WO2001007087A2 - Enzyme catalyzed anti-infective therapeutic agents - Google Patents
Enzyme catalyzed anti-infective therapeutic agents Download PDFInfo
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- WO2001007087A2 WO2001007087A2 PCT/US2000/019844 US0019844W WO0107087A2 WO 2001007087 A2 WO2001007087 A2 WO 2001007087A2 US 0019844 W US0019844 W US 0019844W WO 0107087 A2 WO0107087 A2 WO 0107087A2
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- 0 *C(OC1=N2)=CC1=CN(*)C2=O Chemical compound *C(OC1=N2)=CC1=CN(*)C2=O 0.000 description 3
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/555—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
- A61K47/556—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells enzyme catalyzed therapeutic agent [ECTA]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- TECHNICAL FIELD This invention relates to the field of therapies for infectious diseases, and in particular, compositions and methods for the treatment of therapy-resistant infectious diseases.
- This invention provides a method for selectively inhibiting the proliferation of an infectious agent or a cell infected by the infectious agent.
- Infectious agents suitably treated by the method of this invention express an activating agent that selectively activates or converts a prodrug to a toxin.
- the enzyme is not inactivated or inhibited by the substrate prodrug compound.
- the method requires contacting the cell or the agent with an effective amount of the substrate compound thereby selectively inhibiting the proliferation of the infectious agent, the cell or the infectious agent within the cell.
- This invention also provides a method for screening for prodrugs selectively converted to a toxin in a cell by an activating enzyme expressed by an infectious agent.
- the screen requires contacting an infectious agent or a cell infected with the infectious agent with a candidate prodrug and assaying for activation of the prodrug into toxic agents by the activating enzyme.
- activation of the prodrug is determined by noting inhibition of the proliferation or growth of the infectious agent or the cell infected with the agent.
- FIGURES Figure 1 schematically shows the mechanism of action of the ECTA prodrugs of this invention.
- Figure 2 is a graph showing fluorescent products from incubation of bromovinyl 2'-deoxyuridine monophosphate (BVdUMP) with recombinant human thymidylate synthase (rHUTS). Incubation of BVdUMP with thymidylate synthase results in a time and enzyme dependent generation of fluorescent product(s).
- BVdUMP was incubated with the indicated amounts of rHuTS in the standard reaction mixture at 30° C (See Materials and Methods below), except that N5, N10- methylenetetrahydrofolate was omitted from the reaction.
- the numbers adjacent to each data curve refer to TS enzyme units.
- Figure 3 shows that BVdUMP is competitive with deoxyuridine monophosphate (dUMP) in rHuTS.
- Thymidylate synthase catalyzed reaction of converting dUMP into dTMP was run in vitro in the absence (triangles) and in the presence of 20 ⁇ M BVdUMP (squares).
- dUMP concentration was varied from 10 to 100 ⁇ M, N5, NlO-methylene tetrahydrofolate concentration was 140 ⁇ M and the enzyme concentration was 0.1 ⁇ M.
- Enzyme activity was determined by measuring the increase in A 340 .
- Figure 4 is the structures of products o ⁇ in vitro reaction of BVdUMP catalyzed by rHuTS. Structures I and II are consistent with mass ions identified in cell free reaction mixtures.
- Figure 5 is a proposed mechanism of NB1011 activation.
- NBIOI 1 must be able to enter cells and convert to BVdUMP before interacting with TS.
- Structures generated following transformation by TS are proposed to be exocyclic pyrimidine nucleotide monophosphates. These compounds may be cytotoxic to cells by a variety of mechanisms including interference with nucleotide and nucleic acid metabolism.
- Figure 6 shows detection of BVdUMP in H630R10 cells treated with
- NBIOI 1 H630 R10 cells were treated with 100 ⁇ M NB1011 for 5 days, then analyzed by liquid chromatograph mass spectroscopy as described in the Materials and Methods, below.
- FIG 7 demonstrates that NBIOI 1 does not irreversibly inactivate TS in vivo.
- the effect of NBIOI 1 on TS activity in intact cells is completely reversible.
- TS activity was measured in intact RKO cells by release of [ 3 H] 2 O from 5-[ 3 H] deoxyuridine as described in Materials and Methods.
- NB1011 was washed out of cells by replacing with fresh media, incubating for 60 minutes at 37 °C, then repeating this procedure. Control and untreated cells were subjected to the same washing procedure.
- Figure 8 shows TS expression level in cells selected with Tomudex or NB1011, as estimated by SDS PAGE Western blot developed with antibody against thymidylate synthase and tubilin.
- Lane 1 shows MCF7 cells, no selection with drug
- lane 2 shows MCF7 cells selected with 2 ⁇ M tomudex
- lane 3 shows MCF7 cells as in lane 2, but after a subsequent selection using NBIOI 1 as the selective agent
- lane 4 shows MCF7 cells as in lane 2, after a subsequent passaging without tomudex.
- compositions and methods include the recited elements, but not excluding others.
- Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination.
- a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
- Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
- An "infectious agent” or a "pathogen” is a organism that is pathological to a cell or organism that it infects. Examples of pathogenic organisms include, but are not limited to, bacteria, parasites, viruses or yeast. Examples of viruses include but are not limited to Herpes, Varicella zoster, Hepatitis C and Epstein Barr virus. Examples of parasites include but are not limited to T. brucei, T. cruzi, and
- Plasmodium falcipurum examples include, but are not limited to, all gram positive and gram negative bacteria, especially, Staphylococcus, sp., Enterococcus sp., Myoplasma sp., E. coli sp., Psudomonas sp., Nisseria sp.. And, from among these, preferred pathogens are those which have become resistant to common antibiotics (see reveiw by Murray, BE "Antibiotic Resistance” (1997) Adv. Int. Med. 42:339-367.)
- composition is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label or a pharmaceutically acceptable carrier) or active, such as an adjuvant.
- pharmaceutical composition is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
- the term "pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
- the compositions also can include stabilizers and preservatives.
- stabilizers and adjuvants see Martin, REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975)).
- an “effective amount” is an amount sufficient to effect beneficial or desired results.
- An effective amount can be administered in one or more administrations, applications or dosages.
- a "subject,” “individual” or “patient” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
- a “control” is an alternative subject or sample used in an experiment for comparison purpose. A control can be "positive” or "negative”.
- the purpose of the experiment is to determine a correlation of an altered expression level of a gene with a particular type of pathogenic agent
- a positive control a subject or a sample from a subject, carrying such alteration and exhibiting syndromes characteristic of that infection
- a negative control a subject or a sample from a subject lacking the altered expression and clinical syndrome of that disease
- activating enzyme means an enzyme that is expressed by a pathogen in its native or natural environment. It is intended to distinguish enzymes or other agents that are administered to activate a prodrug.
- pathological cells encompass cells characterized by the presence of an activating enzyme.
- the expression of the activating enzyme occurs as a consequence of infection by a pathogenic organism, as defined above.
- Enzymes expressed by the pathogen or within an infected cell providing targets for this therapy include, but are not limited to thymidylate synthase and dihydrofolate reductase. Additional examples are listed below.
- this invention is directed to methods for inhibiting the proliferation or growth of an infectious agent or a cell infected with the agent by contacting the agent or infected cell with a substrate prodrug that is selectively converted to a toxin in the cell by an activating enzyme expressed by the infectious agent.
- the methods and compositions of this invention are useful to preferentially inhibit the growth or proliferation of cells that express or contain activating enzyme, for example microbial cells, virally infected cells or cells infected with other pathogens. Overexpression of the enzyme is not required, as specificity is related to the species-specificity of the prodrug to the activating enzyme expressed by the pathogen.
- the activating enzyme may or may not be expressed by the host cell.
