Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
WATER-SOLUBLE COMPOUND
Document Type and Number:
WIPO Patent Application WO/2005/043159
Kind Code:
A1
Abstract:
A process for treating a biological organism, comprising the steps in which a cell cycle arresting drug is administered to the organism to produce synchronized cells, the microtubules within the synchronized cells are stabilized by means of a microtubule stabilizing agent, and the synchronized cells with the stabilized microtubules are then contacted with mechanical vibrational energy.

Inventors:
TUSZYNSKI JACK (CA)
GREENWALD HOWARD J (US)
CURRY STEPHEN H (US)
GOSS KENDRICK (US)
Application Number:
PCT/US2004/038076
Publication Date:
May 12, 2005
Filing Date:
October 29, 2004
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
TECHNOLOGY INNOVATIONS LLC (US)
TUSZYNSKI JACK (CA)
GREENWALD HOWARD J (US)
CURRY STEPHEN H (US)
GOSS KENDRICK (US)
International Classes:
A61K41/00; A61K45/06; A61K49/00; G01N33/53; G01N33/566; A61N7/00; (IPC1-7): G01N33/53; A61K49/00; G01N33/566
Other References:
GEORGOULIAS V. ET AL.: "Docetaxel (texotere) in the treatment on non-small cell lung cancer", CURRENT MEDICINAL CHEMISTRY, vol. 9, 2002, pages 869 - 877, XP008071572
LINDER J.R. ET AL.: "Noninvasive ultrasound imaging of inflammation using microtubules targeted to activated leukocytes", CIRCULATION, vol. 102, no. 22, 28 November 2000 (2000-11-28), pages 2745 - 2750, XP008071569
Attorney, Agent or Firm:
O'shaughnessy, Brian P. (P.O. Box 1404 Alexandria, VA, US)
Download PDF:
Claims:
We claim:
1. A process for treating a biological organism, comprising the steps of : (a) administering a cell cycle arresting drug to said organism, thereby producing synchronized cells within such organism, (b) administering a microtubule stabilizing drug to said organism, thereby producing synchronized cells whose microtubules have been stabilized within said organism, and (c) contacting said synchronized cells whose microtubules have been stabilized with mechanical vibrational energy.
2. The process as recited in claim 1, wherein said mechanical vibrational energy has an excitation source frequency in the range of from about 1 hertz to about 10 Gigahertz.
3. The process as recited in claim 1, wherein said cell cycle arresting drug synchronizes tumor cells with respect to cell cycle progression.
4. The process as recited in claim 3, wherein said cell cylcle arresting drug is selected from the group consisting of gemcitabine, cisplatin, carboplatin, cyclophosphamide, topoisomerase inhibitor, etopside, 5fluoroacil, doxorubicin, methotrexate, hydroxyurea, 3'azido3' deoxythymidine, and mixtures thereof.
5. The process as recited in claim 3, wherein said cell cycle arresting drug is gemcitabine.
6. The process as recited in claim 1, wherein said cell cycle arresting drug synchronizes said cells in metaphase.
7. The process as recited in claim 1, wherein said cell cycle arresting. drug synchronizes said cells in anaphase.
8. The process as recited in claim 6, wherein at least about 30 percent of said cells are synchronized in metaphase.
9. The process as recited in claim 6, wherein at least about 50 percent of said cells are synchronized in metaphase.
10. The process as recited in claim 6, wherein at least about 70 percent of said cells are synchronized in metaphase.
11. The process as recited in claim 1, wherein said mechanical vibrational energy is ultrasound.
12. The process as recited in claim 11, wherein said synchronized cells are contacted withsaid ultrasound only aftr at least 25 minutes after said cell cycle arresting drug has been administered to said organism.
13. The process as recited in claim 11, wherein said synchronized cells are contacted withsaid ultrasound only after at least 60 minutes after said cell cycle arresting drug has been administered to said organism.
14. The process as recited in claim 11, wherein said synchronized cells are contacted withsaid ultrasound only after at least 240 minutes after said cell cycle arresting drug has been administered to said organism.
15. The process as recited in claim 11, wherein said synchronized cells are contacted withsaid ultrasound only after at least 48 hours after said cell cycle arresting drug has been administered to said organism.
16. The process as recited in claim 11, wherein said microtubule stabilizing drug is a laulimalidge microtubule stabilizing agent.
17. The process as recited in claim 11, wherei said microtubule stabilizing drug is a coumarin compouond.
18. The process as recited in claim 11, wherein said ultrasound has a frequency of from about 270 to about 420 kilohertz.
19. The process as recited in claim 11, wherein said ultrasound has an intensity of from about 10 to about 30 watts per square meter.
20. The process as recited in claim 11, wherein said microtubule stabiling drug is paclitaxel.
21. The process as recited in claim 11, wherein said ultrasound has a frequency of from about 50 megahertz to about 2 gigahertz.
22. The process as recited in claim 11, wherein said ultrasound has a frequency of from about 100 megahertz to about 1 gigahertz.
23. The process as recited in claim 11, wherein the power of said ultrasound is at least about 0.01 watts per square meter.
24. The process as recited in claim 11, wherein the power of said ultrasound is at least about 0.1 watts per square meter.
25. The process as recited in claim 11, wherein said ultrasound has a frequency of form about 100 kilohertz to about 500 kilohertz.
26. The process as recited in claim 11, wherein said ultrasound has a frequency of from about 110 to about 200 kilohertz.
27. The process as recited in claim 11, wherein said ultrasound has a frequency of from about 130 to about 170 kilohertz.
28. The process as recited in claim 11, wherein the power of said ultrasound is from about 1 to about 30 watts per square meter.
29. The process as recited claim 11, wherein the power of said ultrasound is form about 5 to about 15 watts per square meter.
Description:
WATER-SOLUBLE COMPOUND Technical Field A water-soluble magnetic anti-mitotic compound with a water-solubility of at least 100 micrograms per milliliter, a molecular weight of at least 150 grams per mole, a mitotic index factor of at least 10 percent, a positive magnetic susceptibility of at least 1,000 x 10-6 cgs, and a magnetic moment of at least 0.5 bohr magnetrons, wherein said compound is comprised of at least 7 carbon atoms and at least one inorganic atom with a positive magnetic susceptibility of at least 200 x 10-6 cgs.

Background Art Paclitaxel is a complex diterpenoid that is widely used as an anti-mitotic agent; it consists of a bulky, fused ring system and an extended side chain that is required for its activity.

See, e. g. , page 112 of Gunda I. Georg's"Taxane Anticancer Aents: Basic Science and Current Status, "ACS Symposium Series 583 (American Chemical Society, Washington, D. C. , 1995).

The aqueous solubility of paclitaxel is relatively low. Thus, as is disclosed at page 112 of such Georg text, estimates of paclitaxel solubility vary widely, ranging from about 30 micrograms per milliliter and about 7 micrograms per milliliter to less than 0.7 micrograms per milliter.

The molecular weight of paclitaxel is in excess of 700; this relatively high molecular weight is one factor that, according to the well-known"rule of 5,"contributes to paclitaxel's poor water solubility.

The"rule of 5"was set forth by Christopher A. Lipinski et al. in an article entitled "Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings, "Adv. Drug Delivery Rev. , 1997,23 (1-3), 3-25. In this article, it was disclosed that :"In the USAN set we found that the sum of Ns and Os in the molecular formula was greater than 10 in 12% of the compounds. Eleven percent of compounds had a MWT of over 500.... The'rule of 5'states that: poor absorption of permeation is more likely where: A. There are more than 5 H-bond donors (expressed as the sum of OHs and NHs); B. The MWT is over 500; C. The LogP is over 500...; D. There are more than 10 H- bond acceptors (expressed as the sum of Ns and Os)." The Lipinksi"rule of 5"has also erroneously been referred to as the"Pfizer rule of 5," as is illustrated by United States patent 6,675, 136, the entire disclosure of which is hereby incorporated by reference into this specification. As is disclosed in such patent, "To further

illustrate the versatility of the present technique, we also introduce the concept of'anchor' objects. Anchor objects are molecules situated at the corners of a region of the drug space that is defined by Pfizer's'rule of 5'. This rule has been empirically derived by a computer analysis of known drugs, as described by Christopher A. Pfizer and co-workers in Adv. Drug Delivery Rev. , vol. 23, pp. 3-25 (1997). The'rule of 5"is focused on drug permeability and oral absorption.... According to Pfizer's"rule of 5", LIPO and HBDON are between 0 and 5, HBACC is between 0 and 10, and M. W. has a maximum of 500." The problems that high molecular weight compounds have with poor water solubility are discussed in United States patent 6,667, 048 of Karel J. Lambert et al. , which discloses an "emulsion vehicle for a poorly soluble drug. "In the"background of the invention"section of this patent, it is disclosed that:"Hundreds of medically useful compounds are discovered every year, but clinical use of these drugs is possible only if a drug delivery vehicle is developed to transport them to their therapeutic target in the human body. This problem is prticularly critical for drugs requiring intraveneous injection in order to reach their therapeutic target or dosage but which are water insoluble or poorly water insoluble. For such hydrophobic compounds, direct injection may be impossible or highly dangerous, and can result in hemolysis, phlebitis, hypersensitivity, organ failure and/or death. Such compounds are termed by pharmacists 'lipophilic,''hydrophobic,'or in their most difficult form,'aamphiphobic'.... A few examples of therapeutic substances in these categories are ibuprofen, diazepam, grisefulvin, cyclosporin, cortisone, proleukin, cortisone, proleukin, etoposide and paclitaxel...." As is also disclosed in United States patent 6,667, 048,"Administration of chemotherapuetic or anti-cancer agents is particularly problematic. Low solubility anti-cancer agents are difficult to solubulize and supply at therapeutically useful levels. On the other hand, water-soluble anti-cancer agents are generally taken up by both cancer and non-cancer cells thereby exhibiting non-specificity.... Efforts to improve water-solubility and comfort of administration of such agents have not solved, and may have worsened, the two fundamental problems of cancer chemotherapy: 1) non-specific toxicity, and 2) rapid clearance from the bloodstream by non-specific mechanisms. In the case of cytotoxins, which form the majority of currently available chemotherapies, these two problems are clearly related. Whenever the therapeutic is taken up by noncancerous cells, a diminished amount of thedrug remains available to treat the cancer, and more importantly, the normal cell ingesting the drug is killed." As is also disclosed in United States patent 6,667, 048, "The chemotherapeutic must be present throughout the affected tissue (s) at high concentration for a sustained period of time so that it may be taken up by the cancer cells, but not at so high a concentration that normal cells

are injured beyond repair. Obviously, water-soluble molecules can be admistered in this way, but only by slow, continuous infusion and monitoring, aspects which entail great difficulty, expense and inconvenience." It does not appear that the prior art has provided a water-soluble anti-mitotic agent that is capable of solving the problems discussed in United States patent 6,667, 048. It is an object of this invention to provide such an agent. In particular, and in one embodiment, it is an object of this invention to provide a magentic anti-mitotic composition that can be directed to be more toxic to cancer cells than normal cells. Furthermore, and in another embodiment, it is another object of this invention to provide a delivery system that will provide a chemotherapeutic agent at a high concentration for a sustained period of time but not at such a high concentration that a substantial number of normal cells are injured beyond repair.

Disclosure of the invention In accordance with one embodiment of this invention, there is provided a water-soluble magnetic anti-mitotic compound with a water-solubility of at least 100 micrograms per milliliter, a molecular weight of at least 150 grams per mole, a mitotic index factor of at least 10 percent, a positive magnetic susceptibility of at least 1,000 x 10-6 cgs, and a magnetic moment of at least 0. 5 bohr magnetrons, wherein said compound is comprised of at least 7 carbon atoms and at least one inorganic atom with a positive magnetic susceptibility of at least 200 x 10-6 cgs.

In accordance with yet another embodiment of this invention, there is provided a compound with molecular weight of at least about 550, a water solubility of at least about 10 micrograms per milliliter, a pKa dissociation constant of from about 1 to about 15, and a partition coefficient of from about 1.0 to about 50.

Brief description of the drawings The invention will be described with referene to the specification and the enclosed drawings, in which like numerals refer to like elements, and wherein: Figure 1 is a schematic illustration of one preferred implantable assembly of the invention; Figure 2 is a schematic illustration of a flow meter that may be used in conjunction with the implantable assembly of claim 1; Figure 3 is a flow diagram of one preferred process of the invention; Figure 4 is a flow diagram of another preferred process of the invention; and Figure 5 is a flow diagram of yet another preferred process of the invention.

Best Mode for Carrying Out the Invention

The magnetic anti-mitotic compound of this invention is particularly well-adapted to bind either to tubulin isotypes and/or microtubules comprised of such isotypes and/or various proteins that are involved in microtubule dynamics..

In this section of the specification, a preferred compound is discussed. The preferred compound of this embodiment of the invention is an anti-mitotic compound. Anti-mitotic compounds are known to those skilled in the art. Reference may be had, e. g. , to United States patents 6,723, 858 (estrogenic compounds as anti-mitotic agents), 6,528, 676 (estrogenic compounds as anti-mitotic agents), 6,350, 777 (anti-mitotic agents which inhibit tubulin polyumerization), 6,162, 930 (anti-mitotic agents which inhibit tubulin polymerization), 5,892, 069 (estrogenic compounds as anti-mitotic agents), 5,886, 025 (anti-mitotic agents which inhibit tubulin polymerization), 5,661, 143 (estrogenic compounds as anti-mitotic agents), 3,997, 506 (anti-mitotic derivatives of thiocolchicine), and the like.

These prior art anti-mitotic agents may be modified, in accordance with the process of this invention, to make them"magnetic, "as that term is defined in this specification, In the next section of this specification, a process for modifying prior art taxanes to make them "magnetic"is described.

Preparation and use of magnetic taxanes hi this portion of the specification, applicant will describe the preparation of certain magnetic taxanes that may be used in one or more of the processes of his invention. The process that is ued to make such taxanes magnetic and/or water soluble may also be used to make other anti-mitotic compounds magnetic and/or water soluble.

In one embodiment of the invention, a biologically active substrate is linked to a magnetic carrier particle. An external magnetic field may then be used to increase the concentration of a magnetically linked drug at a predetermined location. Drug-Linker Magnetic Carrier One method for the introduction of a magnetic carrier particle involves the linking of a drug with a magnetic carrier. While some naturally occurring drugs inherently carry magnetic particles (ferrimycin, albomycin, salmycin, etc. ), it is more common to generate a synthetic analog of the target drug and attach the magnetic carrier through a linker.

FUNCTIONALIZED TAXANES Paclitaxel and docetaxel are members of the taxane family of compounds. A variety of taxanes have been isolated from the bark and needles of various yew trees In one embodiment of the invention, such a linker is covalently attached to at least one of the positions in taxane. R, O 0 OH RO 0 OH 10 R2 NH 0 ./ 13 15 i 6 PhO"" : H : o NO""' HO = Ac0 : H O 14 oh _ _ H 1 2 H 5 PhC00 HD OB co 20 Ri=Ac, R2= PhCO, paclitaxel R = H, 10-DEACETYLBACCATIN III taxa-4 (20), 11 (12)-diene R1=H, R2=Boc, docetaxel R=Ac, BACCATINIII

It is well known in the art that the northern hemisphere of taxanes has been altered without significant impact on the biological activity of the drug. Reference may be had to Chapter 15 of Taxane Anticancer Agents, Basic Science and Current Status, edited by G.

George et al. , ACS Symposium Series 583, 207th National Meeting of the American Chemical Society, San Diego, CA (1994). Specifically the C-7, C-9, and C-10 positions of paclitaxel have been significantly altered without degrading the biological activity of the parent compound. Likewise the C-4 position appears to play only a minor role. The oxetane ring at C-4 to C-5 has been shown to be critical to biological activity. Likewise, certain functional groups on the C-13 sidechain have been shown to be of particular importance.

In one embodiment of the invention, a position within paclitaxel is functionalized to link a magnetic carrier particle. A number of suitable positions are presented below. It should be understood that paclitaxel is illustrated in the figures below, but other taxane analogs may also be employed. Ac o AcO<O OMagnetic Carrier Ph''NH O/ph/'NH O zu Ph'v 0"" : Fi : Ph'v _O"" : Fi _ O OH H _ pH HO = Ac0 ÕH HO _ 1° ÕH HO AcÕ PhCOO Magnetic Carrier PhCOO Attachment at C-4 Attachment at C-7 Magnetic Carrier Magnetic Carrier 1 OH O pH Ac0 O''O Ac (5 C) H PHCOO PHCOO PhCOÕ PhCOO PhC00 PhC00 Attachment at C-9 Attachment at C-7 and C-9 Magnetic Carrier OH Ph''NN O/ Ph-'NH 0 \1 j Ph/\4Ot ~ H ° OH HO-AcÕ PhCO0 Attachment at C-10 0 AcO 0 OH Magnetic Carder Ac0 O ON Ph''IJH/ Ph'v-O""' : H ; O Ph'_NH O/ OH AcO ph'v _pv" : hi ; Ph NH 0 Magnetic Carrier PhCOO Attachment at C-19 Attachment at C-20

Attachment at C-4 C-4 taxane analogs have been previously generated in the art. A wide range of methodologies exist for the introduction of a variety of substituents at the C-4 position. By way of illustration, reference may be had to"Synthesis and Biological Evaluation of Novel C-4 Aziridine-Bearing Paclitaxel Analogs"by S. Chen et al. , J. Med. Chem. 1995, vol 38, pp 2263. AcO 0 OTES AcO ° OTES Bz, NH 0 HO% 0 HO-AcÒ ÕTES HO _ O Bzw Bzw 7-TES baccatin R J. Med. Chem. 1995, vol 38, pp 2263 AcO 0 OTES (1) p-NO2C6H40COCI W4 t 1 (2) Removal of C1, C7, and C13 protecting groups (3) Protection of C7 HO"" : H _ O TESO%",... 0 (4) ethanolamine OH 0 OH = O DMSO-HÕ OAN~ BzÕ H

The secondary (C-13) and tertiary (C-1) alcohols of 7-TES baccatin were protected using the procedure of Chen (J. Org. Chem. 1994, vol 59, p 6156) while simultaneously unmasking the alcohol at C-4. The resulting product was treated with a chloroformate to yield the corresponding carboxylate. Removal of the silyl protecting groups at C-1, C-7, and C-13, followed by selective re-protection of the C-7 position gave the desired activated carboxylate.

The compound was then treated with a suitable nucleophile (in the author's case, ethanolamine) to produce a C-4 functionalized taxane. The C-13 sidechain was installed using standard lactam methodology.

This synthetic scheme thus provides access to a variety of C-4 taxane analogs by simply altering the nucleophile used. In one embodiment of the instant invention, the nucleophile is selected so as to allow the attachment of a magnetic carrier to the C-4 position.

Attachment at C-7 The C-7 position is readily accessed by the procedures taught in United States Patent 6,610, 860. The alcohol at the C-10 position of 10-deacetylbaccatin III was selectively protected. The resulting product was then allowed to react with an acid halide to produce the corresponding ester by selectively acylating the C-7 position over the C-13 alcohol. Standard lactam methodology allowed the installation of the C-13 sidechain. In another embodiment, baccatin III, as opposed to its deacylated analog, is used as the starting material. 0 NO O pH 0 HO 0 OH 0 OTES 0 \4 ?/I I > </1. \X1, 1 1 (I) C-10 Protection lactam coupling (2) Propionyl chloride (2) Propionyl chloride Ru%""0 HO = Ac0 HO°"' : H ; O HO _ Ac0 OBz 10-DEACETYLBACCATIN m ÕBZ Naturally Occuring United States Patent 6, 610, 860

Other C-7 taxane analogs are disclosed in United States Patents 6,610, 860 ; 6,359, 154; and 6,673, 833, the contents of which are hereby incorporated by reference.

Attachment at C-9 It has been established that the C-9 carbonyl of paclitaxel is relatively chemically inaccessible, although there are exceptions (see, for example, Tetrahedron Lett. Vol 35, p 4999). However, scientists gained access to C-9 analogs when 13-acetyl-9-dihydrobaccatin III was isolated from Taxus candidensis (see J. Nat. Products, 1992, vol 55, p 55 and Tetrahedron Lett. 1992, vol 33, p 5173). This triol is currently used to provide access to a variety of such C- 9 analogues.

In chapter 20 of Taxane Anticancer Agents, Basic Science and Current Status, (edited by G. George et al. , ACS Symposium Series 583, 207th National Meeting of the American Chemical Society, San Diego, CA (1994) ) Klein describes a number of C-7/C-9 taxane analogs.

One of routes discussed by Klein begins with the selective deacylation of 13-acetyl-9- dihydrobaccatin III, followed by the selective protection of the C7 alcohol as the silyl ether. A standard lactam coupling introduced the C-13 sidechain. The alcohols at C-7 and C-9 were sufficiently differentiated to allow a wide range of analogs to be generated."hi contrast to the sensitivity of the C-9 carbonyl series under basic conditions, the 9 (R) -dihydro system can be treated directly with strong base in order to alkylate the C-7 and/or the C-9 hydroxyl groups." Ac0 OH pH Ac0 OR OR O Ph'_NH . Ph'Uoll"'0 HO E Ac0 Ph o : H-O OBz OH HO, Ac6 HO AC6 13-ACETYL-9-DIHYDROBACCATIN III Naturally Occuring (1) MeLi (2) TESCI AcO OH OTES lactam coupling Boc, UH / Ph "". : Fi ; HO""' ; H : O pp HO'Ac0 OBz HO-AcÒ OBz One skilled in the art may adapt Klein's general procedures to install a variety of magnetic carriers at these positions. Such minor adaptations are routine for those skilled in the art.

Attachment at C-7 and C-9 Klein also describes a procedure wherein 13-acetyl-9-dihydrobaccatin III is converted to 9-dihydrotaxol. Reference may be had to"Synthesis of 9-Dihydrotaxol: a Novel Bioactive Taxane"by L. L. Klein in Tetrahedron Lett. Vol 34, pp 2047-2050. An intermediate in this synthetic pathway is the dimethylketal of 9-dihydrotaxol. AcO OH OH p Ac0 O''O \ r (1) 1, 3-diol protection Ph NH 0 /Ph NH O \g/ Ac0""' Å 2\ O (2) deacylation of C (3) lactam coupling 0 OBz OH HO AcO- 13-ACETYL. 9-DIHYDROBACCATIN III OBz Natumlly Occuring

In one embodiment, the procedure of Klein is followed with a carbonyl compound other than acetone to bind a wide variety of groups to the subject ketal. Supplemental discussion of C-9 analogs is found in"Synthesis of 9-Deoxotaxane Analogs"by L. L. Klein in Tetrahedron Lett. Vol 35, p 4707 (1994).

