of sufficient size to contain acidic areas; repeat steps (e) and (f) if at least one tumor is detected in step (d). A method of detecting and treating a tumor in an individual containing an acidic area, comprising the steps of: (a) introducing into the individual a first effective substance to raise the urine pH in an individual; (c) providing an effective diagnostic composition by being selectively isolated in the acidic area of the tumor and having a radioactive isotope that emits a signal that can be detected from outside the subject; introduce the diagnostic composition into the individual (e) scan the subject with an effective device to detect the signal emitted from the diagnostic composition (f) provide at least one effective therapeutic composition to be selectively isolated in the acidic areas of the tumor and contain an isotope effective radioactive cell killer; and (g) introducing at least one of the therapeutic compositions within the subject. Method according to claim 31, characterized in that the diagnostic composition is an oncological instrument. A method according to claim 31, characterized in that the therapeutic composition is an oncological instrument. Method according to claim 31, characterized in that the diagnostic composition and therapeutic composition are an oncological instrument. A method according to claim 31, further comprising the steps of: (a) waiting for the diagnostic composition to be isolated in an acidic tumor area upon introduction of the diagnostic composition into the subject; individual an additional amount of a first effective substance to increase the urine pH in the individual; and (c) introducing within the subject an additional amount of a second substance effective to decrease opH in the tumor.