- the prodrug is selective on the basis that it is preferentially activated by the version of the enzyme expressed by the infectious agent as compared to the version of the enzyme expressed by the host cell.
- the activating enzyme can be the wild-type or a mutated version which has developed resistance to prior art therapeutics (Hooker, et al. (1996) J. Virol. 70(11):8010-8018).
- activating enzymes that are selective targets for the prodrugs and methods of this invention include, but are not limited to, thymidylate synthase (TS), dihydrofolate reductase (DHFR) and ⁇ -lactamase activating enzymes.
- TS thymidylate synthase
- DHFR dihydrofolate reductase
- ⁇ -lactamase activating enzymes include, but are not limited to, thymidylate synthase (TS), dihydrofolate reductase (DHFR) and ⁇ -lactamase activating enzymes.
- TS activating enzyme
- TS is merely illustrative and the claims are not to be construed as limited to systems which target TS.
- Thymidylate synthase was used herein as the target, activating enzyme because of the high degree of characterization of its structure and function (Carreras and Santi (1995) Annu. Rev. Biochem. 64:721-726), the fact that it is encoded by a single gene, not a gene family (compare for example the family of enzymes noted as glutathione-S-transferase (GST)).
- GST glutathione-S-transferase
- TS overexpression is the result of acquired resistance to chemotherapeutics.
- the activating enzyme can be expressed as a result of resistance to prior therapy.
- Other target activating enzymes include, but are not limited to viral reverse transcriptases and proteases.
- viruses that encode these enzymes include the retroviruses (eg. HIV-1, both enzymes, see Turner B.G. and Summers M.F. (1999) J. Mol. Biol. 285:1-32), the picornaviruses (eg., Hepatitis A virus, Wang Q.M. (1999) Prog. Drug Res. 52:197-219), and Hepatitis C virus (Kwong A.D. et al. (1999) Antiviral Res. 41:67-84).
- retroviruses eg. HIV-1, both enzymes, see Turner B.G. and Summers M.F. (1999) J. Mol. Biol. 285:1-32
- the picornaviruses eg., Hepatitis A virus, Wang Q.M. (1999) Prog. Drug Res. 52:197-219
- the prodrug is a compound having a structure as defined in more detail herein.
- the term prodrug refers to precursors of active therapeutics.
- the perfect prodrug is one that is pharmacologically inert until activated by the intended mechanism.
- Prodrug strategies are meant to target potentially toxic therapies to the site of disease, thereby avoiding systemic toxicity. A number of approaches have been made to this goal.
- One of the first attempts at a prodrug for cancer therapy was reported by Mead et al. (1966) Cancer Res. 26:2374-2379 and Nichol and Hakala (1966) Biochem. Pharmacol. 15:1621-1623.
- GST glutathione-s-transferase
- the prodrugs are essentially non-toxic to normal, uninfected cells. This aspect further enhances the selectivity of the prodrugs and increases the overall safety of the therapy.
- the prodrug can selectively kill the cell because only infected cells provide an effective amount of the toxic metabolite of the prodrug to inhibit proliferation of the pathogen or the cell infected with the pathogen.
- the ultimate efficacy of the prodrugs of this invention are related to the origin of the activating enzyme.
- the efficacy of NB1011 is unexpectedly more potent as a substrate for human TS than microbial TS, as would be expected from published studies. (Barr (1983) J. Biol. Chem. 258(22): 13637- 13631) summarized in Table 2, infra.
- Applicant also has discovered that cells which have been treated with the substrate prodrugs may revert to a prior phenotype which is suitably treated by conventional therapies.
- TS as an example, Applicant has shown that tumor cells treated with 5-FU became resistant to the drug. At that time, the cells were treated with NB 1011. A subpopulation survived and became resistant to NB 1011 but regained sensitivity to 5-FU (see Table 9 and Figure 8).
- this invention provides the methods described above wherein an effective amount of another anti- infective agent is co-administered with the substrate prodrug of this invention.
- the second or third agent is the drug to which the pathogen had previously developed resistance.
- the additional agent can be administered concurrently or subsequenct to administration of the substrate prodrug.
- This invention further provides prodrugs that are selectively converted by an activating enzyme produced or expressed by an infectious agent or pathogen as compared to the uninfected cell, e.g., an animal cell, a mammalian cell, or a human cell.
- an infectious agent or pathogen e.g., an animal cell, a mammalian cell, or a human cell.
- Applicant has discovered several preferential prodrugs for the practice of this invention. The structures and synthetic methods for these compounds are provided in Materials and Methods, below.
- the term "contacting” includes in vitro, ex vivo and in vivo administration of prodrug. When done in vivo, the prodrug is administered to a subject in an effective amount.
- the term “subject” is intended to include any appropriate animal model, e.g., mouse, rat, rabbit, simian. It also includes administration to humans patients.
- Another aspect of this invention is a method for treating a subject infected with a pathogen by administering to the subject a therapeutically effective amount of a prodrug that is selectively converted to a toxin in a cell by an activating enzyme as defined herein.
- the enzyme is not necessarily overexpressed.
- an effective amount of at least one additional therapeutic agent is co-administered concurrently, previously or subsequently to administration of the substrate prodrug.
- the agent can be added to a pharmaceutically acceptable carrier and systemically or topically administered to the subject.
- Administration in vivo can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents can be found below.
- the pharmaceutical compositions can be administered orally, intranasally, parenterally or by inhalation therapy, and may take the form of tablets, lozenges, granules, capsules, pills, ampoules, suppositories or aerosol form.
- compositions can also contain other pharmaceutically active compounds or a plurality of compounds of the invention.
- a compound of the formula of the present invention also referred to herein as the active ingredient, may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parental (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated.
- a suitable dose for each of the above-named compounds is in the range of about 1 to about 100 mg per kilogram body weight of the recipient per day, preferably in the range of about 1 to about 50 mg per kilogram body weight per day and most preferably in the range of about 1 to about 25 mg per kilogram body weight per day. Unless otherwise indicated, all weights of active ingredient are calculated as the parent compound of the formula of the present invention for salts or esters thereof, the weights would be increased proportionately.
- the desired dose is preferably presented as two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day.
- sub-doses may be administered in unit dosage forms, for example, containing about 1 to about 100 mg, preferably about 1 to above about 25 mg, and most preferably about 5 to above about 25 mg of active ingredient per unit dosage form. It will be appreciated that appropriate dosages of the compounds and compositions of the invention may depend on the type and severity and stage of the disease and can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention.
- the prodrug should be administered to achieve peak concentrations of the active compound at sites of disease. This may be achieved, for example, by the intravenous injection of the prodrug, optionally in saline, or orally administered, for example, as a tablet, capsule or syrup containing the active ingredient. Desirable blood levels of the prodrug may be maintained by a continuous infusion to provide a therapeutic amount of the active ingredient within disease tissue.
- operative combinations is contemplated to provide therapeutic combinations requiring a lower total dosage of each drug that may be required when each individual therapeutic compound or drug is used alone, thereby reducing adverse effects.
- prodrugs described herein are preferentially combined with drugs that exert their toxic effect by a means other that that of the invention prodrugs.
- the prodrug ingredient While it is possible for the prodrug ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation comprising at least one active ingredient, as defined above, together with one or more pharmaceutically acceptable carriers therefor and optionally other therapeutic agents.
- Each carrier must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
- Formulations include those suitable for oral, recta, nasal, topical (including transdermal, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary administration.
- the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
- Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
- the active ingredient may also be presented a bolus, electuary or paste.
- a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
- Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g., povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent.
- Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
- the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
- Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
- compositions for topical administration may be formulated as an ointment, cream, suspension, lotion, powder, solution, past, gel, spray, aerosol or oil.
- a formulation may comprise a patch or a dressing such as a bandage or adhesive plaster impregnated with active ingredients and optionally one or more excipients or diluents.