Attachment at C-10 In one embodiment of the invention, the C-10 position is functionalized using the procedure disclosed in United States Patent 6,638, 973. This patent teaches the synthesis of paclitaxel analogs that vary at the C-10 position. A sample of 10-deacetylbaccatin III was acylated by treatment with propionic anhydride. The C-13 sidechain was attached using standard lactam methodology after first performing a selective protection of the secondary alcohol at the C-7 position. In one embodiment of the invention, this procedure is adapted to allow access to a variety of C-10 analogues of paclitaxel. 0 HO OH OH -0 0 ODMPS po 0 ODMPS Wnnnoo, y, tr ; a CeCi Ixtamcouping --/ ' (2) DMPSCI _ HO'H 0 RO""0 HO j Ac0 H° : H _ HO ; Ac0 ÕBz HO AcÕ ÕBz 10-DGACETYLI3ACCATIN in OBZ NatwallyDcwdng

In one embodiment an anhydride is used as an electrophile. In another embodiment, an acid halide is used. As would be apparent to one of ordinary skill in the art, a variety of electrophiles could be employed. 0 0 0 gMagnetic Carrler FMagnetic Carrier magnetic Carrier lu H LG=leaving group LG H R

SIDEROPHORES In one embodiment, a member of the taxane family of compounds is attached to a magnetic carrier particle. Suitable carrier particles include siderophores (both iron and non-iron containing), nitroxides, as well as other magnetic carriers.

Siderophores are a class of compounds that act as chelating agents for various metals.

Most organisms use siderophores to chelate iron (III) although other metals may be exchanged for iron (see, for example, Exchange of Iron by Gallium in Siderophores by Emergy, Biochemistry 1986, vol 25, pages 4629-4633). Most of the siderophores known to date are either catecholates or hydroxamic acids.

R--N--X' B, 11 "y ? N R 3 Hydroxamic acid-based siderophores catecholate Representative examples of catecholate siderophores include the albomycins, agrobactin, parabactin, enterobactin, and the like. HO HA oh ho O NH OH N, I OH 1 enterobactin (enterchelin) / HN, _,,, N HN N OH O N O HO N O OH X=OH, agrobacn H X=H, parabactin O O OH

Examples of hydroxamic acid-based siderophores include ferrichrome, ferricrocin, the albomycins, ferrioxamines, rhodotorulic acid, and the like. Reference may be had to Microbial Iron Chelators as Drug Delivery Agents by M. J. Miller et al., Acc. Chem. Res. 1993, vol 26, pp 241-249; Structure of Des (diserylglycyl) ferrirhodin, DDF, a Novel Siderophore from Aspergillus ochraceous by M. A. F. Jalal et al. , J. Org. Chem. 1985, vol 50, pp5642-5645; Synthesis and Solution Structure of Microbial Siderophores by R. J. Bergeron, Chem. Rev.

1984, vol 84, pp 587-602; and Coordination Chemistry and Microbial Iron Transport by K. N.

Raymond, Acc. Chem. Res. , 1979, vol 12, pp 183-190. The synthesis of a retrohydroxamate analog of ferrichrome is described by R. K. Olsen et al. in J. Org. Chem. 1985, vol 50, pp 2264- 2271. 0 H . c H N"IlelFe HO OH O O-N N_H O O "IN'o O-N N 0 o , y"r"° oH "0 R/ N vH Oh H O R=H, Y=O albomycin Sl R=H, ferrichrome Ranz Y=NCONH2 albomycin 82 R=H, Y=NH, albomycin e

In"Total Synthesis of Desferrisalmycin" (M. J. Miller et al. in J. Am. Chem. Soc. 2002, vol 124 pp 15001-15005), a natural product is synthesized that contains a siderophore. The author states"siderophores are functionally defined as low molecular mass molecules which acquire iron (III) from the environment and transport it into microganisms. Because of the significant roles they play in the active transport of physiologically essentially iron (III) through microbe cell members, it is not surprising that siderophores-drug conjugates are attracting more and more attention from both medicinal chemists and clinical researchers as novel drug delivery systems in the war against microbial infections, especially in an area of widespread emergency of multidrug-resistance (MDR) strains. There have been three families of compounds identified as natural siderophore-drug conjugates, including ferrimycin, albomycin, and salmycin."In a related paper, Miller describes the use of siderophores as drug delivery agents (Acc. Chem. Res.

1993, vol 26, pp 241-249. Presumably, the siderophore acts as a"sequestering agents [to] facilitate the active transport of chelated iron into cells where, by modification, reduction, or siderophore decomposition, it is released for use by the cell. "Miller describes the process of tethering a drug to a sidrophore to promote the active transport of the drug across the cell membrane.

In"The Preparation of a Fully Differentiated'Multiwarhead'Sidrophore Precursor", by M. J. Miller et al (J. Org. Chem. 2003, vol 68, pp 191-194) a precursor is disclosed which allows for a drug to be tethered to a sidrophore. In one embodiment, the route disclosed by Miller is employed to provide a variety of siderophores of similar structure. The synthesis of similar hydroxamic acid-based siderophores is discussed in J. Org. Chem. 2000, vol 65 (Total Synthesis of the Siderophore Danoxamine by M. J. Miller et al. ), pp 4833-4838 and in the J. of Med. Chem. 1991, vol 32, pp 968-978 (by M. J. Miller et al.).

A variety of fluorescent labels have been attached to ferrichrome analogues in"Modular Fluorescent-Labeled Siderophore Analogues"by A. Shanzer et al. in J. Med. Chem. 1998, vol 41,1671-1678. The authors have developed a general methodology for such attachments. I fluorescent I----R I- o ---'--' 0 3 ii O 3

As discussed above, functionalized ferrichrome analogs have been previous generated, usually using basic amine acids (glycine). In one embodiment, functionality is introduced using an alternative amine acid (such as serine) in place of the central glycine residue. This provides a functional group foothold from which to base a wide variety of analogs. Using traditional synthetic techniques, various linkers are utilized so as to increase or decrease the distance between the magnetic carrier and the drug.

As would be apparent to one of ordinary skill in the art, the above specified techniques are widely applicable to a variety of substrates. By way of illustration, and not limitation, a number of magnetic taxanes are shown below. 0 0 H H zon O N V _N O 0 H O <t N O %'/I LN, e O Np ; ; Fe. OO N HN OO L 0''16 /O O NW N ' N/o o 0 0 OH RIO 00 O/N /O O O a OH RO O p p "NH 0 \ t NH 0 \/J h OH HO AcO HO Ac (5 BzO Bzb R2= PhCO, paclitaxel analog R1=Ac, R2= PhCO, paclitaxel analog R2=Boc, docetaxel analog R1=H, R2=Boc, docetaxel analog H O Ri0 O OH NEZ I'o 7 R2,, UH R1o olO O} Nh to OHA 0-N ; e 0 N 0 0- Ri0 O 0 H RaNH O / RIO 0 1 0 0 Ph'v-O""' : H _\ HN N'O ÕH HO-AcÕ o= ; oO-Fé t\o Bu0 //,. BzO/I., ; 4/N 6-N N-H R1=Ac, R2= PhCO, paclitaxel analog H'SZNX R1=H, R2=Boc, docetaxel analog o h o R1=Ac, R2= PhCO, paclitaxel analog R1 =H, R2=Boc, docetaxel analog

NITROXIDES Another class of magnetic carriers is the nitroxyl radicals (also known as nitroxides).

Nitroxyl radicals a"persistent"radials that are unusually stable. A wide variety of nitroxyls are commercially available. Their paramagnetic nature allows them to be used as spin labels and spin probes.

6 6 6 N N N N N'\ N I N-O OH O NH2 C02Me TEMPO TEMPOL TENTONS TEMPAMINE TMOZ TEMPAMINE TMOZ In addition to the commercially available nitroxyls, other paramagnetic radical labels have been generated by acid catalyzed condensation with 2-Amino-2-methyl-1-propanol followed by oxidation of the amine.

0 HOHZ mCPBA 0 N-H 0 N-0. Ri TsOH FRz RRz Cl I N p O O N Rq0 O p - OH RzNH O RzNH O/ bozo Ph'v O"'° : H : O Ph = O"" : hi O HO = Ac0 OH HO = Ac0 OH _ Bz0 Bz0 R2= PhCO, paclitaxel analog R1=Ac, R2= PhCO, paclitaxel analog R2=Boc, docetaxel analog R1=H, R2=Boc, docetaxel analog RIO 0OH O RZ NH O/ 'nu R O p OH HO = Ac0 O Ph ol 0 / OH HO AcÕ r Bz0 R =Ac, R2= PhCO, paclitaxel analog R1=Ac, R2= PhCO, paciitaxel analog R1=Ac, R2= PhCO, paclitaxel analog R1 =H, R2=Boc, docetaxel analog R1 =H, R2=Boc, docetaxel analog Nit +N N O R. O, N t) n HO 0 RX Ph z 0 RNH O R, 00'"'Y" Ph' ; O"" : Fi ; O : OH HO = Ac0 Ph'v'O""' : H ? O BzO ÕH HO AcO BzO R, =Ac, R2= PHCO, paclitaxel analog RI=AC, R2= PHCO, paclitaxel analog R1=H, R2=Boc, docetaxel analog R1=H, R2=Boc, docetaxel analog "''R1=H, R2=Boc, docetaxe) ana) og One of ordinary skill in the art could use the teachings of this specification to generate a wide variety of suitable carrier-drug complexes. The following table represents but a small sampling of such compounds.

R1 R2 I x=o, NH, NR, etc. N3 N2 F1, Y=CH2, n=0 to 20 H Ac COPh F1, Y=CH2, Ac n=0 to 20 Ac COPh F1, Y=CH2, Ac H n=0 to 20 COPh F1, Y=CH2, Ac H Ac n=0 to 20 H H Ac Boc F1, Y=CH2, n=0 to 20 H Ac Boc F1, Y=CH2, H n=0 to 20 Ac Boc F1, Y=CH2, H H n=0 to 20 Boc F1, Y=CH2, H H Ac n=0 to 20 F1, Y=NH or NR, n=0 to 20 H Ac COPh F1, Y=NH or Ac NR, n=0 to 20 Ac COPh F1, Y=NH or Ac NR, n=0 to 20 COPh R1 R2 R3 R4 F1, Y=NH or Ac H Ac NR, n=0 to 20 H H Ac Boc F1, Y=NH or NR, n=0 to 20 H Ac Boc F1, Y=NH or H NR, n=0 to 20 Ac Boc F1, Y=NH or H H NR, n=0 to 20 Boc F1, Y=NH or H H Ac NR, n=0 to 20 N1, n=0 to 20 H Ac COPh Ac N1, n=0 to 20 Ac COPh Ac H N1, n=0 to 20 COPh Ac H Ac N1, n=0 to 20 H H Ac Boc N1, n=0 to 20 H Ac Boc H N1, n=0 to 20 Ac Boc H H N1, n=0 to 20 Boc H H Ac N1, n=0 to 20 N2, n=0 to 20, X=O or NH H Ac COPh N2, n=0 to 20, X=O or Ac NH Ac COPh N2, n=0 to 20, X=O or Ac H NH COPh N2, n=0 to 20, X=O or Ac H Ac NH H H Ac Boc N2, n=0 to 20, X=O or NH H Ac Boc N2, n=0 to 20, X=O or H NH Ac Boc R1 R2 R3 R4 N2, n=0 to 20, X=O or H H NH Boc N2, n=0 to 20, X=O or H H Ac NH N3, n=0 to 20, X=O or NH H Ac COPh N3, n=0 to 20, X=O or Ac NH Ac COPh N3, n=0 to 20, X=O or Ac H NH COPh N3, n=0 to 20, X=O or Ac H Ac NH H H Ac Boc N3, n=0 to 20, X=O or NH H Ac Boc N3, n=0 to 20, X=O or H NH Ac Boc N3, n=0 to 20, X=O or H H NH Boc N3, n=0 to 20, X=O or H H Ac NH F2 or F3 H Ac COPh Ac F2 or F3 Ac COPh Ac F2 or F3 COPh Ac H Ac F2 or F3 F2 or F3 H Ac Boc R1 R2 R3 R4 H F2 or F3 Ac Boc H H F2 or F3 Boc H H Ac F2 or F3

The prior disclosure illustrates how one may modify prior art taxanes to make them magnetic. As will be apparent to those skilled in the art, one may similarly modify other modifiable prior art anti-mitotic compounds to make them magnetic.

Other modifiable prior art compounds Many anti-mitotic compounds that may be modified in accordance with the process of this invention are described in the prior art. One of these compounds is discodermolide ; and it is described in United States patent 6,541, 509, the entire disclosure of which is hereby incorporated by reference into this specification. Reference may be had, e. g. , to column 10 of such paent and to the references 10,11, 12, and 13 cited in such patent.

The reference 12 in United States patent 6,541, 509 is to an article by R. J. Kowalski et al. , "The Microtubule-Stabilizing Agent Discodermolide Competitively Inhibits the Binding of Paclitaxel (Taxol) to Tubulin Monomers,..."Mol. Pharacol. 52: 613-22,1997. At page 2 of the Kowalski et al. patent, a formula for discodermolide is presented with 29 numbered carbon atoms (see Figure 1).

Elsewhere in this specification, applicants teach how to make"magnetic taxanes"by incorporating therein various linker groups and/or siderophores. The same linker groups and/or siderphores may be utilized via subsgtantially the same process to make the discodermolide magnetic in the same manner.

As is disclosed elsewhere in this specification, siderphores are a class of compounds that act as chelating agents for various metals. When used to make"magnetic taxanes, "they are preferably bound to either the C7 and/or the C10 carbons of the paclitaxels. They can similarly be used to make"magnetic discodermolides,"but in this latter case they should be bonded at the C 17 carbon of discodermolide, to which a hydroxyl group is bound. The same linker that is used to link the C7/C 10 carbon of the taxane to the siderphore may also be sued to link the C 17 carbon of the discodermolde to the siderphore.

In one embodiment, the"siderohophoric group"disclosed in United States patent 6,310, 058, the entire disclosure of which is hereby incorporated by reference into this specification, is utilized. The siderophoric group is of the formula

--(CH2) m--N (OH)--C (O)--(CH2) n--(CH=CH) o--CH3, wherein m is an integer of from 2 to 6, n is 0 or an integer of from 1 to 22, and o is 0 or an integer 1 to 4, provided that m+o is no greater than 25.

In another embodiment, "magentic epothilone A"and/or"magentic epotilone B"is also made by a similar process. As is also disclosed in the Figure 1 of the Kowalski et al. article (see page 614), and in the formula depicted, the epothilone A exists when, in such formula, the alkyl group ("R") is hydrogen, whereas the epothilone B exists when, in such formula, the alkyl group is methyl. In either case, one can make magnetic analogs of these compounds by using the same siderophores and the same linkers groups but utilzing them at a different site. One may bind such siderophores at either the number 3 carbon (which which a hydroxyl group is bound) and/or the number 7 carbon (to which another hydroxyl group is bound.).

Without wishing to be bound to any particular theory, applicants believe that the binding of the siderphores at the specified carbon sites imparts the required magnetic properties to such modified materials without adversely affecting the anti-mitotic properteis of the material. In fact, in some embodiment, the anti-mitotic properties of the modified magnetic materials surpass the anti-mitotic properties of the unmodified materials.

This is unexpected; for, if the same linker groups and/or siderophores are used to bind to other than the specified carbon atoms, materials with no or subtantially poorer anti-mitotic properties are produced.

Thus, e. g. , and referring to the magnetic taxanes described elsewhere in this speficification (and also to Figure 1 of the Kowalski et al. article), one should not link such siderphores to to any carbons on the pendant aromatic rings. Thus, e. g. , and referring to the discodermolide structure, one shouldnot link siderphores to any of 1,2, 3, or 4 carbon atoms.

Thus, e. g. , and referring to the epothilones, one should not link the siderphores to any carbonon the ring structure containing sulfur and nitrogen.

By way of further illustration, and referring to United States patents 5,504, 074, 5,661, 143,5, 892,069, 6, 528, 676, and 6,723, 858 (the entire disclosure of each of which is hereby incorporated by reference into this specification), one may modify estradiol and estradiol metabolites to make them magnetic in accordance with the process of this invention.

As is disclosed in United States patent 6,723, 858 (the entire disclosure of which is hereby incorporated by reference into this specification, "Cell mitosis is a multi-step process that includes cell division and replication (Alberts, B. et al. In The Cell, pp. 652-661 (1989); Stryer, E. Biochemistry (1988)). Mitosis is characterized by the intracellular movement and segregation of organelles, including mitotic spindles and chromosomes. Organelle movement

and segregation are facilitated by the polymerization of the cell protein tubulin. Microtubules are formed from. alpha. and B tubulin polymerization and the hydrolysis of guanosine triphosphate (GTP). Microtubule formation is important for cell mitosis, cell locomotion, and the movement of highly specialized cell structures such as cilia and flagella." As is also disclosed in United States patent 6,723, 858, "Microtubules are extremely labile structures that are sensitive to a variety of chemically unrelated anti-mitotic drugs. For example, colchicine and nocadazole are anti-mitotic drugs that bind tubulin and inhibit tubulin polymerization (Stryer, E. Biochemistry (1988) ). When used Cell mitosis is a multi-step process that includes cell division and replication (Alberts, B. et al. In The Cell, pp. 652-661 (1989); Stryer, E. Biochemistry (1988)). Mitosis is characterized by the intracellular movement and segregation of organelles, including mitotic spindles and chromosomes. Organelle movement and segregation are facilitated by the polymerization of the cell protein tubulin.

Microtubules are formed from. alpha. and B tubulin polymerization and the hydrolysis of guanosine triphosphate (GTP). Microtubule formation is important for cell mitosis, cell locomotion, and the movement of highly specialized cell structures such as cilia and flagella.

Microtubules are extremely labile structures that are sensitive to a variety of chemically unrelated anti-mitotic drugs. For example, colchicine and nocadazole are anti-mitotic drugs that bind tubulin and inhibit tubulin polymerization (Stryer, E. Biochemistry (1988)). When used alone or in combination with other therapeutic drugs, colchicine may be used to treat cancer (WO-9303729-A, published Mar. 4, 1993 ; J 03240726-A, published Oct. 28, 1991), alter neuromuscular function, change blood pressure, increase sensitivity to compounds affecting sympathetic neuron function, depress respiration, and relieve gout (Physician's Desk Reference, Vol. 47, p. 1487, (1993))." As is also disclosed in United States patent 6,723, 858,"Estradiol and estradiol metabolites such as 2-methoxyestradiol have been reported to inhibit cell division (Seegers, J.

C. et al. J. Steroid Biochem. 32,797-809 (1989); Lottering, M-L. et al. Cancer Res. 52,5926- 5923 (1992); Spicer, L. J. and Hammond, J. M. Mol. and Cell. Endo. 64,119-126 (1989); Rao, P. N. and Engelberg, J. Exp. Cell Res. 48, 71-81 (1967) ). However, the activity is variable and depends on a number of in vitro conditions. For example, estradiol inhibits cell division and tubulin polymerization in some in vitro settings (Spicer, L. J. and Hammond, J. M. Mol. and Cell. Endo. 64,119-126 (1989) ; Ravindra, R. , J. Indian Sci. 64 (c) (1983)), but not in others (Lottering, M-L. et al. Cancer Res. 52,5926-5923 (1992); Ravindra, R. , J. Indian Sci. 64 (c) (1983)). Estradiol metabolites such as 2-methoxyestradiol will inhibit cell division in selected in vitro settings depending on whether the cell culture additive phenol red is present and to what

extent cells have been exposed to estrogen. (Seegers, J. C. et al. Joint NCI-IST Symposium.

Biology and Therapy of Breast Cancer. Sep. 25, Sep. 27,1989, Genoa, Italy, Abstract A 58). alone or in combination with other therapeutic drugs, colchicine may be used to treat cancer (WO-9303729-A, published Mar. 4,1993 ; J 03240726-A, published Oct. 28,1991), alter neuromuscular function, change blood pressure, increase sensitivity to compounds affecting sympathetic neuron function, depress respiration, and relieve gout (Physician's Desk Reference, Vol. 47, p. 1487, (1993)).

As is also disclosed in United States patent 6,723, 858, estradiol and estradiol metabolites such as 2-methoxyestradiol have been reported to inhibit cell division (Seegers, J.

C. et al. J. Steroid Biochem. 32,797-809 (1989); Lottering, M-L. et al. Cancer Res. 52,5926- 5923 (1992); Spicer, L. J. and Hammond, J. M. Mol. and Cell. Endo. 64,119-126 (1989); Rao, P. N. and Engelberg, J. Exp. Cell Res. 48, 71-81 (1967) ). However, the activity is variable and depends on a number of in vitro conditions. For example, estradiol inhibits cell division and tubulin polymerization in some in vitro settings (Spicer, L. J. and Hammond, J. M. Mol. and Cell. Endo. 64,119-126 (1989); Ravindra, R. , J. Indian Sci. 64 (c) (1983)), but not in others (Lottering, M-L. et al. Cancer Res. 52,5926-5923 (1992); Ravindra, R. , J. Indian Sci. 64 (c) (1983)). Estradiol metabolites such as 2-methoxyestradiol will inhibit cell division in selected in vitro settings depending on whether the cell culture additive phenol red is present and to what extent cells have been exposed to estrogen. (Seegers, J. C. et al. Joint NCI-IST Symposium.

Biology and Therapy of Breast Cancer. Sep. 25, Sep. 27,1989, Genoa, Italy, Abstract A 58).

In one preferred embodiment, the modifiable anti-mitotic agent is an anti-microtubule agent. In one aspect of this embodiment, and referring to United States patent 6,689, 803 at columns 5-6 thereof (the entire disclosure of which patent is hereby incorporated by reference into this specification), representative anti-microtubule agents include, e. g.,".... taxanes (e. g., paclitaxel and docetaxel), campothecin, eleutherobin, sarcodictyins, epothilones A and B, discodermolide, deuterium oxide (D2 O), hexylene glycol (2-methyl-2,4-pentanediol), tubercidin (7-deazaadenosine), LY290181 (2-amino-4- (3-pyridyl)-4H-naphtho (1, 2-b) pyran-3- cardonitrile), aluminum fluoride, ethylene glycol bis- (succinimidylsuccinate), glycine ethyl ester, nocodazole, cytochalasin B, colchicine, colcemid, podophyllotoxin, benomyl, oryzalin, majusculamide C, demecolcine, methyl-2-benzimidazolecarbamate (MBC), LY195448, subtilisin, 1069C85, steganacin, combretastatin, curacin, estradiol, 2-methoxyestradiol, flavanol, rotenone, griseofulvin, vinca alkaloids, including vinblastine and vincristine, maytansinoids and ansamitocins, rhizoxin, phomopsin A, ustiloxins, dolastatin 10, dolastatin 15, halichondrins and halistatins, spongistatins, cryptophycins, rhazinilam, betaine, taurine,

isethionate, HO-221, adociasulfate-2, estramustine, monoclonal anti-idiotypic antibodies, microtubule assembly promoting protein (taxol-like protein, TALP), cell swelling induced by hypotonic (190 mosmol/L) conditions, insulin (100 nmol/L) or glutamine (10 mmol/L), dynein binding, gibberelin, XCH01 (kinesin-like protein), lysophosphatidic acid, lithium ion, plant cell wall components (e. g. , poly-L-lysine and extensin), glycerol buffers, Triton X-100 microtubule stabilizing buffer, microtubule associated proteins (e. g. , MAP2, MAP4, tau, big tau, ensconsin, elongation factor-1-alpha (EF-l. alpha. ) and E-MAP-115), cellular entities (e. g. , histone H1, myelin basic protein and kinetochores), endogenous microtubular structures (e. g. , axonemal structures, plugs and GTP caps), stable tubule only polypeptide (e. g. , STOP145 and STOP220) and tension from mitotic forces, as well as any analogues and derivatives of any of the above.

Within other embodiments, the anti-microtubule agent is formulated to further comprise a polymer." The term"anti-microtubule, "as used in this specification (and in the specification of United States patent 6,689, 803), refers to any"... protein, peptide, chemical, or other molecule which impairs the function of microtubules, for example, through the prevention or stabilization of polymerization. A wide variety of methods may be utilized to determine the anti-microtubule activity of a particular compound, including for example, assays described by Smith et al.

(Cancer Lett 79 (2): 213-219,1994) and Mooberry et al. , (Cancer Lett. 96 (2): 261-266,1995) ; " see, e. g. , lines 13-21 of column 14 of United States patent 6, 689, 803. One preferred method, utilizing the anti-mitotic factor, is described in this specification.

An extensive listing of anti-microtubule agents is provided in columns 14,15, 16, and 17 of United States patent 6, 689, 803 ; and one or more of them may be modified them in accordance with the process of this invention to make them magnetic. These anti-microtubule agents include"... taxanes (e. g., paclitaxel (discussed in more detail below) and docetaxel) (Schiff et al. , Nature 277: 665-667,1979 ; Long and Fairchild, Cancer Research 54: 4355-4361, 1994; Ringel and Horwitz, J. Natl. Cancer Inst. 83 (4): 288-291, 1991; Pazdur et al., Cancer Treat. Rev. 19 (4) : 351-386,1993), campothecin, eleutherobin (e. g. , U. S. Pat. No. 5,473, 057), sarcodictyins (including sarcodictyin A), epothilones A and B (Bollag et al. , Cancer Research 55: 2325-2333,1995), discodermolide (ter Haar et al., Biochemistry 35: 243-250,1996), deuterium oxide (D2 O) (James and Lefebvre, Genetics 130 (2): 305-314,1992 ; Sollott et al. , J.

Clin. Invest. 95: 1869-1876, 1995), hexylene glycol (2-methyl-2,4-pentanediol) (Oka et al. , Cell Struct. Funct. 16 (2): 125-134, 1991), tubercidin (7-deazaadenosine) (Mooberry et al., Cancer Lett. 96 (2): 261-266,1995), LY290181 (2-amino-4- (3-pyridyl)-4H-naphtho (1, 2-b) pyran-3- cardonitrile) (Panda et al. , J. Biol. Chem. 272 (12): 7681-7687, 1997; Wood et al. , Mol.

Pharmacol. 52 (3): 437-444,1997), aluminum fluoride (Song et al. , J. Cell. Sci. Suppl. 14: 147- 150, 1991), ethylene glycol bis- (succinimidylsuccinate) (Caplow and Shanks, J. Biol. Chem.

265 (15): 8935-8941,1990), glycine ethyl ester (Mejillano et al. , Biochemistry 31 (13): 3478- 3483,1992), nocodazole (Ding et al. , J. Exp. Med. 171 (3): 715-727,1990 ; Dotti et al. , J. Cell Sci. Suppl. 15: 75-84,1991 ; Oka et al., Cell Struct. Funct. 16 (2): 125-134,1991 ; Weimer et al., J. Cell. Biol. 136 (1), 71-80,1997), cytochalasin B (Illinger et al. , Biol. Cell 73 (2-3): 131-138, 1991), colchicine and CI 980 (Allen et al., Am. J. Physiol. 261 (4 Pt. 1) : L315-L321,1991 ; Ding et al. , J. Exp. Med. 171 (3): 715-727,1990 ; Gonzalez et al. , Exp. Cell. Res. 192 (1) : 10-15, 1991 ; Stargell et al. , Mol. Cell. Biol. 12 (4): 1443-1450,1992 ; Garcia et al. , Antican. Drugs 6 (4): 533- 544,1995), colcemid (Barlow et al., Cell. Motil. Cytoskeleton 19 (1) : 9-17, 1991 ; Meschini et al. , J. Microsc. 176 (Pt. 3): 204-210,1994 ; Oka et al. , Cell Struct. Funct. 16 (2): 125-134, 1991), podophyllotoxin (Ding et al. , J. Exp. Med. 171 (3): 715-727,1990), benomyl (Hardwick et al. , J.

Cell. Biol. 131 (3): 709-720,1995 ; Shero et al., Genes Dev. 5 (4): 549-560,1991), oryzalin (Stargell et al. , Mol. Cell. Biol. 12 (4): 1443-1450,1992), majusculamide C (Moore, J. Ind.

Microbiol. 16 (2): 134-143,1996), demecolcine (Van Dolah and Ramsdell, J. Cell. Physiol.

166 (1) : 49-56, 1996; Wiemer et al. , J. Cell. Biol. 136 (1) : 71-80,1997), methyl-2- benzimidazolecarbamate (MBC) (Brown et al. , J. Cell. Biol. 123 (2): 387-403,1993), LY195448 (Barlow & Cabral, Cell Motil. Cytoskel. 19: 9-17,1991), subtilisin (Saoudi et al. , J.

Cell Sci. 108: 357-367,1995), 1069C85 (Raynaud et al., Cancer Chemother. Pharmacol. 35: 169-173,1994), steganacin (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), combretastatins (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), curacins (Hamel, Med. Res. Rev. 16 (2): 207- 231,1996), estradiol (Aizu-Yokata et al., Carcinogen. 15 (9): 1875-1879, 1994), 2- methoxyestradiol (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), flavanols (Hamel, Med. Res.

Rev. 16 (2): 207-231,1996), rotenone (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), griseofulvin (Hamel, Med. Res. Rev. 16 (2): 207-231 ; 1996), vinca alkaloids, including vinblastine and vincristine (Ding et al. , J. Exp. Med. 171 (3): 715-727,1990 ; Dirk et al., Neurochem. Res. 15 (11): 1135-1139,1990 ; Hamel, Med. Res. Rev. 16 (2): 207-231,1996 ; Illinger et al. , Biol. Cell 73 (2-3): 131-138, 1991 ; Wiemer et al. , J. Cell. Biol. 136 (1) : 71-80, 1997), maytansinoids and ansamitocins (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), rhizoxin (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), phomopsin A (Hamel, Med. Res. Rev.

16 (2): 207-231,1996), ustiloxins (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), dolastatin 10 (Hamel, Med Res. Rev. 16 (2): 207-231,1996), dolastatin 15 (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), halichondrins and halistatins (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), spongistatins (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), cryptophycins (Hamel, Med. Res.

Rev. 16 (2): 207-231,1996), rhazinilam (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), betaine (Hashimoto et al. , Zool. Sci. 1: 195-204,1984), taurine (Hashimoto et al. , Zool. Sci. 1 : 195-204, 1984), isethionate (Hashimoto et al. , Zool. Sci. 1: 195-204,1984), HO-221 (Ando et al., Cancer Chemother. Pharmacol. 37: 63-69,1995), adociasulfate-2 (Sakowicz et al., Science 280: 292- 295, 1998), estramustine (Panda et al. , Proc. Natl. Acad. Sci. USA 94: 10560-10564,1997), monoclonal anti-idiotypic antibodies (Leu et al. , Proc. Natl. Acad. Sci. USA 91 (22): 10690- 10694,1994), microtubule assembly promoting protein (taxol-like protein, TALP) (Hwang et al. , Biochem. Biophys. Res. Commun. 208 (3): 1174-1180,1995), cell swelling induced by hypotonic (190 mosmol/L) conditions, insulin (100 nmol/L) or glutamine (10 mmol/L) (Haussinger et al. , Biochem. Cell. Biol. 72 (1-2): 12-19,1994), dynein binding (Ohba et al., Biochim. Biophys. Acta 1158 (3): 323-332,1993), gibberelin (Mita and Shibaoka, Protoplasma 119 (1/2): 100-109,1984), XCHO1 kinesin-like protein) (Yonetani et al. , Mol. Biol. Cell 7 (suppl): 211A, 1996), lysophosphatidic acid (Cook et al. , Mol. Biol. Cell 6 (suppl): 260A, 1995), lithium ion (Bhattacharyya and Wolff, Biochem. Biophys. Res. Commun. 73 (2): 383- 390,1976), plant cell wall components (e. g. , poly-L-lysine and extensin) (Akashi et al. , Planta 182 (3): 363-369,1990), glycerol buffers (Schilstra et al. , Biochem. J. 277 (Pt. 3): 839-847, 1991; Farrell and Keates, Biochem. Cell. Biol. 68 (11): 1256-1261,1990 ; Lopez et al. , J. Cell.

Biochem. 43 (3): 281-291, 1990), Triton X-100 microtubule stabilizing buffer (Brown et al. , J.

Cell Sci. 104 (Pt. 2): 339-352,1993 ; Safiejko-Mroczka and Bell, J. Histochem. Cytochem.

44 (6): 641-656,1996), microtubule associated proteins (e. g. , MAP2, MAP4, tau, big tau, ensconsin, elongation factor-l-alpha EF-l. alpha. ) and E-MAP-115) (Burgess et al. , Cell Motil.

Cytoskeleton 20 (4): 289-300,1991 ; Saoudi et al. , J. Cell. Sci. 108 (Pt. I) : 357-367,1995 ; Bulinski and Bosser, J. Cell. Sci. 107 (Pt. 10): 2839-2849,1994 ; Ookata et al. , J. Cell Biol.

128 (5): 849-862,1995 ; Boyne et al. , J. Comp. Neurol. 358 (2): 279-293,1995 ; Ferrera and Caceres, J. Neurosci. 11 (2): 392400,1991 ; Thurston et al. , Chromosoma 105 (1) : 20-30,1996 ; Wang et al., Brain Res. Mol. Brain Res. 38 (2): 200-208,1996 ; Moore and Cyr, Mol. Biol. Cell 7 (suppl): 221-A, 1996; Masson and Kreis, J. Cell Biol. 123 (2), 357-371,1993), cellular entities (e. g. histone H1, myelin basic protein and kinetochores) (Saoudi et al. , J. Cell. Sci. 108 (Pt. 1) : 357-367,1995 ; Simerly et al. , J. Cell Biol. 111 (4): 1491-1504,1990), endogenous microtubular structures (e. g. , axonemal structures, plugs and GTP caps) (Dye et al., Cell Motil. Cytoskeleton 21 (3): 171-186,1992 ; Azhar and Murphy, Cell Motil. Cytoskeleton 15 (3): 156-161,1990 ; Walker et al. , J. Cell Biol. 114 (1) : 73-81,1991 ; Drechsel and Kirschner, Curr. Biol. 4 (12): 1053-1061,1994), stable tubule onlypolypeptide (e. g., STOP145 and STOP220) (Pirollet et al., Biochim. Biophys. Acta 1160 (1) : 113-119,1992 ; Pirollet et al., Biochemistry 31 (37): 8849-

8855,1992 ; Bosc et al. , Proc. Natl. Acad. Sci. USA 93 (5): 2125-2130,1996 ; Margolis et al., EMBO J. 9 (12): 4095-4102,1990) and tension from mitotic forces (Nicklas and Ward, J. Cell Biol. 126 (5): 1241-1253,1994), as well as any analogues and derivatives of any of the above.

Such compounds can act by either depolymerizing microtubules (e. g. , colchicine and vinblastine), or by stabilizing microtubule formation (e. g., paclitaxel)." United States patent 6,689, 803 also discloses (at columns 16 and 17 that, "Within one preferred embodiment of the invention, the anti-mitotic compound is paclitaxel, a compound which disrupts microtubule formation by binding to tubulin to form abnormal mitotic spindles.

Briefly, paclitaxel is a highly derivatized diterpenoid (Wani et al. , J. Am. Chem. Soc. 93: 2325, 1971) which has been obtained from the harvested and dried bark of Taxus brevifolia (Pacific Yew) and Taxomyces Andreanae and Endophytic Fungus of the Pacific Yew (Stierle et al., Science 60: 214-216, -1993). "Paclitaxel" (which should be understood herein to include prodrugs, analogues and derivatives such as, for example, TAXOLO, TAXOTEREO, Docetaxel, 10-desacetyl analogues of paclitaxel and 3'N-desbenzoyl-3'N-t-butoxy carbonyl analogues of paclitaxel) may be readily prepared utilizing techniques known to those skilled in the art (see e. g. , Schiff et al., Nature 277: 665-667,1979 ; Long and Fairchild, Cancer Research 54: 4355-4361,1994 ; Ringel and Horwitz, J. Natl. Cancer Inst. 83 (4): 288-291, 1991; Pazdur et al. , Cancer Treat. Rev. 19 (4): 351-386,1993 ; WO 94/07882 ; WO 94/07881 ; WO 94/07880 ; WO.

94/07876 ; WO 93/23555; WO 93/10076; W094/00156 ; WO 93/24476; EP 590267; WO 94/20089 ; U. S. Pat. Nos. 5,294, 637; 5,283, 253; 5,279, 949; 5,274, 137; 5,202, 448; 5,200, 534; 5,229, 529; 5,254, 580; 5,412, 092; 5,395, 850; 5,380, 751; 5,350, 866 ; 4, 857, 653; 5,272, 171; 5,411, 984; 5, 248, 796; 5,248, 796; 5,422, 364; 5,300, 638 ; 5,294, 637; 5,362, 831; 5,440, 056; 4,814, 470; 5,278, 324; 5,352, 805 ; 5,411, 984; 5,059, 699; 4,942, 184; Tetrahedron Letters 35 (52): 9709-9712,1994 ; J. Med. Chem. 35: 4230-4237, 1992 ; J. Med. Chem. 34: 992-998, 1991; J. Natural Prod. 57 (10): 1404-1410,1994 ; J. Natural Prod. 57 (11): 1580-1583,1994 ; J.

Am. Chem. Soc. 110: 6558-6560,1988), or obtained from a variety of commercial sources, including for example, Sigma Chemical Co. , St. Louis, Mo. (T7402--from Taxus brevifolia)." As is also disclosed in United States patent 6, 689, 893, "Representative examples of such paclitaxel derivatives or analogues include 7-deoxy-docetaxol, 7, 8-cyclopropataxanes, N- substituted 2-azetidones, 6, 7-epoxy paclitaxels, 6, 7-modified paclitaxels, 10-desacetoxytaxol, 10-deacetyltaxol (from 10-deacetylbaccatin III), phosphonooxy and carbonate derivatives of taxol, taxol 2', 7-di (sodium 1,2-benzenedicarboxylate, 10-desacetoxy-11, 12-dihydrotaxol- 10,12 (18) -diene derivatives, 10-desacetoxytaxol, Protaxol (2'-and/or 7-O-ester derivatives), (2'- and/or 7-0-carbonate derivatives), asymmetric synthesis of taxol side chain, fluoro taxols, 9-

deoxotaxane, (13-acetyl-9-deoxobaccatine III, 9-deoxotaxol, 7-deoxy-9-deoxotaxol, 10- desacetoxy-7-deoxy-9-deoxotaxol, Derivatives containing hydrogen or acetyl group and a hydroxy and tert-butoxycarbonylamino, sulfonate 2'-acryloyltaxol and sulfonate 2'-O-acyl acid taxol derivatives, succinyltaxol, 2'-. gamma. -aminobutyryltaxol formate, 2'-acetyl taxol, 7- acetyl taxol, 7-glycine carbamate taxol, 2'-OH-7-PEG (5000) carbamate taxol, 2'-benzoyl and 2', 7-dibenzoyl taxol derivatives, other prodrugs (2'-acetyl taxol ; 2', 7-diacetyltaxol; 2'succinyltaxol; 2'- (beta-alanyl)-taxol) ; 2'gamma-aminobutyryltaxol formate; ethylene glycol derivatives of 2'-succinyltaxol ; 2'-glutaryltaxol; 2'- (N, N-dimethylglycyl) taxol ; 2'- (2- (N, N- dimethylamino) propionyl) taxol; 2'orthocarboxybenzoyl taxol; 2'aliphatic carboxylic acid derivatives of taxol, Prodrugs {2'(N, N-diethylaminopropionyl) taxol, 2' (N, N- dimethylglycyl) taxol, 7 (N, N-dimethylglycyl) taxol, 2', 7-di- (N, N-dimethylglycyl) taxol, 7 (N, N- diethylaminopropionyl) taxol, 2', 7-di (N, N-diethylaminopropionyl) taxol, 2'- (L-glycyl) taxol, 7- (L-glycyl) taxol, 2', 7-di (L-glycyl) taxol, 2'- (L-alanyl) taxol, 7- (L-alanyl) taxol, 2', 7-di (L- alanyl) taxol, 2'- (L-leucyl) taxol, 7- (L-leucyl) taxol, 2', 7-di (L-leucyl) taxol, 2'- (L-isoleucyl) taxol, 7- (L-isoleucyl) taxol, 2', 7-di (L-isoleucyl) taxol, 2'- (L-valyl) taxol, 7- (L-valyl) taxol, 2'7-di (L- valyl) taxol, 2'- (L-phenylalanyl) taxol, 7- (L-phenylalanyl) taxol, 2', 7-di (L-phenylalanyl) taxol, 2'- (L-prolyl) taxol, 7- (L-prolyl) taxol, 2', 7-di (L-prolyl) taxol, 2'- (L-lysyl) taxol, 7- (L-lysyl) taxol, 2', 7-di (L-lysyl) taxol, 2'- (L-glutamyl) taxol, 7- (L-glutamyl) taxol, 2', 7-di (L-glutamyl) taxol, 2'- (L- arginyl) taxol, 7- (L-arginyl) taxol, 2', 7-di (L-arginyl) taxol}, Taxol analogs with modified phenylisoserine side chains, taxotere, (N-debenzoyl-N-tert- (butoxycaronyl)-10-deacetyltaxol, and taxanes (e. g. , baccatin III, cephalomannine, 10-deacetylbaccatin III, brevifoliol, yunantaxusin and taxusin)." By way of yet further illustration, one may use one or more of the anti-mitotic agents disclosed in United States patents 6,673, 937 (syntheses and methods of use of new antimitotic agents), 6,624, 317 (taxoid conjugates as antimitotoic and antitumor agents), 6,593, 334 (camptothecin-taxoid conjugates as antimitotic and agents), 6,593, 321 (2- alkoxyestradiiol analogs with antiproliferative and antimitotic activity), 6,569, 870 (fluorinated quinolones as antimitotic and antitumor agent), 6, 528, 489 (mycotoxin derivatives as antimitotic agents), 6,392, 055 (synthesis and biological evaluation of analogs of the antimitotic marine natural product curacin A), 6,127, 377 (vinka alkaloid antimitotic halogenated derivatives), 5,695, 950 (method of screening for antimitotic compounds using the cdc25 tyrosine phosphatase), 5,620, 985 (antimitotic binary alkaloid derivatives from catharanthus roseus), 5,294, 538 (method of screening for antimitotic compounds using the CDC tyrosine

phosphatase), and the like. The entire disclosure of each of these United States patents is hereby incorporated by reference into this specification.

As will be apparent, one or more of the aforementioned anti-mitotic and/or anti- microtubule agents may be modified to make them magnetic in accordance with this invention.

Properties of the preferred anti-mitotic compounds In one preferred embodiment, the compound of this invention has a mitotic index factor of at least about 10 percent and, more preferably, at least about 20 percent. In one aspect of this embodiment, the mitotic index factor is at least about 30 percent. In another embodiment, the mitotic index factor is at least about 50 percent.

In another embodiment of the invention, the compound of this invention has a mitotic index factor of less than about 5 percent.

As is known to those skilled in the art, the mitotic index is a measure of the extent of mitosis. Reference may be had, e. g. , to United States patents 5,262, 409 (binary tumor therapy), 5,443, 962 (methods of indentifying inhibitors of cdc25 phosphatase), 5,744, 300 (methods and reagents for the indentificatioin and regulation of senescence-related genes), 6,613, 318, 6,251, 585 (assay and reagents for indentifying anti-proliferative agents), 6,252, 058 (sequences for targeting metastatic cells), 6,387, 642 (method for indentifying a reagent that modulates Mytl activity), 6,413, 735 (method of screening for a modulator of angiogenesis), 6,531, 479 (anti-cancer compounds), 6,599, 694 (method of characterizing potential therapeutics by determining cell-cell interactions), 6,620, 403 (in vivo chemosensitivity screen for human tumors), 6,699, 854 (anti-cancer compounds), 6,743, 576 (database system for predictive cellular bioinformatics), and the like. The entire disclosure of each of these United States patents is hereby incorporated by reference into this specification.

Reference may also be had, e. g. , to United States patent 5,262, 409, which discloses that: "Determination of mitotic index: For testing mitotic blockage with nocodazole and taxol, cells were grown a minimum of 16 hours on polylysinecoated glass coverslips before drug treatment.

Cells were fixed at intervals, stained with antibodies to detect lamin B, and counterstained with propidium iodide to assay chromosome condensation. To test cell cycle blocks in interphase, cells were synchronized in mitosis by addition of nocodazole (Sigma Chemical Co. ) to a final concentration of 0.05 llg/ml from a 1 mg/ml stock in dimethylsulfoxide. After 12 hours arrest, the mitotic subpopulation was isolated by shakeoff from the culture plate. After applying cell cycle blocking drugs and/or 2-AP, cells were fixed at intervals, prepared for indirect immunofluorescence with anti-tubulin antibodies, and counterstained with propidium iodide.