- the formulations are preferably applied as a topical ointment or cream containing the active ingredient in an amount of, for example, about 0.075 to about 20% w/w, preferably about 0.2 to about 25% w/w and most preferably about 0.5 to about 10% w/w.
- the prodrug may be employed with either a paraffinic or a water-miscible ointment base.
- the prodrug ingredients may be formulated in a cream with an oil-in-water cream base.
- the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane- 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof.
- the topical formulations may desirably include a compound which enhances absorption or penetration of the prodrug ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.
- the oily phase of the emulsions of this invention may be constituted from known ingredients in an known manner. While this phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at lease one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat.
- the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax
- the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
- Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate.
- the choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is very low.
- the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers.
- Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
- Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the prodrug ingredient.
- the prodrug ingredient is preferably present in such formulation in a concentration of about 0.5 to about 20%, advantageously about 0.5 to about 10% particularly about 1.5% w/w.
- Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
- Formulations suitable for vaginal administration may be presented as suppositories, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the prodrug ingredient, such carriers as are known in the art to be appropriate.
- Formulations suitable for nasal administration wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
- Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebulizer include aqueous or oily solutions of the prodrug ingredient.
- Formulations suitable for parenteral administration include aqueous and non- aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs.
- the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
- Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
- Preferred unit dosage formulations are those containing a daily dose or unit, daily subdose, as herein above-recited, or an appropriate fraction thereof, of a prodrug ingredient.
- formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable of oral administration may include such further agents as sweeteners, thickeners and flavoring agents.
- Prodrugs and compositions of the formula of the present invention may also be presented for the use in the form of veterinary formulations, which may be prepared, for example, by methods that are conventional in the art.
- the agents and compositions of the present invention can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as an active ingredient in pharmaceutical compositions.
- This invention further provides a method for screening for prodrugs that are selectively converted to a toxin by an activating enzyme by providing cells that express an activating enzyme and contacting the cells with a candidate prodrug. At least one test cell expresses the pathogen's version of the enzyme (wild-type or mutated) and another test cell is a cell sample from the host organism which may, or may not express its own version of the enzyme. One then assays for conversion of the prodrug into toxic agents by the activating enzyme produced by the pathogen.
- the test cells can be procaryotic or eucaryotic cells infected with the pathogen or alternatively, transformed to express the activating enzyme. For example, a procaryotic E.
- the test cell can be an infected cell isolated from the subject, or a cultured cell infected with the pathogen.
- the cell can have a control counterpart (lacking the target enzyme), or in a separate embodiment, a counterpart genetically modified to differentially express the target enzyme, or enzymes (containing the appropriate species of target enzyme). More than one species of enzyme can be used to separately transduce separate host cells, so that the effect of the candidate drug on a target enzyme can be simultaneously compared to its effect on another enzyme or a corresponding enzyme from another species.
- a third target cell is used as a positive control because it receives an effective amount of a compound, such as, for example, the compounds shown below, which have been shown to be potent prodrugs.
- transformed cell lines such as ras-transformed NTH 3T3 cells (ATCC, 10801 University Boulevard., Manassas, VA 20110-2209, U.S.A.) are engineered to express variable and increasing quantities of the target enzyme of interest from cloned cDNA coding for the enzyme. Transfection is either transient or permanent using procedures well known in the art and described in Sambrook, et al., supra. Suitable vectors for insertion of the cDNA are commercially available from Stratagene, La Jolla, CA and other vendors.
- the level of expression of enzyme in each transfected cell line can be monitored by immunoblot and enzyme assay in cell lysates, using monoclonal or polyclonal antibody previously raised against the enzyme for immuno-detection.
- the amount of expression can be regulated by the number of copies of the expression cassette introduced into the cell or by varying promoter usage. Enzymatic assays to detect the amount of expressed enzyme also can be performed as reviewed by Carreras and Santi (1995), supra, or the methods described below.
- the test cells can be grown in small multi-well plates and is used to detect the biologic activity of test prodrugs. For the purposes of this invention, the successful candidate drug will block the growth or kill the pathogen but leave the control cell type unharmed.
- the candidate prodrug can be directly added to the cell culture media or previously conjugated to a ligand specific to a cell surface receptor and then added to the media. Methods of conjugation for cell specific delivery are well known in the art, see e.g., U.S. Patent Nos. 5,459,127; 5,264,618; and published patent specification WO 91/17424 (published November 14, 1991).
- the leaving group of the candidate prodrug can be detectably labeled, e.g., with tritium.
- the target cell or the culture media is then assayed for the amount of label released from the candidate prodrug.
- cellular uptake may be enhanced by packaging the prodrug into liposomes using the method described in Lasic, D.D. (1996) Nature 380:561-562 or combined with cytofectins as described in Lewis, J.G. et al. (1996) Proc. Natl. Acad. Sci. USA 93:3176-3181.
- each embodiment can be further modified by providing a separate target cell to act as a control by receiving an effective amount of a compound, such as, for example, the compounds shown below, which have been shown to be potent prodrugs. Agents identified by this method are further provided herein.
- the assay of the effect of the prodrug is provided by analysis of intracellular metabolites of the prodrug, as described in the Materials and Methods and Experimental Section below; the results of which are shown in Figure 4.
- the prodrug contains a detectable label that is monitored during conversion of the prodrug to toxic agent by the activating enzyme.
- the candidate substrate prodrug is detectably labeled, e.g., e.g., fluorescent marker, or a radioisotope.
- the detectable label comprises at least two or more variable isotopes of the same atom, e.g., bromine.
- NB1011 ((E)-5-(2-BromovinyI)-2'-deoxy-5 , -uridyl phenyl L- alaninylphosphoramidate (BVdU-PA, "NB1011")) was prepared as follows. A solution of BVdU (420 mg, 1.26 mmol) and imidazole (103 mg, 1.51 mmol) in 2 mL of anhydrous DMF under argon was treated dropwise with phenyl L- methoxyalaninyl phosphorochloridate (McGuigan et al. (1996) J. Med. Chem.
- Jvinyl CH-vinyl CH 13.5, JH1'-H2' ⁇ 6.8, JH2'-H3* ⁇ 5, JH3'-H4' ⁇ 0, Jalaninyl CH-alaninyl NH ⁇ 6 Hz. Spectral assignments were confirmed by 1H/1H COSY 2D NMR analysis.
- R (at the 5 -position) is or contains a leaving group which is a chemical entity that has a molecular dimension and electrophilicity compatible with extraction from the pyrimidine ring by the activating enzyme, e.g., thymidylate synthase, and which upon release from the pyrimidine ring by the enzyme, has the ability to inhibit the proliferation of the agent or cell.
- Q can be a moiety such as a sugar, carbocylic or acyclic compound, a masked phosphate or phosphoramidate derivative containing a chemical entity selected from the group consisting of sugar groups, thio-sugar groups, carbocyclic groups, and derivatives thereof.
- sugar groups include, but are not limted to, monosaccharide cyclic sugar groups such as those derived from oxetanes (4-membered ring sugars), furanoses (5-membered ring sugars), and pyranoses (6- membered ring sugars).
- furanoses examples include threo-furanosyl (from threose, a four-carbon sugar); erythro-furanosyl (from erythrose, a four-carbon sugar); ribo- furanosyl (from ribose, a five-carbon sugar); ara-furanosyl (also often referred to as arabino-furanosyl; from arabinose, a five-carbon sugar); xylo-furanosyl (from xylose, a five-carbon sugar); and lyxo-furanosyl (from lyxose, a five-carbon sugar).
- threo-furanosyl from threose, a four-carbon sugar
- erythro-furanosyl from erythrose, a four-carbon sugar
- ribo- furanosyl from ribose, a five-carbon sugar
- ara-furanosyl also often
- sugar group derivatives include “deoxy”, “keto”, and “dehydro” derivatives as well as substituted derivatives.
- thio sugar groups include the sulfur analogs of the above sugar groups, in which the ring oxygen has been replaced with a sulfur atom.
- carbocyclic groups include C 4 carbocyclic groups, C 5 carbocyclic groups, and C 6 carbocyclic groups that may further have one or more subsituents, such as -OH groups.
- Q is a ⁇ -D-ribofuranosyl group of the formula:
- R 7 is attached to the furane at the 5' position and is selected from the group consisting of H, a masked phosphate or a phosphoramidate and derivatives thereof, and wherein R 2 and R 3 are the same or different and are independently -H or - OH.
- R 4 is or contains a group selected from the group consisting of H, a halogen, alkyl, alkene, alkyne, hydroxy, -O-alkyl, -O-aryl, O-heteroaryl, -S-alkyl, -S-aryl, a cyanide, cyanate and thiocyanate halovinyl group, a halomercuric group, -S-heteroaryl, -NH 2 , -NH-alkyl, -N(alkyl) 2 , -NHCHO, -NHOH, -NHO-alkyl, NH 2 CONHO-, and NHNH 2 .
- further aspects include: wherein R 2 and R 3 are H; wherein R 2 is OH and R 3 is H; herein R 2 is H and R 3 is OH; or wherein R 2 and R 3 are OH.
- a preferred embodiment for the substituent in the R j position is one that could undergo an allylic interchange.
- the candidate therapeutic agent is a compound of the formula:
- n is 0 or an integer from 1 to 10; wherein A is a phosphorous derivative, or a compound of the formula: o P — N(CH 2 CH 2 CI) 2
- the candidate therapeutic agent is a compound of the formula:
- R 2'-deoxy-5-uridyl
- m is 0 or 1
- n is an integer from 0 to 10.
- the compounds can be in any of their enantiomeric, diasteriomeric, or stereoisomeric forms, including, for example, D- or L-forms, and can be in any stereochemical configuration, including, for example, ⁇ - or ⁇ -anomeric form.
- C5- modification of pyrimidine nucleosides and nucleotides is the formation of C5-trans- styryl derivatives by treatment of unprotected nucleotide with mercuric acetate followed by addition of styrene or ring-substituted styrenes in the presence of Li 2 PdCl 4 . Bigge et al. (1980) J. Am. Chem. Soc. 102(6):2033-2038. Pyrimidine deoxyribonucleoside triphosphates were derivatized with mercury at the 5 position of the pyrimidine ring by treatment with mercuric acetate in acetate buffer at 50° for 3 hours. Dale et al.
- 5-bromodeoxyuridine, 5-iododeoxyuridine, and their monophosphate derivatives are available commercially from Glen Research, Sterling, VA (USA), Sigma- Aldrich Corporation, St. Louis, MO (USA), Moravek Biochemicals, Inc., Brea, CA (USA), ICN, Costa Mesa, CA (USA) and New England Nuclear, Boston, MA (USA).
- Commercially-available 5-bromodeoxyuridine and 5- iododeoxyuridine can be converted to their monophosphates either chemically or enzymatically, though the action of a kinase enzyme using commercial available reagents from Glen Research, Sterling, VA (USA) and ICN, Costa Mesa, CA (USA).
- These halogen derivatives could be combined with other substituents to create novel and more potent antimetabolites.
- the present invention involves four classes of compounds activated by enzymes such as TS.
- Each class is defined by the structure of the uricil base, or modified uricil base present.
- These classes are ECTA compounds wherein: I) the base is a furano-pyrimidinone derivative of uracil; II) the base is 6-fluoro uracil; and III) the base is 4-hydrazone substituted uracil derivative, or IV) the base is uracil.
- the uracil or modified uracil derived base is used to synthesize compounds substituted with toxic leaving groups at the 5 position, attached by an electron conduit tether at this 5 position, and including an appropriate spacer moiety between the electron conduit and the toxic leaving group.
- the ECTA compounds can be unphosphorylated, 5' monophosphate, 5' phosphodiester, or 5' protected ("masked") deoxyuridines or comparable derivatives of alternative carbohydrate moieties, as described below.
- Protected 5-substituted deoxyuridine monophosphate derivatives are those in which the phosphate moiety has been blocked through the attachment of suitable chemical protecting groups. Protection of 5-substituted deoxyuridine monophosphate derivatives can improve solubility, facilitate cellular penetration, facilitate passage across the blood-brain barrier, and prevent action of cellular or extracellular phosphatases, which might otherwise result in loss of the phosphate group.
- 5-substituted uracil or uridine derivatives are administered to cells containing nucleoside kinase activity, wherein the 5-substituted uracil uridine derivative is converted to a 5-substituted uridine monophosphate derivative.
- Uridine derivatives may also be modified to increase their solubility, cell penetration, and/or ability to cross the blood-brain barrier.
- Action of thymidylate synthase upon 5-substituted uridine monophosphate derivatives can release the substituent attached to the 5-position ("leaving group") of the pyrimidine ring.
- the released substituent is then capable, either inherently or following reaction with another cellular component, of acting as a toxin or an inhibitor of cellular proliferation.
- the L and D isomers of the compounds of this invention are selected from the group consisting of the compounds having the structures shown below:
- R 1 has the formula:
- R 2 is or contains a divalent electron conduit moiety.
- R 2 is or contains a mono- or polyunsaturated electron conduit acting to conduct electrons away from the pyrimidine ring and toward the leaving group R 1 with the proviso that in compounds of class I, n can be zero.
- R 2 is selected from the group consisting of: an unsaturated hydrocarbyl group; an aromatic hydrocarbyl group comprising one or more unsaturated hydrocarbyl groups; and, a heteroaromatic group comprising one or more unsaturated hydrocarbyl groups.
- R 2 is an unsaturated hydrocarbyl group having a structure selected from the group consisting of:
- R 2 and R 3 taken together form a structure selected from the group consisting of:
- R 2 is an aromatic hydrocarbyl group having a structure selected from the group consisting of:
- R 2 is a heteroaromatic group having a structure selected from the group consisting of:
- J is a heteroatom, such as -O-, -S-, or -Se-, or a heteroatom group, such as -NH- or -NR ALK -, where ⁇ is a linear or branched alkyl having 1 to 10 carbon atoms or a cycloalkyl group having 3 to 10 carbon atoms.
- R 3 is a divalent spacer moiety, also referred to as a spacer unit.
- R 3 is a divalent spacer moiety having a structure selected from the group consisting of: R J
- R 5 is the same or different and is independently a linear or branched alkyl group having from 1 to 10 carbon atoms, or a cycloalkyl group having from 3 to 10 carbon atoms or R 5 is a halogen (F, CI, Br, I).
- R 3 is a divalent spacer moiety having a structure selected from the group consisting of:
- R 3 is a divalent spacer moiety having a structure selected from the group consisting of: R J
- n is an integer from 0 to 10 and, m is 0 or 1. In one embodiment, n is 0 or an integer from 0 to 10 and, m is 1. In one embodiment, n is 0 and m is 0. In one embodiment, when R 7 is -H, then n is not zero. In one embodiment, when R 7 is -H, then m is not zero. In one embodiment, when R 7 is -H, then n is not zero and m is not zero. In one embodiment, when R 7 is -H, then R 4 is not a halogen (i.e., -F, -CI, -Br, -I).
- R 4 when R 7 is -H, and m is zero, then R 4 is not a halogen (i.e., -F, -CI, -Br, -I). In one embodiment, when R 7 is - H, and m is zero and n is zero, then R 4 is not a halogen (i.e., -F, -CI, -Br, -I).
- R 4 is a toxophore moiety.