All data timepoints represent averages of three counts of greater than 150 cells each. Standard deviation was never more than 1. 5% on the ordinate scale." Reference may be had, e. g. , to United States patent 6,413, 735 which discloses that :" The mitotic index is determined according to procedures standard in the art. Keram et al., Cancer Genet. Cytogenet. 55: 235 (1991). Harvested cells are fixed in methanol: acetic acid (3: 1, v: v), counted, and resuspended at 106 cells/ml in fixative. Ten microliters of this suspension is placed on a slide, dried, and treated with Giemsa stain. The cells in metaphase are counted under a light microscope, and the mitotic index is calculated by dividing the number of metaphase cells by the total number of cells on the slide. Statistical analysis of comparisons of mitotic indices is performed using the 2-sided paired t-test." By means of yet further illustration, one may measure the mitotic index by means of the procedures described in, e. g. , articles by Keila Torres et al. ("Mechanisms of Taxol-Induced Cell Death are Concentration Dependent, "Cancer Research 58,3620-3626, August 15,1998), and Jie-Gung Chen et al. ("Differential Mitosis Responses to Microtubule-stabilizing and destablilizng Drugs, "Cancer Research 62,1935-1938, April 1,2002).

The mitotic index is preferably measured by using the well-known HeLa cell lines. As is known to those skilled in the art, HeLa cells are cells that have been derived from a human carcinoma of the cervix from a patient named Henrietta Lack; the cells have been maintained in tissued culture since 1953.

Hela cells are described, e. g. , in United States patents 5,811, 282 (cell lines useful for detection of human immunodeficiency virus), 5,376, 525 (method for the detection of mycoplasma), 6,143, 512,6, 326,196, 6,365, 394 (cell lines and constructs useful in production of E-1 deleted adenoviruses), 6,440, 658 (assay method for determining effect on aenovirus infection of Hela cells), 6,461, 809 (method of improving inflectivity of cells for viruses), 6,596, 535,6, 605,426, 6,610, 493 (screening compounds for the ability to alter the production of amyloid-beta-peptide), 6,699, 851 (cytotoxic compounds and their use), and the like; the entire disclosure of each of these United States patents is hereby incorporated by reference into this specification. By way of illustration, United States patent 6,440, 658 This patent discloses that, for the experiments described in such patent, "The HeLa cell line was obtained from the American Type Culture Collection, Manassas Va." In one preferred embodiment, the mitotic index of a"control cell line" (i. e. , one that omits that drug to be tested) and of a cell line that includes 50 nanomoles of such drug per liter of the cell line are determined and compared. The"mitotic index factor"is equal to (Mt-

Mc/Mc) x 100, wherein Me is the mitotic index of the"control cell line, "and Mt is the mitotic index of the cell line that includes the drug to be tested.

The compound of this invention preferably has a molecular weight of at least about 150 grams per mole. In one embodiment, the molecular weight of such compound is at least 300 grams per mole. In another embodiment, the molecular weight of such compound is 400 grams per mole. In yet another embodiment, the molecular weight of such compound is at least about 550 grams per mole. In yet another embodiment, the molecular weight of such compound is at least about 1,000 grams per mole. In yet another embodiment, the molecular weight of such compound is at least 1,200 grams per mole.

The compound of this invention preferably has a positive magnetic susceptibility of at least 1,000 x 10-6 centimeter-gram-seconds (cgs). As is known to those skilled in the art, magnetic susceptibility is the ratio of the magnetization of a material to the magnetic filed strength. Reference may be had, e. g. , to United States patents 3,614, 618 (magnetic susceptibility tester), 3,644, 823 (nulling coil apparatus for magnetic susceptibility logging), 3,657, 636 (thermally stable coil assembly for magnetic susceptibility logging), 3,665, 297 (apparatus for determining magnetic susceptibility in a controlled chemical and thermal environment), 3,758, 847 (method and system with voltage cancellation for measuring the magnetic susceptibility of a subsurface earth formation), 3, 758, 848 (magnetic susceptibility well logging system), 3,879, 658 (apparatus for measuring magnetic susceptibility), 3,890, 563 (magnetic susceptibility logging apparatus for distinguishing ferromagnetic materials), 3,980, 076 (method for measuring externally of the human body magnetic susceptibility changes), 4,079, 730 (apparatus for measuring externally of the human body magnetic susceptibility changes), 4,277, 750 (induction probe for the measurement of magnetic susceptibility), 4,359, 399 (taggands with induced magnetic susceptibility), 4,507, 613 (method for identifying non-magnetic minerals in earth formations utilizing magnetic susceptibility measurements), 4,662, 359 (use of magnetic susceptibility probes in the treatment of cancer), 4,701, 712 (thermoregulated magnetic susceptibility sensor assembly), 5,233, 992 (MRI method for high liver iron measurement using magnetic susceptibility induced field distortions), 6,208, 884 (noninvasive room temperature instrument to measure magnetic susceptibility variations in body tissue), 6,321, 105 (contrast agents with high magnetic susceptibility), 6,477, 398 (resonant magnetic susceptibility imaging), and the like. The entire disclosure of each of these United States patent applications is hereby incorporated by reference into this specification.

In one embodiment, the compound of this invention has a positive magnetic susceptibility of at least 5,000 x 10-6 cgs. In another embodiment, such compound has a positive magnetic susceptibility of at least 10,000 x 10-6 cgs.

The compound of this invention is preferably comprised of at least 7 carbon atoms and, more preferably, at least about 10 carbon atoms. In another embodiment, such compound is comprised of at least 13 carbon atoms and at least one aromatic ring; in one aspect of this embodiment, the compound has at least two aromatic rings. In another embodiment, such compound is comprised of at least 17 carbon atoms.

In one embodiment, the compound of this invention is comprised of at least one oxetane ring. As is disclosed, e. g. , on page 863 of N. Iving Sax's"Hawley's Condensed Chemical Dictionary, "Eleventh Edition (Van Nostrand Reinhold Company, New York, New York, 1987), the oxetane group, also known as"trimethylene oxide), is identified by chemical abstract number CAS: 503-30-0. The oxetane group present in the preferred compound preferably is unsubstituted. In one embodiment, however, one ore more of the ring carbon atoms (either carbon number one, or carbon number two, or carbon number 3), has one or more of its hydrogen atoms substituted by a halogen group (such as chlorine), a lower alkyl group of from 1 to 4 carbon atoms, a lower haloalkyl group of from 1 to 4 carbon atoms, a cyanide group (CN), a hydroxyl group, a carboxyl group, an amino group (wich can be primary, secondary, or teriarary and may also contain from 0 to 6 carbon atoms), a substituted hydroxyl group (such as, e. g. , an ether group containing from 1 to 6 carbon atoms), and the like. In one aspect of this embodiment, the substituted oxetane group is 3,3-bis (chlormethyl) oxetane.

In one embodiment, the compound of this invention is comprised of from about 1 to 10 groups of the formula-OB, in which B is selected from the group consisting of hydrogen, alkyl of from about 1 to about 5 carbon atoms, and a moiety of the formula R- (C=0)-O--, wherein R is selected from the group consisting of hydrogen and alkyl of from about 1 to about 6 cabon atoms, and the carbon is bonded to the R moiety, to the double-bonded oxygen, and to the single bonded oxygen, thereby forming what is commonly known as an acetyl group. This acetyl group preferably is linked to a ring structure that is unsaturated and preferably contains from about 6 to about 10 carbon atoms.

In one embodiment, the compound is comprised of two unsaturated ring structures linked by an amide structure, which typically has an acyl group,--CONRI--, wherein Rl is selected from the group consisting of hydrogen lower alkyl of from 1 to about 6 carbon atoms.

In one preferred embodiment, the N group is bonded to both to the Rl group and also to radical that contains at least about 20 carbon atoms and at least about 10 oxygen atoms.

In one embodiment, the compound of this invention contains at least one saturated ring comprising from about 6 to about 10 carbon atoms. By way of illustration, the saturated ring structures may be one or more cyclohexane rings, cyclopheptane rings, cyclooctane rings, cylclononane rings, and/or cylcodecane rings. In one preferred aspect of this embodiment, at least one saturated ring in the compound is bonded to at least one quinine group. Referring to page 990 of the"Hawley's Condensed Chemical Dictionary"described elsewhere in this specification, quinine is 1,4-benzoquinone and is identified as"CAS: 106-51-4. " In one embodiment, the compound of this invention may comprise a ring structure with one double bond or two double bonds (as opposed to the three double bonds in the aromatic structures). These ring structures may be a partially unsaturated material selected from the group consisting of partially unsaturated cyclohexane, partially unsaturated cyclopheptane, partially unsaturated cyclooctane, partially unstaruated cyclononane, partially unsaturated cyclodecane, and mixtures thereof.

The compound of this invention is also preferably comprised of at least one inorganic atom with a positive magnetic susceptibility of at least 200 x 10-6 cgs. Thus, and referring to the"CRC Handbook of Chemistry and Physics,"63rd Edition (CRC Press, Inc. , Boca Raton, Florida, 1982-83), the magnetic susceptibility of elements are described at pages E-118 to E- 123. Suitable inorganic (i. e. , non-carbon containing) elements with a positive magnetic susceptibility greater than about 200 x 10-6 cgs include, e. g. , cerium (+5,160 x 10-6 cgs), cobalt (+11, 000 x 10-6 cgs), dysprosium (+89, 600 x 10-6 cgs), europium (+34,000 x 10-6 cgs), gadolinium (+ 755,000 x 10-6 cgs), iron (+13,600 x 10-6 cgs), manganese (+529 x 10-6 cgs), palladium (+567.4 x 10-6 cgs), plutonium (+610 x 10-6 cgs), praseodymium (+5010 x 10-6 cgs), samarium (+2230 x 10-6 cgs), technetium (+250 x 10-6 cgs), thulium (+51,444 x 10-6 cgs), and the like. In one embodiment, the positive magnetic susceptibility of such element is preferably greater than about +500 x 10-6 cgs and, even more preferably, greater than about +1,000 x 10-6 cgs.

In one preferred compound, the inorganic atom is radioactive. As is known to those skilled in the art, radioactivity is a phenomenon characterized by spontaneous disintegration of atomic nuclei with emission of corpuscular or electromagnetic radiation.

In another preferred embodiment, one or more inorganic or organic atoms that do not have the specified degree of magnetic suscpeptibility are radioactive. Thus, e. g. , the radioactive atom may be,. e. g, radioactive carbon, radioactive hydrogen (tritium), radioactive phosphorus, radioactive sulfur, radioactive potassium, or any other of the atoms that exist is radioactive isotope form.

One preferred class of atoms is the class of radioactive nuclides. As is known to those skilled in the art, radioactive nuclides are atoms disintegrate by emission of corpuscular or electromagnetic radiatons. The rays most commonly emitted are alpha or beta gamma rays.

See, e. g. , page F-109 of the aforementioned"CRC Handbook of Chemistry and Physics." Radioactive nuclides are well known and are described, e. g. , in United States patents 4,355, 179 (radioactive nuclide labeled propiophenone compounds), 4,625, 118 (device for the elution and metering of a radioactive nuclide), 5,672, 876 (method and apparatus for measuring distribution of radioactive nuclide in a subject), and 6,607, 710 (bisphosphonic acid derivative and compound thereof labeled with radioactive nuclide. ). The entire disclosure of each of these United States patents is hereby incorporated by reference into this specification.

Referring again to the aforementioned"CRC Handbook of Chemistry and Physics,"and to pages and in particular to pages B340-B378 thereof, it will be seen that the inorganic atom may be, e. g. , cobalt 53, cobalt 54, cobalt 55, cobalt 56, cobalt 57, cobalt 58, cobalt 59, cobalt 60, cobalt 61, cobalt 62, cobalt 63, gadolinium 146, iron 49, iron 51, iron 52, iron 53, iron 54, iron 57, iron 58, iron 59, iron 60, iron 61, iron 62, manganese 50, praseodymium 135, samarium 156, and the like.

The compound of this invention preferably has a magnetic moment of at least about 0.5 Bohr magnetrons per molecule and, more preferably, at least about 1.0 Bohr magnetrons per molecule. In one embodiment, the compound has a magnetic moment of at least about 2 Bohr magnetrons per molecule.

As is known to those skilled in the art, a Bohr magnetron is the amount he/4 (pi) mc, wherein he is Plank's constant, e and m are the charge and mass of the electron, c is the speed of light, and pi is equal to about 3.14567. Reference may be had, e. g. , to United States patents 4,687, 331,4, 832, 877,4, 849, 107,5, 040,373 (" (One Bohr magnetron is equal to 9. 273X10-24 Joules/Tesla"), 5,169, 944,5, 323,227 ("uo is a constant known as the Bohr magnetron at 9. 274x10-21 erg/Gauss"), 5,352, 979 6,383, 597,6, 725,668, 6,739, 137 ("One Bohr magnetron gB is equal to 9. 273x 10-24 Joules/Tesla"), and the like. The entire disclosure of each of these United States patents is hereby incorporated by reference into this specification.

In one preferred embodiment, the magnetic compound of this invention is water soluble. As is known to those skilled in the art, solubility of one liquid or solid in another is the mass of the substance cotnained in a solution which is in equilibrium with an excess of the substance. Under such conditions, the solution is said to be saturated. Reference may be had, e. g. , to page F-95 of the CRC"Handbook of Chemistry and Physics,"53 Edition (The

Chemical Rubber Company, CRC Press Division, 18901 Cranwood Parkway, Cleveland, Ohio, 44128, 1972-1973).

As used in this specification, the term"water soluble"refers to a solubility of at least 10 micrograms per milliliter and, more preferably, at least 100 micrograms per milliliter; by way of comparison, the solubility of paclitaxel in water is only about 0.4 micrograms per milliliter.

One may determine water solubulity by conventional means. Thus, e. g. , one may mix 0.5 milliters of water with the compound to be tested under ambient conditions, stir for 18 hours under ambient conditions, filter the slurry thus produced to remove the non-solubulized portion of the fitrand, and calculae how much ofthe filtrand was solubilized. From this, one can determine the number of micrograms that went into solution.

In one embodiment, the magnetic compound of this invention has a water solubility of at least 500 micrograms per milliliter, and more preferably at least 1,000 micrograms per milliliter. In yet another embodiment, the magnetic compound of this invention has a water solubility of at least 2500 micrograms per milliliter. li yet another embodiment, the magnetic compound of this invention has a water solubility of at least 5,000 micrograms per milliliter. In yet another embodiment, the magnetic compound of this invention has a water solubility of at least 10,000 micrograms per milliliter.

In another embodiment, the magnetic compound of this invention has a water solubility of less than about 10 micrograms per milliliter and, preferably, less than about 1. 0 micrograms per milliliter.

Without wishing to be bound to any particular theory, applicants believe that the presence of a hydrophilic group in the compound of their invention helps render such compound water-soluble. Thus, e. g. , it is believed that the siderophore group that is present in their preferred compounds aids in creating such water-solubility. As is known to those skilled in the art, a siderophe is one of a number of low molecular weight, iron-containing, or iron binding organic compounds or groups. Siderophores have a storng affinity for Fe (which they chelate) and function in the solubilization and transport of iron. Siderophores are classified as belonging to either the phenol-catechol type (such as enterobactin and agrobactin), or the hydroxyamic acid type (such as ferrichome and mycobactin). Reference may be had, e. g. , to page 442 of J. Stenesh's"Dictionary of Biochemistry and Molecular Biology, "Second Edition (John Wiley & Sons, New York, New York, 1989).

In one preferred embodiment, the compound of this invention is comprised of one or more siderophore groups bound to a magnetic moiety (such as, e. g. , an atom selected from the group consisting of iron, cobalt, nickel, and mixtures thereof).

As will be apparent, the inclusion of other hydrophilic groups into otherwise water- insoluble compounds is contemplated. Thus, by way of illustration and not limitation, and in place of or in addition to such siderophore group, one use hydrophilic groups such as the siderophore group (s) described hereinabove, hydroxyl groups, carboxyl groups, amino groups, organometallic ionic structures, phosphate groups, and the like. In one preferred aspect of this embodiment, the hydrophilic group utilized should preferably be biologically inert.

In one embodiment, the magnetic compound of this invention has an association rate with microtubules of at least 3,500, 000/mole/second. The association rate may be determined in accordance with the procedure described in an article by J. F. Diaz et al. ,"Fast Kinetics of Taxol Binding to Microtubules, "Journal of Biological Chemistry, 278 (10) 8407-8455.

Reference also may be had, e. g. , to a paper by J. R. Strobe et al. appearing in the Journal of Biological Chemistry, 275: 26265-26276 (2000). As is disclosed, e. g. , in the Diaz et al. paper, "The kinetics of binding and dissociation of Flutax-1 and Flutax-2 were measured by thechange of fluorescence intensity using an SS-51 stopped flow device (High-Tech Scientific, UK) equipped with a fluorescence detetion system, using an excitation wavelenght of 492 and a 530- nmcut-off filter in the emission pathway. Thefitting of the kinetic curves was done with a non- linear least squares sfitting program based upon the Marquardt algorithm... where pseudo-firt order conditions were used...." In another embodiment of the invention, the magnetic compound of this invention has a dissociation rate with microubules, as measured in accordance with the procedure desribed in such Diaz et al. paper, of less than about 0. 08/second, when measured at a temperature of 37 degrees Celsius and under atmospheric conditions. Thus, in this embodiment, the magnetic compound of this invention binds more durably to microtubules than does paclitaxel, which has a dissociation rate of at least 0. 91/second.

In one embodiment, the dissociation rate of the magnetic compound of this invention is less than 0.7/second and, more preferably, less than 0.6/second.

In one embodiment of this invention, the anti-mitotic compound of the invention has the specified degree of water-solubility and of anti-mitotic activity but does not necessarily possess one or more of the magnetic properties described hereinabove.

Another preferred compound of the invention In another embodiment of this invention, there is provided a compound that, in spite of having a molecular weight in excess of 550, still has a water solubility in excess of about 10 micrograms per milliliter. In particular, there is provided a compound with a molecular weight of at least about 550, a water solubility of at least about 10 micrograms per milliliter, a pKa

dissociation constant of from about 1 to about 15, and a partition coefficient of from about 1.0 to about 50.

The compound of this embodiment of the invention has a molecular weight of at least about 550. In one embodiment, this compound has a molecular weight of at least about 700.

The water solubility of this compound is at least about 1 micrograms per milliliter and, more preferably, at least about 10 micrograms per milliliter. In one embodiment, such compound has a water solubility of at least about 100 micrograms per milliliter. In yet another embodiment, such compound has a water solubility of at least about 1,000 micrograms per milliliter.

The compound of this embodiment of the invention has a pKa dissociation constant of from about 1 to about 15. As used herein, the term"pKa dissociation constant"is equal to- log Ka, wherein Ka is equal to [H3 O+] [A-]/ [HA], wherein the square brackets indicate concentration, and wherein A is the counterion. Reference may be had, e. g. , to pages 327-328 of Maitland Jones, Jr. 's"Organic Chemistry" (W. M. Norton & Company, New York, New York, 1997). Reference may also be had, e. g. , to United States patents 5,036, 164; 5,025, 063; 5,767, 066; 5,155, 162; 5,132, 000; and 5,079, 134. The entire disclosure of each of these United States patents is hereby incorporated by reference into this specification As is known to those skilled in the art, and as is disclosed at pages 39 et seq. of Stephen H. Curry et al. 's"Manual of Laboratory Phamaconkinetics" (John Wiley & Sons, New York, New York, 1983),"Many drugs are weak acids and/or bases. The degree of ionization will influence the absorption, distribution, and excretion in vivo, the solubility at a given pH, the distribution of the drug between aqueous and organic pahses the choice of pH in liquid chromatographic separations, etc.... From the above it follows that the pH at which the compound is 50 percent ionized is equal to the pKa. To determine a value of pKa the relative concentrations of ionized and non-ionized forms msut be known at a particular pH. Several methods are available, including potentiometric titration, conductimetry, solubility, and spectrometery...." The compound of this embodiment of the invention preferably has a partition coefficient of from about 1.0 to about 50. This partition coefficient is also dicussed at pages 41 et seq. of the aforementioned Curry book, wherein it is disclosed that:"When a solute is distributed between two immiscible phases, 1 and 2, the ratio of the activities of the solute in the phases is constant. If the solutions are dilute and ideal behavior is assumed, then the ratio of the concentration of the solute will be constant.... The constant is known as the partition (or distribution) coefficient.... The convention with regard to which phase is classed as 1 and which

is as 2 is not entirely clear. Usually, partition coefficients are defined as the concentration in the organic phase divided by the concentration in the aqueous phase." It is preferred to measure the partition coefficient between water and octane. Means for measuring the partition coefficient are well known to those skilled in the art and are described, e. g. , in the patent literature. Reference may be had, e. g. , to United States patents 6,660, 288; 6,645, 479; 6, 585, 953; 6,583, 136; 6,500, 995; 6,475, 961; 6.369. 001; 6,362, 158 ; 6,315, 907; 6,310, 013; 6,271, 665; 6,218, 378 ; 6,203, 817 ; 6,156, 826; 6,124, 086 ; 6,071, 409; 6,045, 835; 6,042, 792; 5,874, 481; 5,763, 146; 5,555, 747; 5,252, 320 (complexes having a partition coefficient above 300); 5,254, 342; 5,252, 320; 5,164, 189; 5,071, 769; 5,041, 523;-5, 013,556 ; 5,011, 982 ; 5,011, 967; 4,986, 917; 4, 980, 453; 4,957, 862; 4,940, 654; 4, 886, 656; 4,859, 584; 4,762, 701; 4,746, 745; 4,743, 550 (method for improving the partition coefficient in enzyme containing systems having at least two phases), 4,736, 016; 4,721, 730; 4,699, 924; 4,619, 939; 4,420, 473; 4,371, 540; 4,363, 793; and the like. The entire disclosure of each of these United States patents is hereby incorporated by reference into this specification.

In one embodiment, the compound of this invention has a tumor uptake of at least about 10 percent and, more preferably, at least about 20 percent. In one embodiment, the tumor uptake is at least about 30 percent. In yet another embodiment, the tumor uptake is at least about 50 percent. In yet another embodiment, the tumor uptake is at least about 70 percent.

Tumor uptake is the extent to which the compound is selectively taken up by tumors from blood. It may be determined by dissolving 1 milligram of the compound to be tested in 1 milliliter of"Cremophor EL, "a 1: 1 (volume/volume) mixture of anhydrous ethanol and polyethoxylated castor oil. For a discussion of such"Cremophor EL, "reference may be had, e. g. , to United States patents 5,591, 715 (methods and compositions for reducing multidrug resistance), 5, 686, 488 (polyethoxylated castor oil products as anti-inflammatory agents), 5,776, 891 (compositions for reducing multidrug resistance), and the like. The entire disclosure4 of each of these United States patents is hereby incorporated by reference into this specification.

The mixture of the compound to be tested and"Cremophor EL"is injected ito the blood supply (artery) of a laboratory rat, near the tumor. Thirty seconds later the rate is sacrificed, the tumor is removed, and it and the blood are analyzed for the presence of the compound. Both the arterial blood and the venous drainage beyond the tumor are analyzed. The percent tumor uptake is equal to ( [Ca-C, I/Ca) x 100, wherein Ca is the concentration of the compound in the arterial blood, and C, is the concentration of the compound in the venous blood.