- the term "toxophore” shall mean a moiety which is or contains a leaving group which is a chemical entity that has a molecular dimension and electrophilicity compatible with extraction from the pyrimidine ring by thymidylate synthase, and which upon release from the pyrimidine ring by thymidylate synthase, has the ability to inhibit the proliferation of the cell or kill the cell.
- the toxophore is or contains a leaving group that is activated or released by an intracellular enzyme overexpressed in the cell.
- R 4 is or contains a group having a structure selected from the group consisting of:
- X is -CI, -Br, -I, or other potent leaving group (including, but not limited to, -CN, -OCN, and -SCN);
- Y is the same or different, and is independently - H or -F; and
- Z is the same or different and is independently -O- or -S-,
- R 8 and R 9 are lower alkyls, and
- R 10 is H or CH 3 .
- R 4 is or contains a chemical entity selected from the group consisting of: -Br, -I, -O-alkyl, -O-aryl, O-heteroaryl, -S-alkyl, -S-aryl, -S-heteroaryl, -CN, -OCN, -SCN, -NH 2 , -NH-alkyl, -N(alkyl) 2 , -NHCHO, -NHOH, -NHO-alkyl, NH 2 CONHO-, NHNH 2 , -N 3 , and a derivative of cis-platin, such as:
- Q is or contains a group which supports functional binding of the prodrug to the enzyme, e.g., TS or TK.
- Q is selected from the group consisting of:
- R is the same or different and is independently -H, F, -OH, -
- R 7 attached at the 5' position of Q, is hydrogen , a phosphate group, a phosphodiester group, a phosphoramidate group, or other phosphorus containing group.
- R 7 is a phosphoramidate group derived from an amino acid, including, for example, the twenty naturally occurring amino acids. In one embodiment, R 7 is a phosphoramidate group derived from alanine. In one embodiment, R 7 is or contains a group having the structure:
- R 7 is a phosphoramidate group derived from tryptophan. In one embodiment, R 7 is or contains a group having the structure:
- R 7 is a phosphate group. In one embodiment, R 7 is or contains a group having a structure selected from the group consisting of:
- R 7 is or contains a group having a structure selected from the group consisting of (where R is an aromatic substituent):
- the R 7 forms a cyclic group within Q.
- DMTr 4,4'-dimethoxytrityl
- Boc is t-butyloxycarbonyl
- DCC is 1,3-dicyclohexylcarbodiimide
- 4-DMAP is 4-dimemylaminopyridine
- the compound may be in any enantiomeric, diasteriomeric, or stereoisomeric form, including, D-form, L-form, ⁇ -anomeric form, and ⁇ -anomeric form.
- the compound may be in a salt form, or in a protected or prodrug form, or a combination thereof, for example, as a salt, an ether, or an ester.
- the above structures are further modified to possess thiophosphodiaziridine instead of phosphodiaziridine groups, using the methods described below.
- 5-bromodeoxyuridine, 5-iododeoxyuridine, and their monophosphate derivatives are available commercially from Glen Research, Sterling, VA (USA), Sigma-Aldrich Corporation, St. Louis, MO (USA), Moravek Biochemicals, Inc., Brea, CA (USA), ICN, Costa Mesa, CA (USA) and New England Nuclear, Boston, MA (USA).
- 5- bromodeoxyuridine and 5-iododeoxyuridine can be converted to their monophosphates either chemically or enzymatically, though the action of a kinase enzyme using commercial available reagents from Glen Research, Sterling, VA (USA) and ICN, Costa Mesa, CA (USA). These halogen derivatives could be combined with other substituents to create novel and more potent antimetabolites.
- the structures at the 5-position of uracil are referred to as the tethers because they connect the proposed leaving group (toxophore) to the heterocycle. Upon activation of the heterocycle by reaction with a Cys residue in the active site of human TS, a negative charge is conducted from the 6-position of uracil into the tether.
- the tether "spacer" between the toxin and dNMP must be unsaturated so that it can conduct the toxin-labilizing negative charge supplied by the TS-Cysteine- sulfhydryl attack.
- the vinyl, allyl, and propargyl units are simple, small, and readily accessible synthetically.
- the vinyl and allyl units have the advantage that they can be prepared in either of two non-interconvertible geometric isomeric forms. Thus, they can be used as "probes" of prodrug accommodation by the TS active site.
- the propargyl unit has the advantage of being cylindrically symmetrical, so that TS-catalyzed toxin release from this type of tether does not depend upon its orientation with respect to dUMP's uracil ring, as is the case with the vinyl and allyl molecules.
- Two distinct approaches have been taken to design several of the nucleotide- based prodrugs of this invention.
- One is based on the structure of BVDU monophosphate and features a leaving group/toxin directly attached to the terminus of a (poly)vinyl substituent at C5 of dUMP. This is the vinyl tether approach.
- the other is based on the structure of TFPe-dUMP and is similar to the first but has a methylene unit separating the leaving group/toxin and the unsaturated unit and thus contains an allyl or propargyl unit. This is the allyl tether approach.
- EdUMP 5-ethynyl-2 '-deoxyuridine 5'- monophosphate
- HOPdUMP 5-(3-hydroxy-l-propynyl)-2'deoxyuridine 5'- monophosphate
- 5-Alkylidenated 5,6-dihydrouracils similar in structure to the intermediate common to both the vinyl and allyl tether approach mechanisms have been synthesized recently (Anglada et al. (1996) J. Heterocycl. Chem. 33:1259-1270). These were shown to be highly electrophilic. Their ready reaction with ethanol to generate 5-(ethoxymethyl)uracils is a precedent for the water addition that regenerates catalytically competent TS. Even more recently, the existence of the long-elusive C5 methylene intermediate produced by TS was demonstrated by trapping studies (Barrett et al. (1998) J. Am. Chem. Soc. 120:449-450).
- TBDMS-protected propargyl- and (Z)-allytic- tethered nucleosides will serve as convenient precursors to some of the toxophore- equipped targets.
- the known O- tetrahydropyranyl ether derivative is prepared by the literature Heck coupling of an (E)-tributylstannylated ethylene (Crisp (1989) supra).
- TEPA Bis-aziridin-l-yl-phosphinic acid 3-[2-deoxyuridin-5-yl]-prop-2-ynyl ester
- furano-pyrimidinones Synthesis of furano-pyrimidinones. Synthesis of furano-pyrimidinones begins with synthesis of a C5 propargylic -alcohol-equipped 2 '-deoxyuridine. Furano-pyrmidinone compounds are then be formed from the O-tetrahydropyranyl ether derivative described above. Synthesis proceeds by reaction of the second carbon of the propargyl bond with the oxygen attached to the C4 position of the pyrimidine ring to yield a fluorescent furano-pyrimidinone which can be readily separated from the reaction mix. Such compounds provide an additional basis for synthesis of ECTA compounds through various combinations of specific electron conduits, spacers and toxic leaving groups.
- furo[2,3-d]pyrimidinone nucleosides were prepared by condensing 2',3'- di-O-p-toluoyl or 2',3'-di-O-acetyl-5-iodo-2'-deoxyuridine with 1- (tetrahydropyranyloxy)-2-propyne (Jones and Mann (1953) J. Am. Chem. Soc. 75:4048-4052) under conditions known to promote the formation of these fluorescent compounds (Barr et al. (1983) supra).
- Salient features ⁇ 8.5 (s, 1, H4), 7.4- 7.1 (m, 5, PhO), 6.36 and 6.30 (each s, each 1, H5), 6.23 (m, 1, HI'), 3.67 and 3.65 (each s, each 3, CO 2 Me), 2.69 (m, 1, H2'a), 2.10 (m, 1, H2'b), 1.35 (m, 3, alaninyl- ⁇ - Me).
- Low-resolution mass spectrum DCI-NH 3 ), m/z 525 (MH + ), 595 (MNH 4 + ).