Other conventional means may be used to determine the tumor uptake. Reference may be had, e. g. , to United States patents 4,448, 762; 5,077, 034; 5,094, 835; 5,135, 717; 5,166, 944; 5, 284, 831; 5,5, 391,547 ; 399,338 ; 5,474, 772; 5,516, 940; 5,578, 287; 5,595, 738; 5,601, 800; 5,608, 060; 5,616, 690; 5,624, 798 ; 5,624, 896; 5,683, 873 ; 5, 688, 501; 5,753, 262; 5,762, 909; 5, 783, 169; 5,810, 888 ; 5,811, 073; 5,820, 873; 5, 847, 121; 5, 869, 248; 5,877, 162; 5,891, 689; 5,902, 604; 5,911, 969; 5,914, 312; 5,955, 605; 5,965, 598; 5,976, 535; 5,976, 874; 6,008, 319; 6,022, 522; 6,022, 966; 6,025, 165; 6,027, 725; 6,057, 153; 6,074, 626; 6,103, 889 ; 6,121, 424; 6,165, 441; 6,171, 577; 6,172, 045; 6,197, 333; 6,217, 869; 6,217, 886 ; 6,235, 264; 6,242, 477; 6,331, 287 ; 6, 348, 214; 6,358, 490; 6,403, 096; 6,426, 400; 6,436, 708; 6,441, 158; 6,458, 336; 6,498, 181; 6,515, 110; 6,537, 521; 6,610, 478; 6,617, 135; 6,620, 805; 6,624, 187; 6, 723,318 ; 6,734, 171 ; 6, 685, 915 ; and the like. The entire disclosure of each of these United States patents is hereby incorporated by reference into this specification.

Guided delivery of the compounds of this invention In one preferred embodiment, the magnetic properties of the anti-mitotic compound of this invention are used in order to preferentially deliver such compound to a specified site. In another embodiment, the magnetic properties of the compounds and compositions of this invention which are not necessarily anti-mitotic but have the desired magnetic properties also may be used to deliver such compounds and/or compositions to a desired site.

Thus, by way of illustration, one may guide delivery of the compound of this invention with conventional magnetic focusing means. In one aspect of this embodiment, a magnetic field of a specified strength is focused onto a desired therapeutic site, such as a tumor to be treated, whereby the compound is selectively drawn to the therapeutic site and binds with tubulin moleuces at the site. In one embodiment, the focused magnetic field has a field strength of at least about 6 Tesla in order to cause microtubules to move linearly. The magnetic field may, e. g. , be focused for a period of at least about 30 minutes following the administration of the compound of this invention.

One may use any of the conventional magnetic field generators known to those skilled in the art to produce such a magnetic field. Thus, e. g. , one may use one or more of the magnetic field generators disclosed in United States patents 6,503, 364,6, 377,149 (magnetic field generator for magnetron plasma generation), 6,353, 375 (magnetostatic wave device), 6,340, 888 (magnetic field generator for MRI), 6,336, 989, 6,335, 617 (device for calibrating a magnetic field generator), 6,313, 632,6, 297,634, 6,275, 128, 6,246, 066 (magnetic field generator and charged particle beam irradiator), 6,114, 929 (magnetostatic wave device), 6,099, 459 (magnetic field generating device and method of generating and applying a magnetic field),

5,795, 212,6, 106,380 (deterministic magnetorheological finishing), 5,839, 944 (apparatus for deterministic magnetorheological finishing), 5,971, 835 (system for abrasive jet shaping and polishing of a surface using a magnetorheological fluid), 5,951, 369,6, 506,102 (system for magnetorheological finishing of substrates), 6,267, 651,6, 309, 285 (magnetic wiper), 5,929, 732 and 6, 488, 615 I (which describe devices and methods for creating a high intensity magnetic field for magnetically guiding a anti-mitotic compoundto a predetermined site within a biological organism), and the like. The entire disclosure of each of these United States patents is hereby incorporated by reference into this specification.

The use of externally applied energy to affect an implanted medical device The prior art discloses many devices in which an externally applied electromagnetic field (i. e. , a field originating outside of a biological organism, such as a human body) is generated in order to influence one or more implantable devices disposed within the biological organism; these may be used in conjunction with anti-mitotic compound of this invention.

Some of these devices are described below.

United States patent 3,337, 776 describes a device for producing controllable low frequency magnetic fields comprising solenoid means for creating the magnetic field..

United States patent 3,890, 953 also discloses an apparatus for promoting the growth of bone and other body tissues by the application of a low frequency alternating magnetic field.

United States patent 4,095, 588 discloses a"vascular cleansing device"adapted to "... effect motion of the red corpuscles in the blood stream of a vascular system... whereby these red cells may cleanse the vascular system by scrubbing the walls thereof... ;"This patent claims (in claim 3) "A means to propel a red corpuscle in a vibratory and rotary fashion, said means comprising an electronic circuit and magnetic means including: a source of electrical energy; a variable oscillator connected to said source; a binary counter means connected to said oscillator to produce sequential outputs; a plurality of deflection amplifier means connected to be operable by the outputs of said binary counter means in a sequential manner, said amplifier means thereby controlling electrical energy from said source ; a plurality of separate coils connected in separate pairs about an axis in series between said deflection amplifier means and said source so as to be sequentially operated in creating an electromagnetic field from one coil to the other and back again and thence to adjacent separate coils for rotation of the electromagnetic field from one pair of coils to another; and a table within the space encircled by said plurality of coils, said table being located so as to place a person along the axis such that the red corpuscles of the person's vascular system are within the electromagnetic field between the coils creating same."The energy used to affect such red blood corpuscles may also be used

affect the anti-mitotic compounds of this invention, and/or tubulin and/or microtubules and/or other moieties.

United States patent 4,340, 038 discloses an implanted medical system comprised of magnetic field pick-up means for converting magnetic energy to electrical energy. In column 1 of United States patent 4,340, 038, at lines 12 et seq. , it is disclosed that"Many types of implantable devices incorporate a self-contained transducer for converting magnetic energy from an externally-located magnetic field generator to energy usable by the implanted device.".

United States patent 4,361, 153 discloses an implantable telemetry system. Such an implantable telemetry system, equipped with a multiplicity of sensors, may be used to report how the anti-mitotic compounds of this invention respond to applied electromagnetic fields.

As is disclosed at column 1 of United States patent 4,361, 153 (see lines 9 et seq.), "Externally applied oscillating magnetic fields have been used before with implanted devices.

Early inductive cardiac pacers employed externally generated electromagnetic energy directly as a power source. A coil inside the implant operated as a secondary transformer winding and was interconnected with the stimulating electrodes. More recently, implanted stimulators with rechargeable (e. g., nickel cadmium) batteries have used magnetic transmission to couple energy into a secondary winding in the implant to energize a recharging circuit having suitable rectifier circuitry...." United States patent 4, 408, 607 discloses a rechargeable, implantable capacitive energy source.

United States patent 4,416, 283 discloses a implantable shunted coil telemetry transponder employed as a magnetic pulse transducer for receiving externally transmitted data.

United States patent 5,487, 760 discloses an implantable signal transceiver disposed in an artificial heart valve.

United States patent 5,702, 430 discloses an implantable power supply. Columns 1 through 5 of United States patent 5,702, 430 presents an excellent discussion of"prior art" implantable pump assemblies that may be used, e. g. , to deliver the anti-mitotic compound of this invention. As is disclosed in such portion of United States patent 5,702, 430, "The most widely tested and commonly used implantable blood pumps employ variable forms of flexible sacks (also spelled sacs) or diaphragms which are squeezed and released in a cyclical manner to cause pulsatile ejection of blood. Such pumps are discussed in books or articles such as Hogness and Antwerp 1991, DeVries et al 1984, and Farrar et al 1988, and in U. S. Pat. No.

4,994, 078 (Jarvik 1991), 4,704, 120 (Slonina 1987), 4,936, 758 (Coble 1990), and 4,969, 864 (Schwarzmann et al 1990). Sack or diaphragm pumps are subject to fatigue failure of compliant

elements and as such are mechanically and functionally quite different from the pump which is the subject of the present invention." United States patent 5,743, 854 discloses a device for inducing and localizing epileptiform activity that is comprised of a direct current (DC) magnetic field generator, a DC power source, and sensors adapted to be coupled to a patient's head, United States patent 5,810, 015 describes an implantable power supply that can convert non-electrical energy (such as mechanical, chemical, thermal, or nuclear energy) into electrical energy. In column 1 of this patent, a discussion of"prior art"rechargeable power supplies is presented. It is disclosed in this column 1 that :"Modern medical science employs numerous electrically powered devices which are implanted in a living body. For example, such devices may be employed to deliver medications, to support blood circulation as in a cardiac pacemaker or artificial heart, and the like. Many implantable devices contain batteries which may be rechargeable by transcutaneous induction of electromagnetic fields in implanted coils connected to the batteries. Transcutaneous inductive recharging of batteries in implanted devices is disclosed for example in U. S. Pat. Nos. 3,923, 060; 4,082, 097; 4,143, 661; 4,665, 896; 5,279, 292; 5,314, 453; 5,372, 605, and many others." United States patent 5,810, 015 also discloses that:"Other methods for recharging implanted batteries have also been attempted. For example, U. S. Pat. No. 4,432, 363 discloses use of light or heat to power a solar battery within an implanted device. U. S. Pat. No. 4,661, 107 discloses recharging of a pacemaker battery using mechanical energy created by motion of an implanted heart valve. "These"other methods"may also be used in the process of this invention.

United States patent 5,810, 015 also discloses that:"A number of implanted devices have been powered without batteries. U. S. Pat. Nos. 3, 486, 506 and 3,554, 199 disclose generation of electric pulses in an implanted device by movement of a rotor in response to the patient's heartbeat. U. S. Pat. No. 3,563, 245 discloses a miniaturized power supply unit which employs mechanical energy of heart muscle contractions to generate electrical energy for a pacemaker. U. S. Pat. No. 3,456, 134 discloses a piezoelectric converter for electronic implants in which a piezoelectric crystal is in the form of a weighted cantilever beam capable of responding to body movement to generate electric pulses. U. S. Pat. No. 3,659, 615 also discloses a piezoelectric converter which reacts to muscular movement in the area of implantation. U. S. Pat. No. 4,453, 537 discloses a pressure actuated artificial heart powered by a second implanted device attached to a body muscle which in turn is stimulated by an electric

signal generated by a pacemaker. "These"other devices"may also be used in the process of this invention.

United States patent 5,810, 015 also discloses that:"In spite of all these efforts, a need remains for efficient generation of energy to supply electrically powered implanted devices." The solution provided by United States patent 5,80, 015 is described in claim 1 thereof, which describes:"An implantable power supply apparatus for supplying electrical energy to an electrically powered device, comprising: a power supply unit including: a transcutaneously, invasively rechargeable non-electrical energy storage device (NESD); an electrical energy storage device (EESD); and an energy converter coupling said NESD and said EESD, said converter including means for converting non-electrical energy stored in said NESD to electrical energy and for transferring said electrical energy to said EESD, thereby storing said electrical energy in said EESD." An implantable ultrasound communicaton system is disclosed in United States patent 5,861, 018 As is disclosed in the abstract of this patent, there is disclosed in such patent"A system for communicating through the skin of a patient, the system including an internal communication device implanted inside the body of a patient and an external communication device. The external communication device includes an external transmitter which transmits a carrier signal into the body of the patient during communication from the internal communication device to the external communication device. The internal communication device includes an internal modulator which modulates the carrier signal with information by selectively reflecting the carrier signal or not reflecting the carrier signal. The external communication device demodulates the carrier signal by detecting when the carrier signal is reflected and when the carrier signal is not reflected through the skin of the patient. When the reflected carrier signal is detected, it is interpreted as data of a first state, and when the reelected carrier signal is not detected, it is interpreted as data of a second state. Accordingly, the internal communication device consumes relatively little power because the carrier signal used to carry the information is derived from the external communication device. Further, transfer of data is also very efficient because the period needed to modulate information of either the first state or the second state onto the carrier signal is the same. In one embodiment, the carrier signal operates in the ultrasound frequency range." United States patent 5,861, 019, discloses a telemetry system for communications between an external programmer and an implantable medical device.

United States patent 5,945, 762 discloses an external transmitter adapted to magnetically excite an implanted receiver coil

United States patent 5,954, 758 claims an implantable electrical stimulator comprised of an implantable radio frequency receiving coil, an implantable power supply, an implantable input signal generator, an implantable decoder, and an implantable electrical stimulator.

United States patent 6,083, 166, the entire disclosure of which is hereby incorporated by reference into this specification, discloses an ultrasound transmitter for use with a surgical device.

United States patent 6,152, 882 discloses an implantable electroporation unit, an implantable proble electrode, an implantable reference electrode, and an an amplifier unit; this electroporation unit may be used to treat, e. g. , cancer cells in conjunction with the anti-mitotic compound of this invention.

United States patent 6,169, 925 describes a transceiver for use in communication with an implantable medical device.

United States patent 6,185, 452 claims a device for stimulating internal tissue, wherein such device is comprised of :"a sealed elongate housing configured for implantation in said patient's body, said housing having an axial dimension of less than 60 mm and a lateral dimension of less than 6 mm; power consuming circuitry carried by said housing including at least one electrode extending externally of said housing, said power consuming circuitry including a capacitor and pulse control circuitry for controlling (1) the charging of said capacitor and (2) the discharging of said capacitor to produce a current pulse through said electrode ; a battery disposed in said housing electrically connected to said power consuming circuitry for powering said pulse control circuitry and charging said capacitor, said battery having a capacity of at least one microwatt-hour; an internal coil and a charging circuit disposed in said housing for supplying a charging current to said battery; an external coil adapted to be mounted outside of said patient's body; and means for energizing said external coil to generate an alternating magnetic field for supplying energy to said charging circuit via said internal coil." United States patent 6,235, 024, discloses an implantable high frequency energy generator. Claim 1 of this patent describes:"A catheter system comprising: an elongate catheter tubing having a distal section, a distal end, a proximal end, and at least one lumen extending between the distal end and the proximal end; a handle attached to the proximal end of said elongate catheter tubing, wherein the handle has a cavity; an ablation element mounted at the distal section of the elongate catheter tubing, the ablation element having a wall with an outer surface and an inner surface, wherein the outer surface is covered with an outer member made of a first electrically conductive material and the inner surface is covered with an inner

member made of a second electrically conductive material, and wherein the wall comprises an ultrasound transducer ; an electrical conducting means having a first and a second electrical wires, wherein the first electrical wire is coupled to the outer member and the second electrical wire is coupled to the inner member of the ablation element; and a high frequency energy generator means for providing a radiofrequency energy to the ablation element through a first electrical wire of the electrical conducting means." An implantable light-generating apparatus is described in claim 16 of United States patent 6,363, 279.

An implantable ultrasound probe is described in claim 1 of United States patent 6,421, 565. Claim 1 of this patent describes:"An implantable cardiac monitoring device comprising: an A-mode ultrasound probe adapted for implantation in a right ventricle of a heart, said ultrasound probe emitting an ultrasound signal and receiving at least one echo of said ultrasound signal from at least one cardiac segment of the left ventricle; a unit connected to said ultrasound probe for identifying a time difference between emission of said ultrasound signal and reception of said echo and, from said time difference, determining a position of said cardiac segment, said cardiac segment having a position which, at least when reflecting said ultrasound signal, is correlated to cardiac performance, and said unit deriving an indication of said cardiac performance from said position of said cardiac segment." An implantable stent that contains a tube and several optical emitters located on the inner surface of the tube is disclosed in United States patent 6, 488, 704, the entire disclosure of which is hereby incorporated by reference into this specification. One may use one or more of the implantable devices described in United States patent 6,488, 704 together with the anti- mitotic compound of this invention and/or tubulin and/or microtubules and/or another in vivo device.

United States patent 6,605, 089 discloses an implantable bone growth promoting device.

United States patent 6,641, 520 discloses a magnetic field generator for providing a static or direct current magnetic field. The magnetic field generator claimed in United States patent 6,641, 520 comprised".... a magnetic field generating coil composed of a wound wire coil generating the static magnetic field in response to electrical power; a mounting member having the coil mounted thereon and having an opening therethrough of a size to permit insertion of a limb of the recipient in order to receive electromagnetic therapy from the magnetic field coil; an electrical power supply furnishing power to the magnetic field coil to cause the coil to generate a static electromagnetic field within the opening of the mounting member for application to the recipient's limb; a level control mechanism providing a reference signal representing a specified

electro-magnetic field strength set point for regulating the power furnished to the magnetic field coil; a field strength sensor detecting the static electromagnetic field strength generated by the magnetic field coil and forming a field strength signal representing the detected electro- magnetic field strength in the opening in the mounting member; a control signal generator receiving the field strength signal from the field strength sensor and the reference signal from the level control mechanism representing a specified electro-magnetic field strength set point; and the control signal generator forming a signal to regulate the power flowing from the electrical power supply to the magnetic field coil." United States patent 6,663, 555, also claims a magnetic field generator.

Precursor anti-microtubule agents The anti-mitotic compound of this invention may be derived from an anti-microtuble agent. As is disclosed in United States patent 6,689, 803 (at columns 5-6), representative anti- microtubule agents include, e. g.,".... taxanes (e. g., paclitaxel and docetaxel), campothecin, eleutherobin, sarcodictyins, epothilones A and B, discodermolide, deuterium oxide (D2 O), hexylene glycol (2-methyl-2,4-pentanediol), tubercidin (7-deazaadenosine), LY290181 (2- amino-4- (3-pyridyl)-4H-naphtho (1, 2-b) pyran-3-cardonitrile), aluminum fluoride, ethylene glycol bis- (succinimidylsuccinate), glycine ethyl ester, nocodazole, cytochalasin B, colchicine, colcemid, podophyllotoxin, benomyl, oryzalin, majusculamide C, demecolcine, methyl-2- benzimidazolecarbamate (MBC), LY195448, subtilisin, 1069C85, steganacin, combretastatin, curacin, estradiol, 2-methoxyestradiol, flavanol, rotenone, griseofulvin, vinca alkaloids, including vinblastine and vincristine, maytansinoids and ansamitocins, rhizoxin, phomopsin A, ustiloxins, dolastatin 10, dolastatin 15, halichondrins and halistatins, spongistatins, cryptophycins, rhazinilam, betaine, taurine, isethionate, HO-221, adociasulfate-2, estramustine, monoclonal anti-idiotypic antibodies, microtubule assembly promoting protein (taxol-like protein, TALP), cell swelling induced by hypotonic (190 mosmol/L) conditions, insulin (100 nmol/L) or glutamine (10 mmol/L), dynein binding, gibberelin, XCHO1 (kinesin-like protein), lysophosphatidic acid, lithium ion, plant cell wall components (e. g., poly-L-lysine and extensin), glycerol buffers, Triton X-100 microtubule stabilizing buffer, microtubule associated proteins (e. g., MAP2, MAP4, tau, big tau, ensconsin, elongation factor-l-alpha (EF-l. alpha. ) and E-MAP-115), cellular entities (e. g. , histone H1, myelin basic protein and kinetochores), endogenous microtubular structures (e. g. , axonemal structures, plugs and GTP caps), stable tubule only polypeptide (e. g., STOP145 and STOP220) and tension from mitotic forces, as well as any analogues and derivatives of any of the above. Within other embodiments, the anti- microtubule agent is formulated to further comprise a polymer."

The term"anti-micrtubule, "as used in this specification (and in the specification of United States patent 6,689, 803), refers to any"... protein, peptide, chemical, or other molecule which impairs the function of microtubules, for example, through the prevention or stabilization of polymerization. A wide variety of methods may be utilized to determine the anti-microtubule activity of a particular compound, including for example, assays described by Smith et al.

(Cancer Lett 79 (2): 213-219,1994) and Mooberry et al. , (Cancer Lett. 96 (2): 261-266,1995) ; " see, e. g. , lines 13-21 of column 14 of United States patent 6,689, 803.

An extensive listing of anti-microtubule agents is provided in columns 14,15, 16, and 17 of United States patent 6,689, 803 ; and one or more of them may be disposed within the polymeric material together with and/or instead of the anti-mitotic compound of this invention.

In one embodiment, these prior art anti-microtubule agents are made magnetic in accordance with the process described earlier in this specification.

These prior art anti-microtubule agents, which may be used to prepare the anti-mitotic compounds of this invention, include"... taxanes (e. g., paclitaxel (discussed in more detail below) and docetaxel) (Schiff et al., Nature 277: 665-667,1979 ; Long and Fairchild, Cancer Research 54: 4355-4361,1994 ; Ringel and Horwitz, J. Natl. Cancer Inst. 83 (4): 288-291, 1991; Pazdur et al., Cancer Treat. Rev. 19 (4): 351-386,1993), campothecin, eleutherobin (e. g. , U. S.

Pat. No. 5,473, 057), sarcodictyins (including sarcodictyin A), epothilones A and B (Bollag et al. , Cancer Research 55: 2325-2333,1995), discodermolide (ter Haar et al., Biochemistry 35: 243-250,1996), deuterium oxide (D2 O) (James and Lefebvre, Genetics 130 (2): 305-314,1992 ; Sollott et al. , J. Clin. Invest. 95: 1869-1876,1995), hexylene glycol (2-methyl-2,4-pentanediol) (Oka et al., Cell Struct. Funct. 16 (2): 125-134,1991), tubercidin (7-deazaadenosine) (Mooberry et al., Cancer Lett. 96 (2): 261-266,1995), LY290181 (2-amino-4- (3-pyridyl)-4H-naphtho (1, 2- b) pyran-3-cardonitrile) (Panda et al. , J. Biol. Chem. 272 (12): 7681-7687,1997 ; Wood et al., Mol. Pharmacol. 52 (3): 437-444,1997), aluminum fluoride (Song et al. , J. Cell. Sci. Suppl. 14: 147-150, 1991), ethylene glycol bis- (succinimidylsuccinate) (Caplow and Shanks, J. Biol.

Chem. 265 (15): 8935-8941,1990), glycine ethyl ester (Mejillano et al. , Biochemistry 31 (13): 3478-3483,1992), nocodazole (Ding et al. , J. Exp. Med. 171 (3): 715-727,1990 ; Dotti et al. , J.

Cell Sci. Suppl. 15: 75-84,1991 ; Oka et al. , Cell Struct. Funct. 16 (2): 125-134,1991 ; Weimer et al. , J. Cell. Biol. 136 (1), 71-80,1997), cytochalasin B (Illinger et al. , Biol. Cell 73 (2-3): 131- 138, 1991), colchicine and CI 980 (Allen et al. , Am. J. Physiol. 261 (4 Pt. 1) : L315-L321, 1991 ; Ding et al. , J. Exp. Med. 171 (3): 715-727,1990 ; Gonzalez et al. , Exp. Cell. Res. 192 (1) : 10-15, 1991; Stargell et al. , Mol. Cell. Biol. 12 (4): 1443-1450,1992 ; Garcia et al. , Antican. Drugs 6 (4): 533-544,1995), colcemid (Barlow et al. , Cell. Motil. Cytoskeleton 19 (1) : 9-17, 1991 ;

Meschini et al. , J. Microsc. 176 (Pt. 3): 204-210, 1994 ; Oka et al., Cell Struct. Funct. 16 (2): 125- 134, 1991), podophyllotoxin (Ding et al. , J. Exp. Med. 171 (3): 715-727,1990), benomyl (Hardwick et al. , J. Cell. Biol. 131 (3): 709-720,1995 ; Shero et al., Genes Dev. 5 (4): 549-560, 1991), oryzalin (Stargell et al. , Mol. Cell. Biol. 12 (4): 1443-1450,1992), majusculamide C (Moore, J. Ind. Microbiol. 16 (2): 134-143,1996), demecolcine (Van Dolah and Ramsdell, J.

Cell. Physiol. 166 (1) : 49-56,1996 ; Wiemer et al. , J. Cell. Biol. 136 (1) : 71-80,1997), methyl-2- benzimidazolecarbamate (MBC) (Brown et al. , J. Cell. Biol. 123 (2): 387-403,1993), LY195448 (Barlow & Cabral, Cell Motil. Cytoskel. 19: 9-17,1991), subtilisin (Saoudi et al. , J.

Cell Sci. 108 : 357-367,1995), 1069C85 (Raynaud et al. , Cancer Chemother. Pharmacol. 35: 169-173,1994), steganacin (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), combretastatins (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), curacins (Hamel, Med. Res. Rev. 16 (2): 207- 231,1996), estradiol (Aizu-Yokata et al., Carcinogen. 15 (9): 1875-1879, 1994), 2- methoxyestradiol (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), flavanols (Hamel, Med. Res.

Rev. 16 (2): 207-231,1996), rotenone (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), griseofulvin (Hamel, Med. Res. Rev. 16 (2): 207-231 ; 1996), vinca alkaloids, including vinblastine and vincristine (Ding et al. , J. Exp. Med. 171 (3): 715-727,1990 ; Dirk et al., Neurochem. Res. 15 (11): 1135-1139,1990 ; Hamel, Med. Res. Rev. 16 (2): 207-231,1996 ; Illinger et al. , Biol. Cell 73 (2-3): 131-138,1991 ; Wiemer et al. , J. Cell. Biol. 136 (1) : 71-80, 1997), maytansinoids and ansamitocins (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), rhizoxin (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), phomopsin A (Hamel, Med. Res. Rev.

16 (2): 207-231,1996), ustiloxins (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), dolastatin 10 (Hamel, Med Res. Rev. 16 (2): 207-231,1996), dolastatin 15 (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), halichondrins and halistatins (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), spongistatins (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), cryptophycins (Hamel, Med. Res.

Rev. 16 (2): 207-231,1996), rhazinilam (Hamel, Med. Res. Rev. 16 (2): 207-231,1996), betaine (Hashimoto et al. , Zool. Sci. 1: 195-204, 1984), taurine (Hashimoto et al. , Zool. Sci. 1: 195-204, 1984), isethionate (Hashimoto et al. , Zool. Sci. 1: 195-204,1984), HO-221 (Ando et al., Cancer Chemother. Pharmacol. 37: 63-69,1995), adociasulfate-2 (Sakowicz et al. , Science 280: 292- 295,1998), estramustine (Panda et al. , Proc. Natl. Acad. Sci. USA 94: 10560-10564,1997), monoclonal anti-idiotypic antibodies (Leu et al. , Proc. Natl. Acad. Sci. USA 91 (22): 10690- 10694,1994), microtubule assembly promoting protein (taxol-like protein, TALP) (Hwang et al. , Biochem. Biophys. Res. Commun. 208 (3): 1174-1180,1995), cell swelling induced by hypotonic (190 mosmol/L) conditions, insulin (100 nmol/L) or glutamine (10 mmol/L) (Haussinger et al. , Biochem. Cell. Biol. 72 (1-2): 12-19,1994), dynein binding (Ohba et al.,

Biochim. Biophys. Acta 1158 (3): 323-332,1993), gibberelin (Mita and Shibaoka, Protoplasma 119 (1/2): 100-109, 1984), XCHO1 kinesin-like protein) (Yonetani et al. , Mol. Biol. Cell 7 (suppl): 211A, 1996), lysophosphatidic acid (Cook et al. , Mol. Biol. Cell 6 (suppl): 260A, 1995), lithium ion (Bhattacharyya and Wolff, Biochem. Biophys. Res. Commun. 73 (2): 383- 390,1976), plant cell wall components (e. g. , poly-L-lysine and extensin) (Akashi et al. , Planta 182 (3): 363-369,1990), glycerol buffers (Schilstra et al. , Biochem. J. 277 (Pt. 3): 839-847, 1991 ; Farrell andKeates, Biochem. Cell. Biol. 68 (11): 1256-1261,1990 ; Lopez et al. , J. Cell.

Biochem. 43 (3): 281-291,1990), Triton X-100 microtubule stabilizing buffer (Brown et al. , J.

Cell Sci. 104 (Pt. 2): 339-352,1993 ; Saflejko-Mroczka and Bell, J. Histochem. Cytochem.

44 (6): 641-656,1996), microtubule associated proteins (e. g. , MAP2, MAP4, tau, big tau, ensconsin, elongation factor-1-alpha EF-l. alpha. ) and E-MAP-115) (Burgess et al., Cell Motil.

Cytoskeleton 20 (4): 289-300, 1991; Saoudi et al. , J. Cell. Sci. 108 (Pt. 1) : 357-367,1995 ; Bulinski and Bosser, J. Cell. Sci. 107 (Pt. 10): 2839-2849,1994 ; Ookata et al. , J. Cell Biol.

128 (5): 849-862, 1995; Boyne et al. , J. Comp. Neurol. 358 (2): 279-293,1995 ; Ferrera and Caceres, J. Neurosci. 11 (2): 392400,1991 ; Thurston et al. , Chromosoma 105 (1) : 20-30,1996 ; Wang et al., Brain Res. Mol. Brain Res. 38 (2) : 200-208,1996 ; Moore and Cyr, Mol. Biol. Cell 7 (suppl): 221-A, 1996; Masson and Kreis, J. Cell Biol. 123 (2), 357-371,1993), cellular entities (e. g. histone H1, myelin basic protein and kinetochores) (Saoudi et al. , J. Cell. Sci. 108 (Pt. 1) : 357-367,1995 ; Simerly et al. , J. Cell Biol. 111 (4): 1491-1504,1990), endogenous microtubular structures (e. g. , axonemal structures, plugs and GTP caps) (Dye et al., Cell Motil. Cytoskeleton 21 (3): 171-186, 1992; Azhar and Murphy, Cell Motil. Cytoskeleton 15 (3): 156-161,1990 ; Walker et al. , J. Cell Biol. 114 (1) : 73-81,1991 ; Drechsel and Kirschner, Curr. Biol. 4 (12): 1053-1061,1994), stable tubule only polypeptide (e. g. , STOP145 and STOP220) (Pirollet et al., Biochim. Biophys. Acta 1160 (1) : 113-119,1992 ; Pirollet et al., Biochemistry 31 (37): 8849- 8855,1992 ; Bosc et al. , Proc. Natl. Acad. Sci. USA 93 (5): 2125-2130,1996 ; Margolis et al. , EMBO J. 9 (12): 4095-4102,1990) and tension from mitotic forces (Nicklas and Ward, J. Cell Biol. 126 (5): 1241-1253,1994), as well as any analogues and derivatives of any of the above.

Such compounds can act by either depolymerizing microtubules (e. g., colchicine and vinblastine), or by stabilizing microtubule formation (e. g., paclitaxel)." United States patent 6, 689, 803 also discloses (at columns 16 and 17 that, "Within one preferred embodiment of the invention, the therapeutic agent is is paclitaxel, a compound which disrupts microtubule formation by binding to tubulin to form abnormal mitotic spindles.

Briefly, paclitaxel is a highly derivatized diterpenoid (Wani et al. , J. Am. Chem. Soc. 93: 2325, 1971) which has been obtained from the harvested and dried bark of Taxus brevifolia (Pacific

Yew) and Taxomyces Andreanae and Endophytic Fungus of the Pacific Yew (Stierle et al., Science 60: 214-216, -1993). "Paclitaxel" (which should be understood herein to include prodrugs, analogues and derivatives such as, for example, TAXOL@, TAXOTEREX, Docetaxel, 10-desacetyl analogues of paclitaxel and 3'N-desbenzoyl-3'N-t-butoxy carbonyl analogues of paclitaxel) may be readily prepared utilizing techniques known to those skilled in the art (see e. g., Schiff et al., Nature 277: 665-667,1979 ; Long and Fairchild, Cancer Research 54: 4355-4361,1994 ; Ringel and Horwitz, J. Natl. Cancer Inst. 83 (4): 288-291,1991 ; Pazdur et al., Cancer Treat. Rev. 19 (4): 351-386,1993 ; WO 94/07882 ; WO 94/07881; WO 94/07880; WO 94/07876; WO 93/23555; WO 93/10076; W094/00156 ; WO 93/24476; EP 590267; WO 94/20089; U. S. Pat. Nos. 5,294, 637; 5,283, 253; 5,279, 949; 5,274, 137; 5,202, 448; 5,200, 534; 5,229, 529; 5,254, 580 ; 5,412, 092; 5,395, 850; 5, 380, 751; 5,350, 866 ; 4,857, 653; 5,272, 171; 5,411, 984; 5,248, 796; 5, 248, 796; 5,422, 364; 5,300, 638; 5,294, 637; 5,362, 831; 5,440, 056; 4,814, 470; 5,278, 324; 5,352, 805; 5,411, 984; 5,059, 699; 4,942, 184; Tetrahedron Letters 35 (52): 9709-9712,1994 ; J. Med. Chem. 35: 4230-4237,1992 ; J. Med. Chem. 34: 992-998, 1991; J. Natural Prod. 57 (10): 1404-1410,1994 ; J. Natural Prod. 57 (11): 1580-1583, 1994; J.

Am. Chem. Soc. 110: 6558-6560, 1988), or obtained from a variety of commercial sources, including for example, Sigma Chemical Co. , St. Louis, Mo. (T7402--from Taxus brevifolia)." As is also disclosed in United States patent 6,689, 893, "Representative examples of such paclitaxel derivatives or analogues include 7-deoxy-docetaxol, 7,8-cyclopropataxanes, N- substituted 2-azetidones, 6, 7-epoxy paclitaxels, 6, 7-modified paclitaxels, 10-desacetoxytaxol, 10-deacetyltaxol (from 10-deacetylbaccatin III), phosphonooxy and carbonate derivatives of taxol, taxol 2', 7-di (sodium 1, 2-benzenedicarboxylate, 10-desacetoxy-11, 12-dihydrotaxol- 10,12 (18) -diene derivatives, 10-desacetoxytaxol, Protaxol (2'-and/or 7-O-ester derivatives), (2'- and/or 7-0-carbonate derivatives), asymmetric synthesis of taxol side chain, fluoro taxols, 9- deoxotaxane, (13-acetyl-9-deoxobaccatine III, 9-deoxotaxol, 7-deoxy-9-deoxotaxol, 10- desacetoxy-7-deoxy-9-deoxotaxol, Derivatives containing hydrogen or acetyl group and a hydroxy and tert-butoxycarbonylamino, sulfonate 2'-acryloyltaxol and sulfonate 2'-O-acyl acid taxol derivatives, succinyltaxol, 2'-. gamma.-aminobutyryltaxol formate, 2'-acetyl taxol, 7- acetyl taxol, 7-glycine carbamate taxol, 2'-OH-7-PEG (5000) carbamate taxol, 2'-benzoyl and 2', 7-dibenzoyl taxol derivatives, other prodrugs (2'-acetyl taxol; 2', 7-diacetyltaxol; 2'succinyltaxol ; 2'- (beta-alanyl)-taxol) ; 2'gamma-aminobutyryltaxol formate; ethylene glycol derivatives of 2'-succinyltaxol ; 2'-glutaryltaxol; 2'- (N, N-dimethylglycyl) taxol; 2'- (2- (N, N- dimethylamino) propionyl) taxol; 2'orthocarboxybenzoyl taxol; 2'aliphatic carboxylic acid derivatives of taxol, Prodrugs {2'(N, N-diethylaminopropionyl) taxol, 2' (N, N-

dimethylglycyl) taxol, 7 (N, N-dimethylglycyl) taxol, 2', 7-di- (N, N-dimethylglycyl) taxol, 7 (N, N- diethylaminopropionyl) taxol, 2', 7-di (N, N-diethylaminopropionyl) taxol, 2'- (L-glycyl) taxol, 7- (L-glycyl) taxol, 2', 7-di (L-glycyl) taxol, 2'- (L-alanyl) taxol, 7- (L-alanyl) taxol, 2', 7-di (L- alanyl) taxol, 2'- (L-leucyl) taxol, 7- (L-leucyl) taxol, 2', 7-di (L-leucyl) taxol, 2'- (L-isoleucyl) taxol, 7- (L-isoleucyl) taxol, 2', 7-di (L-isoleucyl) taxol, 2'- (L-valyl) taxol, 7- (L-valyl) taxol, 2'7-di (L- valyl) taxol, 2'- (L-phenylalanyl) taxol, 7- (L-phenylalanyl) taxol, 2', 7-di (L-phenylalanyl) taxol, 2'- (L-prolyl) taxol, 7- (L-prolyl) taxol, 2', 7-di (L-prolyl) taxol, 2'- (L-lysyl) taxol, 7- (L-lysyl) taxol, 2', 7-di (L-lysyl) taxol, 2'- (L-glutamyl) taxol, 7- (L-glutamyl) taxol, 2', 7-di (L-glutamyl) taxol, 2'- (L- arginyl) taxol, 7- (L-arginyl) taxol, 2', 7-di (L-arginyl) taxol}, Taxol analogs with modified phenylisoserine side chains, taxotere, (N-debenzoyl-N-tert- (butoxycaronyl)-10-deacetyltaxol, and taxanes (e. g. , baccatin III, cephalomamnine, 10-deacetylbaccatin III, brevifoliol, yunantaxusin and taxusin)." A process for delivering the magnetic anti-mitotic compound Figure 1 isa schematic of a preferred process 10 for delivering the magentic anti-mitotic compound described elsewhere in this specification to a specified location. In one embodiment, the magnetic anti-mitotic compound is disposed within a biological organism such as, e. g. , a blood vessel 12, and particles 14 of the anti-mitotic compound are delivered to a drug-eluting stent 16.

Referring to Figure 1, and to the preferred embodiment depicted therein, a bodily fluid, such as blood (not shown for the sake of simplicity of representation) is continuously fed to and through blood vessel 12 in the directions of arrows 20 and 22. In the embodiment depicted, the blood is fed through a generator 26 in order to cause the production of electrical current. In one preferred embodiment, the generator 26 is implanted within an artery 12 or vein 12 of a human being. In another embodiment, not shown, the generator 26 is disposed outside of the artery 12 or vein 12 of the human being.

One may use any of the implanted or implantable generators known to those skilled in the art. Thus, e. g. , one may use the power supply disclosed and claimed in United States patent 3,456, 134 (a piezoelectric converter), (3,486, 506 (an electric pulse generator with stator winding means), 3,554, 199 (heart actuated generator), 3,563, 245 (a miniaturized power supply unit which employs the mechanical energy of heart muscle contractions to produce electrical energy for a pacemaker), 3,659, 615 (a piezoelectric converter activated by organic mucle),.

4,453, 537 (a pressure actuated artificial heart powered by a another implanted device attached to a body muscle), 5, 810, 015 (an implantable power supply that is comprised means for

converting non-electrical energy to electrical energy), and the like. The entire disclosure of each of these United States patents is hereby incorporated by reference into this specification.

Referring again to Figure 1, and to the preferred embodiment depicted therein, the blood preferably flows in the direction of arrow 20, past generator 26, and through stent assembly.

The electrical energy from generator 26 is passed via line 28 to regulator 30.

In one referred emodiment, the generator 26 produces alternating current that is converted into direct current by regulator 30. One may use, e. g. , any of the implantable rectifiers known to those skiled in the art as regulator 30.

These prior art implantable rectifiers are well known and are described, e. g. , in United States patent 5,999, 849, the entire disclosure of which is hereby incorporated by reference into this specification. Thus, by way of further illustration, reference may also be had to United States patent 6,456, 883, the entire disclosure of which is hereby incorporated by reference into this specification, one may use the implantable rectifier disclosed in such patent.

Referring again to Figure 1, and in one preferred embodiment thereof, the regulator 30 is operatively connected to controller 32 by means of a link 34, and the regulator 30 is comprised of an andjustable power supply whose output may be regulated in response to signals fed to such regulator 30 by controller 32.

One may use any of the implantable power supplies known to those in the art as regulator 32. Thus, e. g. , one may use the devices disclosed in United States patents 3,563, 245, 3,757, 995 (see claim 6), 4,143, 661 (implantable power supply for a blood pump), 4,665, 896 (a transcutaneous transformer having an external primary winding means and an implanted secondary winding means), 5,702, 430 (a surgically implantable power supply comprising battery means for providing a source of power and charging means for charging the battery means). 5,949, 632 (a subcutaneous secondary coilconnected to a capacitor/rectifier circuitthat is tuned to the carrier frequency being transmitted transcutaneously to the secondary coil), 5,954, 058 (a rechargeable electrically powered implantable infusion pump), 6,141, 583, and the like. The entire disclosure of each of these United States patents is hereby incorporated by reference into this specification.

Referring again to Figure 1, and in the preferred embodiment depicted therein, the generator 26, in one embodiment, produces alternating current This alternating current is fed via line 28 to regulator 30, which preferably converts the alternating current to direct current and either feeds it in a first direction via line 36 to metallic stent 16, or feeds it in another direction via line 38 to metallic stent 16. As will be apparent to those skilled in the art, the regulator 26 thus has the capability of producing a magnetic field of a first polarity (when the

direct current is fed in a first direction 36) or a second polarity (swhen the direct current is fed in a second direction 38), as dictated by the well-known Lenz's law.

In one embodiment, the regulator 26 is capable not only of changing the direction of the electrical current, but also its amount. It preferably is comprised of a variable resistance circuit that can modulate its output.

In the preferred embodiment depicted, the regulator 26 is comprised of a transceiver (not shown) whose antenna 40 is in telemetric contact with a controller 32. The controller 32 is preferably in telemetric contact with biosensors 42,44, 46, and/or 48; and, depending upon the information received from one or more of such sensors, can direct the regulator 30 to increase the production of electrical current in one direction, or another, to decrease the production of electrical current in one direction, or another, or to cease the production of electrical current in one direction or another.

Biosensors 42,44, 46, and/or 48 may be one or more of the implantable biosensors known to those skilled in the art.

In one embodiment, one of such sensors 42,44, 46, and/or 48 can determine the extent to which two recognition molecules have bound to each other. Thus, e. g. , one may use the process and apparatus described in United States patent 5,376, 556, in which an analyte- mediated ligand binding event is monitored; the entire disclosure of this United States patent is hereby incorporated by reference into this specification..

By way of further illustraton, one may use one or more of the sensors described in United States patents 4,513, 280 (device for detecting toxicants), 4,947, 854 (epicardial multifunctional probe), 5,327, 225 (surface plasmon resonance sensor), 5, 284, 146 (removable implanted device), 5,766, 934 (chemical and biological sensosrs having electroactive polymer thin films attached to microfabricated device and possessing immobilized indicator molecules), 5,972, 638 (biosensor), 6,169, 494 (biotelemetry locator), 6,297, 059 (triggered optical sensors), 6,411, 834 (biological sensor), 6,546, 267 (biological sensor), 6,594, 011 (light based sensors), 6,607, 480 (evaluation system for obtaining diagnostic information from the signals and data of medical sensor systems), and the like. The entire disclosure of each of these United States patents is hereby incorporated by reference into this specification.

One may use any of prior art means for externally applying magnetic field 52. Thus, and referring to published United States patent application 2004/0030379, the entire disclosure of which is hereby incorporated by reference into this specification,"An external electromagnetic source or field may be applied to the patient having an implanted coated medical device using any method known to skilled artisan. In the method of the present

invention, the electromagnetic field is oscillated. Examples of devices which can be used for applying an electromagnetic field include a magnetic resonance imaging ("MRI") apparatus.

Generally, the magnetic field strength suitable is within the range of about 0.50 to about 5 Tesla (Webber per square meter). The duration of the application may be determined based on various factors including the strength of the magnetic field, the magnetic substance contained in the magnetic particles, the size of the particles, the material and thickness of the coating, the location of the particles within the coating, and desired releasing rate of the biologically active material." Referring again to Figure 1, and in the preferred embodiment depicted therein, In the embodiment depicted, a layer of drug eluting polymer 49 is present in the stent assembly; and this polymer may be used to either attract anti-mitotic agent into it, and/or to elute anti-mitotic agent out of it.

In one preferred embodiment, direct current electrical energy is delivered via lines 36/38 to a stent assembly 16. In this embodiment, it is preferred that stent assembly 16 be comprised of conductive material, and that the stent also be comprised of wire-like struts (see, e. g. , Figure 1 of published United States patent application 1004/0030379).

As will be apparent, as the direct current flows through the conductive material, it creates a static magnetic field in accordance with the well-known Lenz's law. In one embodiment, with the blood flow that is typical through the blood vessels of human beings, magnetic fields on the order of about 1 Gauss can readily be created.

Referring again to Figure 1, the stent assembly 16 is preferably comprised of a metallic stent body 16 and, disposed thereon, drug eluting polymer 49. The hydrodynamic forces caused by the flow of blood through the stent assembly 16 causes elution of particles 14 of anti- mitotic agent.

It is preferred that regulator 30 be comprised of either a half wave or a full wave rectifier so that the current flowing from regulator 30 be direct current, i. e. , that such current flow in only one direction. As will be apparent with either"half-wave d. c." and/or"full-wave d. c." being fed to the stent 16, a magnetic field will be induced in such stent that will have a constant polarity but constantly varying intensity. Such a magnetic field with either consistently attract and/or repel the magnetic anti-mitotic particles 14, depending upon the magnetic polarity of such particles. In one preferred embodiment, the magnetized stent 16 consistently attracts the magnetic particles 14.