- the 4-mtrophenyl ether derivative of 5-(3-hydroxy-l-propynyl)-2'- deoxyuridine was prepared according to a standard ether synthesis as shown below.
- TS ECTA compounds based on furano-pyrimidinones.
- Toxic R 4 leaving groups can be attached to the furan-2 methyl alcohol using methods similar to those employed to attach toxic leaving groups to the hydroxyl on the C5 propargyl uridine compound, as explained with the synthesis of the TEPA and ThioTEPA derivatives described above.
- a variety of alternative toxic leaving groups apparent to one skilled in the art, are envisioned.
- modifications to the length and composition of the R 2 electron conduit component and of the composition of the R 3 spacer element are also envisioned.
- TS ECTA compounds based on furano-pyrimidinones can also consist of variously modified "Q" moeities.
- Many 5-substituted 2'-deoxyuridines are not substrates for human TK, but interestingly 5-(4-hydroxy-l-butynyl)-2'-deoxyuridine was found to be an exception (Barr et al. (1981) supra).
- the ECTA compounds can have a free 5' hydroxyl, a 5' monophosphate, or a 5' phosphoramidate group attached to alternative carbohydrate groups.
- a novel method for synthesis of such phosphoramidate compounds is accomplished by reacting a 2-deoxy 3 '-hydroxy, 5 '-hydroxy unprotected nucleotide with a phosphochloridate in the presence of an HC1 scavenger.
- the phosphochloridate comprises a phosphorus substituent which is derived from an amino acid such as alanine.
- the phosphochloridate can be phenyl-L-methoxyalanine phosphorochloridate.
- a 4-hydrazone substitution on the uracil facilitates formation of the thiol with TS. It is important that the resulting nucleotide-thiol (TS) intermediate rearranges in such a way as to release the altered nucleotide which can be accomplished passively via hydrolysis.
- ECTA compounds are synthesized by addition of alternative electron conduits, spacer moieties and toxic leaving groups to either the C6 fluoro-uridine base or the C4 hydrazone modified pyrimidine. Methods described above for synthesis of 2 '-deoxyuridine based ECTA compounds can again be employed for synthesis of such molecules.
- Salts, esters, and ethers of the above compounds disclosed herein are also within the scope of this invention.
- Salts of the prodrugs of the present invention may be derived from inorganic or organic acids and bases.
- acids include hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycollic, lactic, salicyclic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic and benzenesulfonic acids.
- acids such as oxalic
- bases include alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and compounds of formula NW 4 + , wherein W is C, ⁇ alkyl.
- salts include: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pahnoate, pectinate, persulfate, phenylproprionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate
- salts include anions of the compounds of the present invention compounded with a suitable cation such as Na + , NH 4 + , and NW 4 + (wherein W is a C M alkyl group).
- a suitable cation such as Na + , NH 4 + , and NW 4 + (wherein W is a C M alkyl group).
- salts of the compounds of the present invention will be pharmaceutically acceptable.
- salts of acids and bases which are non- pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.
- the phosphate esters may be further esterified by, for example, a C,. 20 alcohol or reactive derivative thereof, or by a 2,3-di-(C 6.24 )acyl glycerol.
- any alkyl moiety present advantageously-contains from 1 to 18 carbon atoms, particularly from 1 to 6 carbon atoms, more particularly from 1 to 4 carbon atoms.
- Any cycloalkyl moiety present in such esters advantageously contains from 3 to 6 carbon atoms.
- Any aryl moiety present in such esters advantageously comprises a phenyl group.
- lyxo-furanosyl prodrug derivatives of the present invention include, for example, those with chemically protected hydroxyl groups (e.g., with O-acetyl groups), such as 2'-O- acetyl-lyxo-furanosyl; 3'-0-acetyl-lyxo-furanosyl; 5'-O-acetyl-lyxo-furanosyl; 2',3'- di-O-acetyl-lyxo-furanosyl and 2',3',5'-tri-O-acetyl-lyxo-furanosyl.
- chemically protected hydroxyl groups e.g., with O-acetyl groups
- Ethers of the compounds of the present invention include methyl, ethyl, propyl, butyl, isobutyl, and sec-butyl ethers.
- the substrate may not be chemically related to pyrimidines or folates, but rather synthesized based upon known parameters of rational drug design. (See Dunn et al. (1996) J. Med. Chem. 39:4825).
- GGGCAGATCCAACACATCC-3' (corresponding to bases 208-226 of thymidylate synthase cDNA sequence, Genbank Accession No. X02308), antisense primer 5'- GGTCAACTCCCTGTCCTGAA-3' (corresponding to bases 564-583), ⁇ -actin sense primer 5'-GCCAACACAGTGCTGTCTG-3' (corresponding to bases 2643-2661 of ⁇ -actin gene sequence, Genbank Accession No. M10277) and antisense primer 5'- CTCCTGCTTGCTGATCCAC-3' (corresponding to bases 2937-2955).
- RNAs were isolated using Tri pure isolation reagent (obtained from Boehringer Mannheim Corp., Indianapolis, IN), followed manufactureis protocol. To monitor for possible DNA contamination, the primers for amplification of ⁇ -actin were designed to span the exon4/intron5/exon5 junction. Genomic DNA template leads to a 313 bp ⁇ -actin fragment, and cDNA template generates a 210 bp product.
- Reverse transcriptions were performed, using Superscript preamplification system (Gibco/BRL, Gaithersburg, MD). 3 ⁇ g total RNA was applied in a volume of 20 ⁇ l buffer to conduct reverse transcription reaction, followed manufacture's protocol.
- PCR reactions were performed in a volume of 96 ⁇ l, containing 5 ⁇ l of cDNA mixture from reverse transcription reaction, 3 mM MgCl 2 , 50 mM KC1, 20 mM Tris- CI, pH 8.4, 0.2 mM of each dNTP, 0.3 ⁇ M of thymidylate synthase sense and antisense primers and 5 units of Tag DNA polymerase (obtained from Promega, Madison, WI).
- the reaction mixtures were incubated at 94°C for 3 min, followed by 9 cycles of 1 min incubation at 94°C , 1 min incubation at 58°C , and then 1 min incubation at 72°C.
- HT1080 cells were grown in PRMI1640 medium supplemented with 10% fetal calf serum, and transfected with GFP-TS expression vector. 48 hours after, transfection cells were tripsinized and replated in culture medium containing 750 ⁇ g ml G418. After selection with G418 for two weeks, surviving cells were sorted based upon fluorescence expression. One clone with higher fluorescence expression (named as TSH/HT1080) and one clone with lower fluorescence expression (named as TSL/HT1080) were selected and expanded into cells lines. The stable HT1080 cells transfected with pEGFP-C3 were used as control.
- GFP-TS expression vector A cDNA fragment encoding conserved region of human thymidylate synthase (amino acids 23 to 313) was obtained by PCR amplification using following primers: Sense primer, 5'- CGGAAGCTTGAGCCGCGTCCGCCGCA-3' and antisense primer, 5'- GAAGGTACCCTAAACAGCCATTTCCA-3'.
- the cDNA was cloned into Hindlll and Kpnl sites of mammalian expression vector pEGFP-C3 ( Clontech Laboratories. Inc., Palo Alto, CA), in-frame with GFP sequence. The cDNA insert was confirmed by DNA sequencing.
- F Western Blot analysis.
- the separated proteins were transferred onto PVDF membrane, followed by immunoblot with human thymidylate synthase monoclonal primary antibody (manufactured by NeoMarkers, Fremont, CA) and horseradish peroxidase linked sheep anti-mouse Ig secondary antibody (obtained from Amersham, England).
- the ECL plus kit (Amersham) was used for detection of immunoreactivity.
- the bands corresponding to thymidylate synthase were quantified and normalized to that of tubulin by Molecular Dynamics Storm. The quantified expression levels were expressed as values relative to that of cell strain CCD 18co.