As will be apparent, the regulator is capable of varying the intensity and/or polarity of its output, preferably in response to a signal from the controller 32. The controller 32 is

preferably equipped with an antenna 50 which is in telemetric contact with both the regulator 30 and the sensors 42,44, 46, and 48.

The sensors 42,44, 46, and 48 may be any of implantable biosensors known to those skilled in the art.

The sensor (s) may comprise a means for sensing the strength of a magnetic field. As is disclosed in claim 4 of United States patent 5,562, 714 (the entire disclosure of which is hereby incorporated by reference into this specification), the sensing means"... comprises a sensing antenna having an electrical connection through diodes to a power supply so that the Q of said transmitting antenna is regulated by draw down of energy by said sense antenna through said diode connection to said power supply.: A process for predicting mutation type and mutation frequency In one embodiment of applicants'invention, there is provided a process for predicting both the type and frequency of mutations in certain protein drug targets.

As is known to those skilled in the art, many mutations are"silent, "i. e. , they do not result in amino acid changes in the protein being expressed. Put another way, a silent mutation is a mutation that does not result in a detectable phenotypic effect. A silent mutation may be due to a transition or a transversion that leads to synonym codon. Additionally, mutations can change a codonto code for an amino acid closely related in terms of shape, hydrophobicity or other properties to that coded for by the original codon. Reference may be had, e. g. , to United States patents 5,240, 846 5,639, 650; 5,840, 493 (mitochondrial DNA mutations); 5,976, 798 (methods for detecting mitochondrial mutations); 6,010, 908 (gene therapy by small fragment homologous replacement); 6,329, 138 (method for the detection of antibiotic resistance); 6,344, 356 (methods for recombining nucleic acids); 6,544, 745 (diagnostic assay for diabetes); 6,699, 479; and the like. The entire disclosure of each of these United States patents is hereby incorporated by reference into this specification.

An additional preferred embodiement is an algorithm using artificial intelligence or computer programs that improve their performance based on information gathered from previous cycles to predict which DNA bases are most likely to be mutated and result in important amino acid changes. This information can be derived empirically from data gathered by the sequencing of tubulin mutants from clinical samples of tumors.

As is also known to those skilled in the art, the active site of a protein is assembled from many amino acids that interact with the substrate of the enzymatic reaction or ligand binding reactions. In one embodiment of applicants'invention, one can anticipate which amino acid

changes will result in a change in drug binding. In one aspect of this embodiment, one anticipates which amino acid changes result in changes in drug binding in paclitaxeal and, thereafter, designs drugs to bind to the modified binding sites. In this aspect, by utilizing such drugs in advance of the mutation event, or concurrently therewith, the incidence of selecting for resistant forms of cancer is minimized.

Applicants'process 200 is schematically illustrated in Figure 3. In step 202 of the process, the structure of the target protein is obtained. The target protein may, e. g. , be a beta- tubulin that is implicated in, e. g. , certain drug resistance.

One may obtain the structure of the target protein by conventional or unconventional means. One, thus, may conduct conventional x-ray crystallography analysis of the protein in question. Alternatively, or additionally, one may obtain and/or confirm the structure of the protein in question by homology modeling, as is discussed elsewhere in this specification.

Thereafter, in step 204 of the process, the binding efficiency of a candidate drug to the target protein is predicted by conventional means. One may use the means disclosed in United States patents 5,854, 992 (system and method for structure-based drug design that includes accurate prediction of binding free energy); 5,933, 819 (prediction of relative biding moits of biologically active peptides and peptide mimetics); 6,226, 603 (method for the prediction of binding targets and the design of ligands) ; 6,772, 073 (method for prediction of binding targets and the design of ligands) ; and the like. The entire disclosure of each of these United States patents is hereby incorporated by reference into this specification.

By way of illustration, and referring to United States patent 5, 854, 992, such patent claims :"1. A method for building molecules for binding at a receptor site, comprising the steps of : (a) evaluating a receptor site for a molecular make up of at least a portion of the receptor site to which a molecule being grown will bind and generating at least a coordinate of at least a portion of the receptor site to which the molecule being grown will bind, and outputting, at least with respect to the molecular make up of the receptor site, the coordinate of the portion of the receptor site to which the molecule being grown will bind; (b) estimating free energy of the molecule being grown using knowledge-based potential data to estimate free energy and outputting the estimated free energy; and (c) building a molecule for binding to the receptor site using the outputs from steps (a) and (b), with the building step including building the molecule by selecting molecular fragments at orientations that will result in free energy estimates for the molecule that may be higher than a lowest free energy estimate possible for the molecule." Thereafter, in step 206 of the process, the key amino acids that are essential for the interaction of the target protein and the candidate drug are identified. This step also may be

conducted by conventional means, such as evaluation of the results of the energy minimization analyses preferably conducted in step 204.

In step 208 of the process, a slight variation in the homology model is made in order to determine how the modified model will function. Thus, e. g. , one may modify the target protein used in step 202, and then the process is repeated to determine the binding efficiency of the candidate drug (in step 204) for the modified target protein. The process is then repeated again, and again, until a multiplicity of sets of data are obtained with a multiplicity of different target proteins for the same drug.

This multiplicity of data will indicate which target protein the drug is most efficiently bound to the candidate drug, and which target protein is least efficiently bound to the target drug. The least efficiently bound target proteins are those proteins that, through natural selection of cells, might cause drug resistance to the candidate drug. Thus, in step 210, the data from repeated runs of process 200 is evaluated to determine which of the target proteins are least likely to bind to the candidate drug.

In step 212, the candidate drug is modified, and the modified drug is then tested again in the cyle of steps 202/204/206/208 to determine its binding efficiency with each of the target proteins initially evaluated as well as other modified target proteins.

This process may lead to other modified candidate drugs. The goal is to test for, and determine, the existence of a modified drug that has a high binding efficiency for all of the targeted protein structures.

As will be apparent, the process depicted in Figure 3 may be used to determine drugs that may minimize drug resistance to anti-mitotic agents; and these"modified drugs"may be used either by themselves and/or in combination with the original cancer drug, depending upon the relative binding efficiencies with regard to particular target proteins and the extent to which the use of such drugs results in synergy. As will also be apparent, the process depicted in Figure 3 may be used to determine drugs that may minimize other drug resistance caused by natural selection, such as antibiotic drug resistance. The process may also be used in cases of herbicide resistance, pesticide resistance, resistance to antiviral drugs, etc.

Figure 4 is a flow diagram of one particular process 220 involving the design of anti- mitotic drugs and, in one embodiment thereof, combinations of antimitotic drugs. Referring to Figure 4, and in step 222 thereof, the mutant proteins that are resistant to certain anti-mitotic agents are identified. These mutant proteins can be identified by conventional means such as, e. g. , those means described hereinbelow, which relate to the identification of mutant tubulin isotypes.

Some of these mutant tubulin isotypes are discussed in published United States patent application 2004/0121351, the entire disclosure of which is hereby incorporated by reference into this specification. This published United States patent application discloses that:"The conservation of structure and regulatory functions among the ß-tubulin genes in three vertebrate species (chicken, mouse and human) allowed the identification of and categorization into six major classes of beta-tubulin polypeptide isotypes on the basis of their variable carboxyterminal ends.... As tubulin molecules are involved in many processes and form part of many structures in the eucaryotic cell, they are possible targets for pharmaceutically active compounds. As tubulin is more particularly the main structural component of the microtubules it may act as point of attack for anticancer drugs such as vinblastin, colchicin, estramustin and taxol which interfere with microtubule function. The mode of action is such that cytostatic agents such as the ones mentioned above, bind to the carboxyterminal end the ß-tubulin which upon such binding undergoes a conformational change. For example, Kavallaris et al. [Kavallaris et al.

1997, J. Clin. Invest. 100: 1282-1293] reported a change in the expression of of specific ß- tubulin isotypes (class I, II, III, and IVa) in taxol resistant epithelial ovarian tumor. It was concluded that these tubulins are involved in the formation of the taxol resistence. Also a high expression of class III (3-tubulins was found in some forms of lung cancer suggesting that this isotype may be used as a diagnostic marker." The function of certain tubulins in paclitaxel resistance was also discussed in United States patent 6, 362, 321, the entire disclosure of which is hereby incorporated by reference into this specification. As is disclosed in this patent, "Taxol is a natural product derived from the bark of Taxus brevafolio (Pacific yew). Taxol inhibits microtubule depolymerization during mitosis and results in subsequent cell death. Taxol displays a broad spectrum of tumorcidal activity including against breast, ovary and lung cancer (McGuire et al. , 1996, N. Engld. J.

Med. 334: 1-6; and Johnson et al. , 1996, J. Clin. Ocol. 14: 2054-2060). While taxol is often effective in treatment of these malignancies, it is usually not curative because of eventual development of taxol resistance. Cellular resistance to taxol may include mechanisms such as enhanced expression of P-glycoprotein and alterations in tubulin structure through gene mutations in the B chain or changes in the ratio of tubulin isomers within the polymerized microtubule (Wahl et al. , 1996, Nature Medicine 2: 72-79; Horwitz et al. , 1993, Natl. Cancer Inst. 15: 55-61; Haber et al. , 1995, J. Biol. Chem. 270: 31269-31275; and Giannakakou et al., 1997, J. Biol. Chem. 272: 17118-17125)..." The increased presence of certain tubulin isotypes associated with certain types of cancers was noted in an article by Tien Yeh et al.,"The Bn Isotype of Tubulin is Present in the

Cell Nuclei of a Variety of Cancers, "Cell Motility and the Cytoskeleton 57: 96-106 (2004).

The Yeh et al. article discloses that both alpha-tubulin and beta-tubulin consist of a series of isotypes differing in amino acid sequence, each one encoded by a different gene ; and it refers to a 1998 article by Richard F. Luduena entitled"The multiple forms oftubulin : different gene products and covalent modifications, "Int. Rev. Cytol 178: 207-275. The Yeh et al. article also disclosed that the BII isotype of tubulin is present in the nuclei of many tumors, stating that "Three quarters (75%) of the tumors we examined contained nuclear the Bn (Table I)."The authors of the Yeh et al. article suggest that (at page 104)"... it would be interesting to expore the possibility of using nuclear BI as a chemotherapeutic target. " The aforementioned articles disclose several conventional means for identifying mutant proteins that are a cause, at least in part, of anti-mitotic drug resistance. Comparable means may be used to identify mutant proteins that are the cause of antibioitic drug resistance, vaccine resistance, herbicide reistance, pesticide resistance, antiviral drug resitance, and the like. In general, one may study specimens of drug resistant orgnanisms to determine the existence of prorteins that are preferentially expressed in the drug resistant organisms as compared with a comparable non-drug resistant organisms. Additionally, or alteratively, one may determine the existence of proteins that are preferentially expressed in the diseased organisms in order to determine whether such proteins are essential for the progress of the disease. Means for making such determinations are well documented in the patent literature. Reference may be had, e. g., to United States pagents 5,853, 995 (large scale genotyping of diseases); 6,162, 604 (methods for determining genetic predisposition to automimmune disease by genotypying apoptotic genes); 6,291, 175 (methods for treating a neurological disease by determining BCHE genotype); 6,303, 307 (large scale genotyping of disease); 6,355, 859; 6,432, 643 (method of determining Alzheimer's disease risk using apolipoprotein E4 genotype analysis); 6,573, 049 (genotyping of the paraoxonase 1 gene for prognosing, diagnosing, and treating a disease); and the like. The entire disclosure of each of these United States patents is hereby incorporated by reference into this specification.

Referring again to Figure 4, and in step 224 thereof, certain candidate drugs are then identified that will bind to the mutant proteins. This can be done with the process depicted in Figure 3.

It is often the case that more than one mutant protein is present in cases of drug resistance. As is known, cancer often has a heterogeneous genotype in which different isotopes preferentially contain different drug-resistant proteins. In such a case, it is often desirable to determine not only which candidate drugs will bind to the particular mutant protein (see step

222), but also what combination of drugs will effectively bind to all the mutant proteins present in the heterogeneous genotype. Furthermore, one should also determine the concentration (s) and/or ratios of such drugs to maximize the possibility of a synergistic therapeutic effect.

After the identity and concentration of the drugs to be used has been determined, one can can either administer these drugs simultaneously (in step 228) and/or administer these drugs sequentially (in step 230). Additionally, or alternatively, in step 232 one may administer non- drug therapy either the same time as the administration of the drug (s), and/or at one or more different times.

One may use one or more of non-drug anti-mitotic therapies that are known to those skilled in the art. Thus, e. g. , in step 234 one may use hyperthermia. With the use of the magnetic anti-mitotic drugs discussed elsewhere in this specification, one may direct these drugs to the site of a tumor with the aid of an external electromagnetic field and thereafter, with the use of one or more other electromagnetic fields, cause such drug (s) to heat up to its Curie temperature and preferentially damage and/or destroy cancer cells. In one aspect of this embodiment, the Curie temperature of the magnetic anti-mitotic compound is less than about 41 degrees Celsius.

One may use radiation therapy in step 236. Thus, e. g. , the magnetic anti-mitotic drug of this invention may contain a radioactive moiety, such as radioactive iron, or radioactive cobalt.

One may use ultrasound therapy is step 238. This step is described in more detail in the next section of this specification.

Treatment of in vivo tumors with high frequency energy Figure 5 is a flow diagram of a preferred process 260 for treating a biological organism with mechanical vibrational energy (such as ultrasound) as set forth in step 238 of Figure 4.

In the process of applicants'invenition, in addition to the ultrasound energy, one may use other forms of mechanical energy, some of which are disclosed in published United States patent application 2004/0030379.

Referring to published United States patent application 2004/0030379, the entire disclosure of which is hereby incorporated by reference into this specification, "The mechanical vibrational energy source includes various sources which cause vibration such as ultrasound energy. Examples of suitable ultrasound energy are disclosed in U. S. Pat. No. 6,001, 069 to Tachibana et al. and U. S. Pat. No. 5,725, 494 to Brisken, PCT publications WO00/16704, WO00/18468, WO00/00095, WO00/07508 and W099/33391, which are all incorporated herein by reference. Strength and duration of the mechanical vibrational energy of the application may be determined based on various factors including the biologically active material contained in

the coating, the thickness of the coating, structure of the coating and desired releasing rate of the biologically active material." As is also disclosed in published United States patent application 2004/0030379, "Various methods and devices may be used in connection with the present invention. For example, U. S. Pat. No. 5, 895, 356 discloses a probe for transurethrally applying focused ultrasound energy to produce hyperthermal and thermotherapeutic effect in diseased tissue. U. S.

Pat. No. 5, 873, 828 discloses a device having an ultrasonic vibrator with either a microwave or radio frequency probe. U. S. Pat. No. 6,056, 735 discloses an ultrasonic treating device having a probe connected to a ultrasonic transducer and a holding means to clamp a tissue. Any of those methods and devices can be adapted for use in the method of the present invention." As is also disclosed in published United States patent application 2004/0030379, "Ultrasound energy application can be conducted percutaneously through small skin incisions.

An ultrasonic vibrator or probe can be inserted into a subject's body through a body lumen, such as blood vessels, bronchus, urethral tract, digestive tract, and vagina. However, an ultrasound probe can be appropriately modified, as known in the art, for subcutaneous application. The probe can be positioned closely to an outer surface of the patient body proximal to the inserted medical device." As is also disclosed in published United States patent application 2004/0030379,"The duration of the procedure depends on many factors, including the desired releasing rate and the location of the inserted medical device. The procedure may be performed in a surgical suite where the patient can be monitored by imaging equipment. Also, a plurality of probes can be used simultaneously. One skilled in the art can determine the proper cycle of the ultrasound, proper intensity of the ultrasound, and time to be applied in each specific case based on experiments using an animal as a model." As is also disclosed in published United States patent application 2004/0030379,"In addition, one skilled in the art can determine the excitation source frequency of the mechanical vibrational energy source. For example, the mechanical vibrational energy source can have an excitation source frequency in the range of about 1 Hertz to about 300 kiloHertz. Also, the shape of the frequency can be of different types. For example, the frequency can be in the form of a square pulse, ramp, sawtooth, sine, triangle, or complex. Also, each form can have a varying duty cycle.: Referring to Figure 5, and in step 261 thereof, the cells of a biological organism to be treated are first preferably synchronized. so that they are experiencing substantially

synchronous growth; in one aspect of this embodiment, such cells are synchronized in metaphase.

As is known to those skilled in the art, synchronous growth is growth in which all (or a substantial porition) of the cells are at the same stage of cell division at a given time; this is also often referred to as"synchronized growth."Reference may be had, e. g., to page 471 of J.

Stensch's"Dictionary of Biochemistry and Molecular Biology, "Second Edition (John Wiley & Sons, New York989). Reference may also be had, e. g. , to United States patent 5, 18, 887, the entire disclosure of which is hereby incorporated by reference into this specification.

Referring to such United States patent 5,158, 887, in claim 15 thereof there is described ". 15. The process as set forth in claim 1, wherein said modified cell elongation and synchronization of growth in the number of said cells and their effective mass is accomplished by: carrying out at least one additional subculture and incubation step between steps (c) and (d) of claim 1 wherein in each instance a batch subculture is prepared which contains a quantity of said slowly metabolizable carbon source in a growth medium and bacterial cells obtained from the immediately preceding batch subculture at a density level no greater than about one half of the density of the bacterial cells present in the immediately preceding batch subculture, and the batch subculture thus prepared incubated for a time to cause the cells therein to multiply only about one to one and one half generations."The"claim 1."of such patent referred to in such claim 15 describes"1.1. A process for producing bacterial cells useful in selective production of spores and a metabolic end product selected from the group consisting of solvents, enzymes, antibiotics and useful toxic proteins, and comprising the steps of providing an initial stock culture containing a carbon source in a growth medium, and at least about 1x106 cells per milliliter of bacteria of the genus Clostridium, said bacterial cells, when treated to inhibit division, being genetically capable of metabolizing a carbon source to produce spores or a metabolic end product selected from the group consisting of said solvents, enzymes, antibiotics and proteins; providing a quantity of a divalent cation source; inducing elongation of said bacterial cells under conditions to produce modified cells of a critical length of at least about 3x while synchronizing the growth in the number of said cells and their effective mass by (a) preparing from the initial stock culture another batch subculture which contains a quantity of a slowly metabolizable carbon source other than glucose in a growth medium by adding to the other batch subculture bacterial cells obtained from the initial stock culture and present at a density level no greater than about one half of the density of the bacterial cells present in the initial stock culture; (b) incubating said other batch subculture within a time to cause the cells therein to multiply for only about one to one and one half generations in said batch subculture

while maintaining the growth medium at a temperature within a range of about-20° C. to +10° C. of the speciesspecific optimum growth temperature, said growth medium being devoid of an amount of cellular metabolites that would be sufficient to substantially interfere with synchronous growth of said cells, (c) preparing from an immediately preceding batch subculture a final batch subculture which contains a quantity of a slowly metabolizable carbon source other than glucose in a growth medium by adding to said final batch subculture bacterial cells obtained from the immediately preceding batch subculture and present at a density level no greater than about one half of the density of the bacterial cells present in said immediately preceding batch subculture; (d) incubating said final batch subculture for a time to cause the cells therein to multiply while maintaining the growth medium at a temperature within the range of step (b), said growth medium being devoid of an amount of cellular metabolites that would be sufficient to substantially interfere with synchronous growth of said cells, and (e) carrying out at least incubation step (d) in the presence of at least about 0. 01M of said divalent cation and which is sufficient to cause cellular incorporation of an amount of said divalent cation into said elongated cells during step (d) to stabilize the cells against death, lysis and aggregation and cause modified cell division in a manner such that, as each cell divides into two cells, the resulting divided cells remain elongated to at least said 3 x length, said slowly metabolizable carbon source being selected in each instance to cause the bacteria to grow in the selected growth medium at a rate of about 10%-90% less than the maximum growth rate Km for the bacteria in an optimum growth medium; and thereafter selectively subjecting the cells resulting from step (d) to treatment conditions which thereafter inhibit cell division and cause the cells to primarily produce either spores or at least one of said metabolic end products." As is well known to those skilled in the art, other means of synchronization of growth of the cells of a biological organism may be used. Reference may be had, e. g. , to United States patents 4,315, 503 (modification of the growth, repair, and maintenance behavior of living tissues and cells by a specific and selective change in electrical environment), 4, 533, 635 (process for stimulating the growth of epidermal cells), 4,931, 053 (method for enhancing vascular and other growth), 5,158, 887 (process for massive conversion of clostridia in synchronized cells), 6,050, 990 (methods and devices for inhibiting hair growth), 6,143, 560 (method of synchronizing epithelial cells into Go phase), 6,149, 495 (human fibroblast diffusible factors), 6,369, 294 (methods comprising apoptosis inhibitors for the generation of transgenic pigs), 6,448, 040 (inhibitor of cellular proliferation), 6,767, 734 (method and apparatus for producing age-synchronized cells), and the like. The entire disclosure of each of these United States patents is hereby incorporated by reference into this specification.

In one especially preferred embodiment, and referring again to Figure 5, in step 261 the cells of biological organisms are synchronized by means of cell cycle arresting drugs. These drugs are well known to those skilled in the art. Reference may be had, e. g. , to European patent publication EP 0 870 506 for"Compositons comprising a cryptophicin compound in combination with a synchronizing or activating agent for treating cancer. "As is disclosed in this patent publication, As used herein, the term"synchronizing agent"refers to an agent that can partially synchronize tumor cells with respect to cell cycle progression. Thus the term shall refer to cell cycle phase specific agents such as Gemcitabine, which is now commercially available and other agents such as multitargeted antifolate (MTA, LY231514), the sulfonylurea LY295501, cisplatin, carboplatin, cyclophosphamide, topoisomerase inhibitor, CPT-11, etoposide, VP-16,5-fluorouracil, doxorubicin, methotrexate, hydroxyurea and 3'-azido-3'- deoxythymidine (AZT). Methods for preparing Gemcitabine are known to the skilled artisan and are described in U. S. patent number 4, 808, 614, herein incorporated by reference in its entirety. See also, European Patent number EP122707 (September 16,1987).... As used herein the term"activating agent"refers to an agent that can activate non-cycling cells so that they enter the cell cycle where they will be sensitive to the cytotoxic activity of Compounds I-V and agents which effect growth factor downstream kinase cascade to activate the cell cycle.

Examples of activating agents are growth factors, interleukins, and agents which modulate the function of cell cycle regulation which control cell cycle checkpoints and progression through the cell cycle. For example, but not limited to cdc25 phosphatase or p21. (sdil, wafl, cipl). Such growth factors and interleukins are known and readily available to the skilled artisan." In one preferred embodiment, the synchronizing agent used is preferably an agent that can partially synchronize tumor cells with respect to cell cycle progression and preferably is a cell cycle phase specific agents such as Gemcitabine, which is now commercially available.