- G. TS Activity Assay by Tritium Release from dUMP- 3 H Cells were plated in 24 well plates to a density of 30,000 cells/plate and incubated for 16 hours to allow adhesion to the plastic surface of the plate. Immediately prior to the thymidylate synthase assay, the media was replaced with RPMI+10% dialyzed fetal calf serum. 0.5 ⁇ Ci of 5-[ 3 H] deoxyuridine was added to each well, and plates were incubated for 60 minutes at 37 °C without additional CO 2 . [ 3 H] release was measured by adsorbing 5-[ 3 H]deoxyuridine to activated charcoal (10% in 1 x PBS) for 5 minutes at room temperature. After centrifugation for 5 minutes at 13,000 RPM, the amount of [ 3 H] in the supernatant was determined by liquid scintillation counting.
- Concentration vs. relative fluorescence units were plotted, and sigmoid curves were fit using the Hill equation.
- IC 50 indicated by the inflection point of the curve, is the concentration at which growth is inhibited by 50%).
- infectious agents of this class include, but are not limited to, Mycobacterium sp., Chlamydia sp., Rickettsia sp. And Pheumocystis sp. pathogenic Enterococcus sp., Moraxella sp., Haemophilis sp., and Staphylococcus sp.
- Colony formation assays can be used to measure cytotoxicty of metabolized ECTA compounds on extracellular pathogenic bacteria or other pathogens on plates or in liquid media (Miller, J.H. A Short Course in Bacterial Genetics: A Laboratory Manual and Hardbook for E. Coli and Related Bacteria, Cold Spring Harbor Press (1992)).
- MCF7-TDX were transferred to a 384 well assay plate at 500 cells in 25 ⁇ L complete medium per well. After 24 hours (day 0), 25 ⁇ L complete medium containing a combination of NB1011 in doubling serial dilutions from lmM and tomudex at discrete concentrations (0,1,10,100,1000 nM) were added in duplicate. Drug exposure time was 120 hours (day 5) after which growth inhibition was measured with alamarBlue as described above in Growth Inhibition Studies.
- the column Ni 2+ His Bind metal chelation column was washed with 20 mM Tris pH 7.9, 5 mM imidazole, 0.5 M NaCl; thymidylate synthase activity was eluted with 20 mM Tris pH 7.9, 60 mM imidazole, 0.5 M NaCl.
- Thymidylate synthase assays were done in 96 well Costar UV transparent plates in a reaction volume of 200 ⁇ l, consisting of 40 mM Tris pH 7.5, 25 mM MgCl 2 , 1 mM EDTA, 25 mM- mercaptoethanol, 125 M dUMP, and 65 ⁇ M N5, NlO-methylene tetrahydrofolate indicated.
- Tetrahydrofolate stock solutions were prepared by dissolving tetrahydrofolic acid (Sigma) directly into 0.2 M Tris pH 7.5, 0.5 M-mercaptoethanol; stock solutions were stored at -80 °C.
- NlO-methylene tetrahydrofolate was prepared by adding 12 ⁇ l of 3.8% formaldehyde to 1 ml of a 0.65 mM solution of tetrahydrofolate and incubating for 5 minutes at 37 °C. N5, NlO-methylene tetrahydrofolate was kept on ice and used within 2 hours of preparation.
- Enzyme kinetic constants K,,, and V ⁇ were determined for the human thymidylate synthase substrates dUMP and BVdUMP using the enzyme assay conditions described above. The initial rates of the enzyme reactions was determined by measuring the increase in A 340 for the reaction with dUMP, and decrease in A 294 for the reaction with BVdUMP. The catalytic efficiency of the enzyme (K ⁇ /K,,,) was calculated from the kinetic constants K Promote, and V, ⁇
- MCF-7 breast cancer cells were selected in vitro for resistance to Tomudex by continuous exposure to stepwise increases in TDX concentrations up to 2.0 ⁇ M.
- a resistant subline was selected for resistance to NBIOI 1 by continuous exposure of the parental MCF7 TDX cell line to medium supplemented without TDX but with 50 ⁇ M NBIOI 1, a concentration approximately 16 times higher than the IC 50 for NB1011 in the parental MCF7 TDX cell line.
- TS protein level and IC 50 for 5-FU, TDX, and NB 1011 were determined for the resultant MCF7 TDX/ 1011 cell line as described in "Materials and Methods" by western blot and the alamarBlue cytotoxicity assay, respectively.
- 373(l):41-4) support the concept that, once BVDU is converted to the monophosphate in cells (e.g. via herpes virus thymidine kinase), then it binds to and inactivates the HuTS enzyme during processing.
- the actual reaction of human TS with BVdUMP has never been adequately characterized.
- Santi and colleagues (Barr et al. (1983) supra) utilized a bacterial TS for their work to show generation of product from the BVdUMP + TS reaction, and DeClercq and colleagues utilized cells and cell lysates, not purified human TS (Balzarini (1987) supra); Balzarini (1993) supra; Balzarini (1995) supra).
- TS The best characterized reaction of TS is the conversion of dUMP to dTMP.
- This reaction involves the transfer of a methylene group from N5,N10-methylene tetrahydrofolate (THF) to the C-5 position of dUMP (Carreras CW (1995) supra).
- THF N5,N10-methylene tetrahydrofolate
- 5F-dUMP the uridylate mimic
- a second well characterized inhibitor of TS activity is Tomudex, which occupies the folate binding site of the TS homodimer, prevents the binding of THF, and blocks TS activity in the cell (Drake et al.
- THF is not required for the conversion of BVdUMP to fluorescent product(s).
- Table 1 demonstrate that THF stimulates the production of fluorescent product(s) in the BVdUMP reaction with rHuTS . This result is not expected from the earlier data reporting that THF has no effect on this reaction (Barr et al. (1983) supra), and illustrates a potentially important possibility that cofactors, or cofactor agonists, like leucovorin , could modulate the reaction of BVdUMP with human TS.
- the increased efficiency of BVdUMP utilization by the human enzyme as compared to the L. casei enzyme also establishes that species specific substrates are possible and can be designed. These substrates are applied in the treatment of infections (either viral or cell-mediated) in which the infectious agent expresses a TS enzyme distinct from that encoded by the host.
- viral infections examples include hepatitis virus, herpesviruses, or other viruses that express their own TS enzyme; and bacterial infections, especially drug resistant bacteria like multiply resistant staphyloccoccus aureus, and other infectious agents for example Pneumocystis carnii and Plasmodium falciparum.
- bacterial infections especially drug resistant bacteria like multiply resistant staphyloccoccus aureus, and other infectious agents for example Pneumocystis carnii and Plasmodium falciparum.
- the ability to specifically inhibit heterologous enzymes via binding to species specific regions on the surface of L. casei vs. human TS has recently been reported (Stout (1999) Biochemistry 38(5):1607-17 and Costi et al. (1999) J. Med. Chem. 42(12) :2112-2124).
- NB 1011 is converted to the monophosphate in tumor cells
- NBIOI 1 is converted from the phosphoramidate to the monophosphate form in cells, as a prerequisite for binding to TS.
- the proposed pathway for unmasking the phosphate of NB 1011 , its binding to TS and conversion to toxic metabolites is shown in Figure 5.
- the tumor/normal cell screen As an initial step in characterizing the biological activity of NB1011, a large series of normal and tumor cell types were tested in the alamar blue assay for sensitivity to both NBIOI 1 and 5-fluorouracil.
- Therapeutic index is calculated as the ratio of the average IC 50 for normal cells to the average IC 50 for tumor cells. All assays were done at least three times.
- NB1011 has met the primary design goal for TS ECTA compounds, i.e. increased potency on tumor cells vs. normal cell types.
- NBIOI 1 is about 2-fold more cytotoxic to tumor cells vs. normal cells, while 5FU is 3-fold more toxic to normal cells than it is to tumor cells. The total benefit of
- a critical tactic that allows for selection of chemotheraputics with a positive therapeutic index is screening of activity on both normal and tumor cell types. This approach has not been consistently employed in the field of new cancer drug discovery. For instance, screening of new candidate compounds on normal cell types is not part of the National Cancer Institute's screening procedure (Curt (1996) Oncologist 1 (3):II-III).
- NB 1011 does not inactivate TS in vivo
- the results described above indicate that BVdUMP, generated intracellularly from NB1011, is unlikely to inactivate TS during its transformation to product(s). However, the cell free system is different from the intracellular milieu.
- cell-based assays for TS activity were performed. In these experiments exogenous 5-( 3 H) deoxyuridine is added to cell culture medium and the release of tritiated water is monitored (Carreras et al. (1995) supra, and Roberts (1966) Biochem. 5(11) 3546-3548).
- Figure 7 shows that the presence of NBIOI 1 in cell culture media reduces the rate at which [ 3 H] 2 O is released from 5-[ 3 H]dUMP.
- NB1011 -treated cells were allowed to briefly recover in fresh culture media, then assayed for TS activity. Cells that have been allowed to recover in culture media lacking NBIOI 1 have the same level of TS activity as untreated cells. This result supports the proposal that NBIOI 1 does not irreversibly inactivate the TS enzyme following intracellular processing.
- TS participates in NBIOI 1 -mediated cytotoxicity was established using several approaches: 1). the activity of NBIOI 1 was examined on normal colon cells vs. high TS expressing, 5FU-resistant, tumor cells; 2). transfection of TS into a tumor cell background, and generating clonal derivatives which differ primarily by TS expression level, but are otherwise very similar; and 3). use of a specific inhibitor of TS, Tomudex, to decrease intracellular TS activity.
- TS in HT1080 tumor cells enhances their sensitivity to NB1011.
- Activation of NB1011 requires several steps. These include cell penetration conversion to the nucleotide monophosphate, binding to TS, and subsequent toxic metabolism. The precise mechanisms of cell penetration and conversion are not fully defined. Cell entry may depend in part on nucleoside transport mechanisms (Cass et al. (1998) Biochem. Cell Biol. 76(5):761-70). Similarly, processing from the phosphoramidat+e to the monophosphate employs poorly defined mechanisms (Abraham et al. (1996) J. Med. Chem. 8:39(23):4589-4575.
- Tomudex is a chemotherapeutic that acts primarily via inhibition of TS. If NBIOI 1 exerts cytotoxicity via the TS enzyme, then inhibition of TS with Tomudex should decrease NBIOI 1 -mediated cytotoxicity.
- Tomudex-resistant MCF7 cells which overexpress TS 11-fold compared to the parental MCF7 cell line, were exposed to NBIOI 1 in the presence of increasing concentrations of TDX. Cells were plated and exposed to indicated concentrations of compound(s) as described in the Materials and Methods, above.
- NB1011 To further characterize the intracellular metabolism of NB1011, combination experiments with leucovorin (LV; 5-formyltetrahydrofolate) were performed. This experiment was initiated because we had observed that THF stimulates production of fluorescent product(s) in the cell-free reaction of BVdUMP and rHuTS. It was hypothesized that if the fluorescent products are related to the cytotoxic effects of NB1011, then enhancing intracellular levels of THF by providing LV in the culture media would also enhance NB1011-mediated cytotoxic effects. Surprisingly, in the presence of 3 ⁇ M LV, NBIOI 1 activity on the H630R10 cell line was diminished by more than 90%, compared to NB1011 alone, as determined in the alamar blue assay.
- LV leucovorin
- LV which supplements intracellular reduced folate pools
- NB1011 may work in part by diminishing these pools.
- LV or a metabolite could directly impact the metabolism of BVdUMP by interfering with its interaction with TS.
- Methotrexate (MTX), LV or Tomudex (TDX).
- NB1011 activity may be modulated by other chemotherapeutics.
- rescue of NBIOI 1 -treated cells is feasible by providing LV, similar to the LV rescue from MTX.
- LV rescue occurs via supplementation of intracellular folate pools, which are diminished via MTX inhibition of dihydrofolate reductase and TS.
- the cell line results shown in Figure 2 may suggest an additional similarity between the TS and HER2/NEU systems.
- the similarity is that each has a similar overexpression requirement (about 4-fold) which predicts more aggressive disease for both TS and HER2/NEU overexpressing patients (Johnston et al. (1994) J. Clin. Oncol. 12:2640-2647).
- NB1011 is active against 5FU and Tomudex-resistant colon and breast tumor cell lines. Because NBIOI 1 has promising anticancer activity, it is important to compare it with other chemotherapeutics with respect to safety. The utility of NBIOI 1 in the treatment of cancer is further strengthened when it is compared with Tomudex, a chemotherapeutic which, like 5FU, is often used to treat colon and breast cancer, among other malignancies. The results show that while NB 1011 is more than 10-fold less toxic than
- TDX vs. normal cells CCDl ⁇ co
- TDX vs. normal cells CCDl ⁇ co
- TDX R tumor cells Similar results have been obtained for other TDX- resistant tumor cell lines.
- the low level of toxicity vs. normal cells and the high activity vs. TDX R tumor cells supports the application of NB1011 to drug resistant cancers that overexpress TS. 3.
- NBIOI 1 is more dependent upon TS protein levels than TS activity as measured by tritium release from dUMP- 3 H.
- Enzyme kinetics were done as described in Methods. Data for Lactobacillus casei are derived from Barr et al. (1983) supra. The rHuTS was prepared as described in Methods, above.
- Thymidylate synthase reactions containing enzyme inhibitors or cofactor were incubated at 30°C as described in Materials and Methods, and the initial rates of the enzyme reaction were determined by measuring the increase in relative fluorescence units at 340 nm excitation, 595 nm emission for the BVdUMP reactions, or increase in A 340 for the dUMP reaction.
- a cDNA encoding rHuTS was subcloned into ventor pEGFP-C3, in-frame with GFP.
- the construct was transfected into HT1080 cells and selected with G418 (750 ug/ml) in order to obtain clones that stably express fusion rHuTS.
- Individual cells were cloned based upon high or low fluorescence expression as described in Methods. *TS levels were determined by using Westem blot analysis, the quantified expression levels were expressed as values relative to that of cell strain CCD18co.
- the Tomudex rescue assay (alamar blue) was done with TDX-resistant MCF7 breast tumor cells as described in Methods, 'Fold Protection" was calculated as the ratio of IC « 50» with and without added TDX.
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WO2001007088A2 (en) * | 1999-07-22 | 2001-02-01 | Newbiotics, Inc. | Methods for treating therapy-resistant tumors |
WO2002007780A2 (en) * | 2000-07-20 | 2002-01-31 | Newbiotics, Inc. | Methods for identifying therapeutic targets |
US6573247B1 (en) * | 1997-04-28 | 2003-06-03 | University College Cardiff Consultants Limited | Anti-viral pyrimidine nucleoside analogues |
US6677314B2 (en) | 1997-10-30 | 2004-01-13 | The United States Of America As Represented By The Department Of Health And Human Services | Nucleosides for imaging and treatment applications |
US6987180B2 (en) | 2002-02-07 | 2006-01-17 | President And Fellows Of Harvard College | Uridine analogs and techniques for making and using |
US8329664B2 (en) | 2006-05-09 | 2012-12-11 | University College Cardiff Consultants Limited | Anti-viral pyrimidine nucleoside derivatives |
US8551965B2 (en) | 2000-04-17 | 2013-10-08 | University College Cardiff Consultants Limited | Anti-viral pyrimidine nucleoside analogues |
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