Gemcitabine, and its synthesis, are well known to those skilled in the art. Reference may be had, e. g. , to United States patent 6,001, 994, the entire disclosure of which is hereby incorporated by reference into this specificqtion. Claim 1 of this patent describes"An improved process to make gemcitabine hydrochloride, the improvement consisting essentially of making the lactone intermediate, 2-deoxy-2, 2-difluoro-D-erythro-pentafuranose-l-ulose-3, 5- dibenzoate: [Figure] from D-erythro-2-Deoxy-2,2-difluoro-4, 5-O-(l-ethylpropyl)-idene) pentoic acid tert-Butyl ester wherein, the D-erythro-2-Deoxy-2,2-difluoro-4, 5-0- (l- ethylpropyl) -idene) pentoic acid tert-Butyl ester is prepared by the process of reacting S-tert- butyl difluoroethane thioate with 2, 3-O (1-ethylpropylidene)-D-glyceraldehyde, in a solvent and

in the presence of a strong base; with the proviso that the process is conducted in the absence of a catalyst and in the absence of a silyl containing" As is known to those skilled in the human, biological organisms have built in"check points"which allow them to effectuate synchronization of cell growth upon the occurrence of various events. Thus, and referring to Chapter 17 of Bruce Alberts et al. 's"Molecular Biology of the Cell, "Fourth Edition (Garland Publishing, New York, New York), it is disclosed that "We can illustrate the importance of an adjustable cell-cycle control system by extending our washing machine analogy. The control system of simple embryonic cell cycles, like the controller in a simple washing machine, is based on a clock. The clock is unaffected by the events it regulates and will progress through the whole sequence of events even if one of those events has not been successfully completed. In contrast, the control system of most cell cycles (and sophisticated washing machines) is responsive to information received back from the processes it is controlling. Sensors, for example, detect the completion of DNA synthesis (or the successful filling of the washtub), and, if some malfunction prevents the successful completion of this process, signals are sent to the control system to delay progression to the next phase. These delays provide time for the machinery to be repaired and also prevent the disaster that might result if the cycle progressed prematurely to the next stage." The Alberts et al. work also discloses that"In most cells there are several points in the cell cycle, called checkpoints, at which the cycle can be arrested if previous events have not been completed (Figure 17-14). Entry into mitosis is prevented, for example, when DNA replication is not complete, and chromosome separation in mitosis is delayed if some chromosomes are not properly attached to the mitotic spindle.... Progression through Gl and G2 is delayed by braking mechanisms if the DNA in the chromosomes is damaged by radiation or chemicals. Delays at these DNA damage checkpoints provide time for the damaged DNA to be repaired, after which the cell-cycle brakes are released and progress resumes." The Alberts et al. work also discloses that"Checkpoints are important in another way as well. They are points in the cell cycle at which the control system can be regulated by extracellular signals from other cells. These signalswhich can either promote or inhibit cell proliferationtend to act by regulating progression through a Gl checkpoint, using mechanisms." As will be apparent from many of the aforementioned United States patents, one may utilize externally applied chemotherapeutic agents to synchronize the cells within a biological organism at a certain stage. Thus, e. g. , reference may again be had to United States patent 6,511, 818, the entire disclosure of which is hereby incorporated by reference into this specification.

In column 1 of United States patent 6,511, 818, it is disclosed that"Precise coordination of the S and M phases of the eukaryotic cell cycle is critical not only for normal cell division, but also for effective growth arrest under conditions of stress. When damaged, a cell must communicate signals to both the mitotic and DNA synthesis machineries so that a mitotic block is not followed by an extra S phase, or vice versa. The biochemical mechanisms regulating this coordination, termed checkpoints, have been identified in lower eukaryotes, but are largely unknown in mammalian cellsl-3."The references cited in this section of the patent include A. W. Murray, Nature 359,599-604, 1992; P. Nurse, Cell 79,547-550, 1994, and L. H. Hartwell et al., Science 266, 1821-1828, 1994.

As is also disclosed in column 1 of such United States patent,"DNA-damaging agents are used in the clinic to preferentially kill cancer cells. However, there is a need in the art to discover additional therapeutic agents which are selectively toxic to cancer cells." United States patent 6,511, 818 describes and claims"1.1. A method of screening for potential anti-tumor agents, comprising the steps of : determining viability of homozygous p53 gene-defective human colonic cells incubated in the presence and in the absence of a test compound; and identifying the test compound as a potential anti-tumor agent if it causes cell death in the homozygous p53 gene-defective human colonic cells." Other United States patents also describe how to identify agents that synchronize cells at specific portions of the cell cycle. Reference may be had, e. g., to United States patents 5, 879, 889 (cancer drug screen based on cell cycle uncoupling), 5,882, 865 (cancer drug screen based on cell cycle uncoupling), 5,888, 735 (cancer drug screen based on cell cycle uncoupling), and 5, 879, 999 (cancer drug screen based on cell cycle uncoupling). The entire disclosure of each of these United States patents is hereby incorporated by reference into this specification.

In one preferred embodiment, a drug is used in such step 261 to synchronize the cells in the orgnanism at the M phase (metaphase), also known as"mitosis. "As is known, mitosis is the divison of the nucleus of euraryotic cells which occurs in four stages designated prophase, metaphase, anaphase, and telophase. In one aspect of this embodiment, the drug used in such step 261 synchronizes the cells in prophase. In one aspect of this embodiment, the drug used in such step 261 synchronizes the cells in metaphase. In one aspect of this embodiment, the drug used in such step 261 synchronizes the cells in anaphase. In one aspect of this emboidiemnt, the drug used in such step 261 synchronizes the cells in telophase.

In one embodiment, it is preferred that the drug used in step 261 stabilize the cells in metaphase. As is known to those skilled in the art, metaphase is the second stage in mitosis, during which the chromosomes arrange themselves in an equatorial region.

In another embodiment, it is preferred that the drug used in step 261 stabilize the cells in the"S Phase. "As is also disclosed in Chapter 17 of the aforementioned Alberts et al. text, "Replication of the nuclear DNA usually occupies only a portion of interphase, called the S. phase of the cell cycle.... The interval between the completion of mitosis and the beginning of DNA synthesis is called the Gl phase. "Reference also may be had, e. g. , to United States patents 4,812, 394 (flow cytometric measurement of DNA and incorporated nucleoside analogs), 5,633, 945 (accuracy in cell mitosis analysis), 5,866, 338 (cell cycle checkpoint genes), 6,172, 194 (ARF-pl9, a novel regulator of the mammalian cell cycle), 6,274, 576 (method of dynamic retardation of cell cycle kinetics to potentiate cell damage), 6,455, 593 (method of dynamic retardation of cell cycle kinetics to potentiate cell damage), and the like. The entire disclosure of each of these United States patents is hereby incorporated by reference into this specification.

As used in this specification, the term"synchronized"means that at least about 30 weight percent of the cells in question are in the desired phase, and preferably, at least about 50 weight percent of the cells in question are in the desired phase. In one embodiment, at least about 70 weight percent of the cells are in the desired phase.

One may determine the extent to which a collection of cells is synchronized by st andard flow cytometry techniques. Thus, and referring to United States patent 4, 812, 394, one may utilize a process wherein, as described by claim 1, there is"1. A non-radioactive method for measuring unaltered cellular DNA and incorporated nucleoside analog, the method comprising the steps of : growing a population of cells in the presence of a non-radioactive predetermined compound, the non-radioactive predetermined compound being capable of assimilation into the DNA of the cells of the population to form an incorporated nucleoside analog whose presence can be detected by an immunochemical stain; altering a portion of the DNA of each cell of the population to substantially the same extent such that a first portion comprising altered DNA is formed and a second portion comprising unaltered DNA remains, the first portion being sufficiently large so that nucleoside analogs incorporated therein can be detected by an immunochemical stain specific for the incorporated nucleoside analog, and the second portion being sufficiently large so that Gl phase cells of the population can be distinguished from the G2 M phase cells of the population by a second signal generated by a second stain specific for the second portion; applying the immunochemical stain to the cells; applying the second stain to the cells; and detecting at substantially the same time and for each cell of a substantial portion of the population, a non-radioactive first signal from the immunochemical stain bound to the incorporated nucleoside analog in the first portion of DNA of each cell and a second signal

from the second stain bound to the second portion of DNA of the same cell such that a first signal and a second signal are associated with each said cell of the substantial portion of the population. "As is disclosed in the specification of this patent, "A broad range of biological and biomedical investigations depends on the ability to distinguish cells that synthesize DNA from those that do not. Oncologists, for example, have devoted substantial effort to establishing correlations between the proportion of human tumor cells synthesizing DNA and treatment prognosis, e. g. Hart et al., Cancer, Vol. 39, pgs. 1603-1617 (1977). Effort has also been devoted to improvement of anticancer therapy with S-phase specific agents by treating when the experimentally determined proportion of tumor cells in S phase is maximal, e. g. Barranco et al., Cancer Research, Vol. 42, pgs. 2894-2898 (1982). In these studies, S-phase cells are usually assumed to be those that appear labeled in autoradiographs prepared immediately after pulse labeling with tritiated thymidine, or those with S-phase DNA content in DNA distributions measured flow cytometrically. Cancer researchers and oncologists have relied heavily on measurements of the proportion of DNA synthesizing cells to determine the cell cycle traverse characteristics of normal and malignant cells. The classical"fraction of labeled mitosis" procedure, Quastler et al., Experimental Cell Research, Vol. 17, pgs. 420-429 (1959), for example, depends on assessment of the frequency of mitotic cells that appear radioactively labeled in autoradiographs of samples taken periodically after labeling with tritiated thymidine.

Studies of the cell cycle traverse characteristics of drug-treated cell populations typically require measurement of the amount of tritiated thymidine incorporated by cells in S phase (e. g., by liquid scintillation spectrometry) or determination of the fraction of cells with S-phase DNA content (e. g. , by DNA distribution analysis), or both, Pallavicini et al. , Cancer Research, Vol.

42, pgs. 3125-3131 (1982). Studies of mutagen-induced genetic damage that use unscheduled DNA synthesis as an index of damage also rely on the detection of low levels of incorporation of tritiated thymidine, e. g. Painter et al. , Biochim. Biophys. Acta, vol. 418, pgs. 146-153 (1976)." As will apparent, one may use other analytical techniques to determine the degree to which the cells are synchronized in a specified phase. In one embodiment, the phase-sensitive flow cytometer described in United States patent 5,270, 548 is used; the entire disclosure of this United States patent is hereby incorporated by reference into this specification. This patent claims"1. A phase-sensitive flow cytometer for resolving fluorescence emissions from fluorochrome labeled cells into two components, comprising: flow cytometer means for providing a flow steam containing said labeled cells; an excitation light for exciting said labeled cells to fluoresce in said flow stream; modulation means for modulating said excitation light

and generating a reference signal at a selected modulation frequency ; detector means for receiving fluorescence emission spectra from said labeled cells as a modulated fluorescence signal and outputting a modulated intensity signal functionally related to said fluorescence emission spectra from said labeled cells; and phase detector means for resolving said modulated intensity signal into two signal components, each functionally related to a different fluorescence decay lifetime of said fluorescent emission spectra." Referring again to Figure 5, and in the preferred embodiment depicted therein, it is preferred to treat the cells with the synchronizing agent for at least about 25 minutes prior to it is contacted with ultrasound in step 266. It is more preferred to wait at least about 60 minutes prior to time one contacts the cells with ultrasound. In one embodiment, one waits at least about 4 hours until after first administration of the synchronizing agent until the cells are contacted with ultrasound. In one embodiment, a period of at at least about 48 hours is allowed to pass from the initial administration of the synchronizing agent before the cells so synchronized are contacted with the ultrasound energy.

Referring again to Figure 5, and in step 262 of this process, microtubules in diseased cells are preferably stabilized by one or more conventional means. As is known to those skilled in the art, stabilization of microtubles at metaphase can result in the synchronization of a population of cells at the metaphase checkpoint of the cell division cycle.

Thus, e. g. , one may effectuate such stabilization by using anti-mitotic or other chemical agents known to affect microtubules, or using chemicals that influence proteins that aid in the stabilization of microtubules (e. g. Rho or FAK), or a process of post-translational modification to the tubulin protein, until the half-life of an individual microtubule in the mitotic spindle of a dividing cell is an average of at least 8 minutes, or more than 10 percent of the microtubules in a non-dividing cell have a half-life of more than 8 minutes. One may use standard means for stabilizing the microtubules to this extent. Thus, e. g. , reference may be had to United States patents 5, 808, 898 (method of stabilizing microtubules); 5,616, 608; 6,403, 635; 6,414, 015 (laulimalide microtubule stabilizing agents); 6,429, 232; 6, 500859 (method for treating atherosclerosis or restenosis using microtubule stabilizing agent); 6,660, 767 (coumarin compounds as microtubules stabilizing agents); 6,740, 751 (methods and compositions for stabilizing microtubules and intermediate filaments); and the like. The entire disclosure of each of these United States patents is hereby incorporated by reference into this specification.

In step 264 of this process, the resonant frequency of the stabilized microtubules in the diseased cells to be treated is determined. As used herein, the term"resonant frequency"is that frequency which, at a power level of 10 milliwatts per square centimeter, a temperature of 37

degrees Celsius, and atmospheric pressure, is sufficient to break at least 50 weight percent of the microtubules in the cell after an exposure time of five (5) minutes. That frequency which breaks the maximum number of microtubules under these conditions is the resonant frequency.

In step 264 of the process depicted in Figure 5, an estimate of the energy and wavelengths associated with the vibration of microtubules from an external source is conducted.

By way of illustration and not limitation, and without being bound to any particular theory, applicants believe that such an estimate may be readily made in accordance with the discussion and the equations presented elsewhere in this specification.

A theoretical approach to estimate the type of ultrasound to be used in the process 260 Without wishing to be bound to any particular theory, it is believed that the critical force required to break a microtubule can be calculated by the equation : Fc-1/L2, which indicates that the critical force is proportional to 1 divided by the square of L. L is the length of the microtubule.

An estimate of the Fc required to buckle a microtubule can be had from the experimentally derived values of flexural rigidity measured for microtubules. For the purposes of this example, and not wishing to be bound to this value, we will assign L to the value of 1 Omicrometers (pm) and Fc to the value of 6pN. This value was determined experimentally by Gittes et al. , 1996 (ref to follow PLEASE FURNISH REFERENCE).

Again, for the purposes of this example, without wanting to be bound to a single value, the flexural rigidity of the non-taxol stabilized microtubule can be described with the equation: EI = 10. 10-24 Nm2. For comparison purposes, actin's critical stress can be described for the purposes of this example: Oc = 5 dyne/cm2 = 0.5 N/m2 Although not wanting to be bound to this value outside of this example, the buckling pressure of a microtubule has been experimentally determined to be 240 dyne/cm2 (Elbaum et al. PLEASE FURNISH THIS CITATION.) The cross sectional area of a hollow tube is described, as in Johnathan Howard's Mechanics of Motor Proteins and the Cytoskeleton (Sinauer Press, 2001), on page 101 to be: A = (-/4) (d22-dla) = 5 x 10-16 ma. This equation, in which A represents area, can be applied to microtubules as they are a polymer in the shape of a cylinder and the values of d2 and dl, in the case of a microtubule, are simply the outer and inner diameters of the cyliner (25 nm and 15 nm, respectively).

Critical force (Fc) can be calculated based on this area in the equation: Fc = Pc x A = 0.4pN, in which Pc represents the critical pressure applied perpendicularly to the cross-sectional area A..

Young's modulus (Y) is a description of the stiffness of a material. Young's modulus for microtubules has been experimentally determined. Y =109 N/m2.

The spring constant (k) for a microtubule can be calculated from the Young modulus as given below: k = (A x Y) /L, in which A is the area of the cylindrical cross-section (described above), Y is the Young's modulus and L is the length of the microtubule, therefore: k 7r (252 -152) xlO9)/10=~4N/m.

This value is important because it is greater than the force of attraction between 2 protofilaments in a microtubule structure (2 N/m).

In general, one should use the formula (see the book by Jonathon Howard) to derive the formula for the critical force: FC = 7r2 (El/L2), and thus calculate the propagation velocity for a standing vibrational wave in a microtubule by way of the following equation: v = (F/PL) I/2 in which PL is the linear mass density of the protein filament (microtubule) and F stands for the tension force that is less or at best equal to the critical force for breaking a microtubule.

One can then calculate the frequency of the vibrational mode according to: f=v/l = (F/pi)/1, where 1 is the wavelength of the standing wave. The fundamental harmonic will have the wavelength 1=2L where L is the length of the microtubule cylinder along its axis.

In general, the n-th harmonic will have the wavelength given by the formula: ln=2L/n. Hence, its frequency is given by: fn=nf, where f stands for the fundamental harmonic. The formula above is applied for the calculation of the fundamental harmonic, second harmonic, or third harmonic, etc by choosing the value of n as 1,2, 3, etc.... For purposes of this example, EI is assigned to be 26 x 10-24Nm2 in its native state while attached at both ends (one to a polar body, the other to a chromosome, as in mitosis). This value increases to 32 x 10-24Nm2 when the microtubule is stabilized with taxol. Using this value, we can estimate the frequency to be in the range of 270-420 kHz for the fundamental harmonic with a second harmonic at twice the frequency to be in the range of 540-840 kHz, etc.

It should be noted that the frequency formula depends inversely proportionally to the length of a given microtubule. In this connection, polar microtubules are almost twice as long as kinetochore microtubules and hence, in order to break them by means of applying high frequency ultrasound, different frequency ranges must be selected (approximately half the values of those applied to break kinetochore microtubules). In general, this application of ultrasound for breaking up the mitotic apparatus in dividing cells requires a prior microscopic observation and analysis of the cell's cytoskeletal apparatus with particular attention to the length of the microtubules to be determined as accurately as possible. Having determined the lengths and elastic constants for all kinetochore and polar microtubules, a weighted

superposition of the fundamental and first harmonic ultrasound modes must be calculated and then generated with a subsequent application to the cellular targets.

The mass density of tubulin is estimated to be approximately 900 kg/m3 while that of the surrounding medium (mainly water) is assumed to be 1000 kg/m3. The linear mass density of a microtubule cylinder is calculated assuming the length L, the outer and inner diameters d2 and dl, respectively, as stated above. Aqueous environment is filling the inner diameter region of the cylinder as well as forming a thin layer of bound water surrounding the outer surface. We assumed that a 3 angstrom layer of bound water is attached. With these assumptions, the linear mass density (mass per length) of a microtubule is approximately 5x 10 ~'3 kg/m. Using the formula for v stated above as a function of the force of tension applied to a microtubule (at most 6 pN) and the above linear mass density, we evaluate the propagation velocity of standing vibrational waves on microtubules to be in the range of 3-4 m/s which is much less than the propagation velocity of ultrasound in an aqueous medium (on the order of 1000 m/s).

The following is an estimate of the ultrasound intensity required to deliver a sufficiently strong amount of energy to break microtubules. The formula for the power delivered per cross- sectional area for a wave traveling at a speed v in a medium of mass density rho and having an amplitude A is given by: Power/Area = A2 v f rho, where f is the frequency of the wave.

Estimating the amplitude A to be in the 3 angstrom range, the frequency in the MHz range and the velocity of propagation as well as mass density as given above, we obtain an estimate of the intensity as 0.1 W/m2. However, this is only the power deposited in the form of microtubule oscillations. Since the ultrasound propagates at a much faster velocity in the medium before it is resonantly absorbed by the microtubules, the actual power generated at the source most be scaled up by the velocity ratio factor, i. e. we expect it to be at least in the range of 10-30 Wem2 which corresponds to the 130-135 dB range on the decibel scale.

It is known that Taxol, and Taxol-type compounds, stabilize microtubules, prevent them from shortening and dividing the cell as a result of their shortening as they segregate the genetic material in chromosomes. Furthermore, Taxol increases the rigidity of microtubules making them susceptible to breaking given the right physical stimuli.

Ultrasound induces mechanical vibrations of microtubules. At the right frequency, and at the right power level, the application of ultrasound will cause the microtubules to first buckle and then break up.

The ultrasound used in the process of this invention preferably has a frequency of from about 50 megahertz to about 2 Gigahertz, and more preferably has a frequency of from about 100 megahertz to about 1 Gigahertz. The power of such ultrasound is preferably at least about

0. 01 watts per square meter and, more preferably, at least about 10 watts per square meter. The ultrasound is preferably focused on the tumor to be treated. One may use any conventional means for focusing the ultrasound. Thus, e. g. , one may use one or more of the devices disclosed in United States patents 6.613, 0055 (systems and methods for steering a focused ultrasound array), 6,613, 004,6, 595,934 (skin rejuvenation using high intensity focused ultrasound), 6,543, 272 (calibrating a focused ultrasound array), 6,506, 154 (phased array focused ultrasound system), 6, 488, 639 (high intensity focused ultrasound treatment apparatus), 6,451, 013 (tonsil reduction using high intensity focused ultrasound to form an ablated tissue area), 6,432, 067 (medical procedures using high-intensity focused ultrasound), 6,425, 867 (noise-free real time ultrasonic imaging of a treatment site undergoing high intensity focused ultrasound therapy), and the like. The entire disclosure of each of these patent applications is hereby incorporated by reference into this specification.

In one embodiment, Taxol (or a similar composition) is delivered to the patient and, as is its wont, makes the microtubules more rigid. Thereafter, when the microtubules are polymerized in a dividing cell and substantially immobilized, the ultrasound is selectively delivered to the microtubules in the tumor, thereby breaking such microtubules and halting the process of cell growth and division, ultimately leading to cell death (apoptosis).

In one aspect of this embodiment, after the Taxol (or similar material) has been delivered to the patient, a high intensity magnetic field is applied to the tumor in order to selectively cause the Taxol to bind the microtubules in the tumor. Thereafter, the ultrasound is applied to break the microtubules so bound to the Taxol enhancing the efficacy of the drug due to a combined effect of the magnetic field, ultrasound and chemotherapeutic action of Taxol itself.

When microtubules have been broken, they tend to reform. Therefore, in one embodiment, and referring again to Figure 5, the ultrasound is periodically or continuously delivered to the synchronized to the typical time elapsed between subsequent cell division processes during which microtubules are polymerized (see, e. g. , steps 261/270/272 of Figure 5).

In one embodiment, a portable device is worn by the patient and applied to the tumor site; and this device periodically and/or continuously delivers ultrasound and/or magnetic energy to the patient. In one aspect of this embodiment, the device first delivers high intensity magnetic energy, and then it delivers the ultrasound energy.

Referring again to Figure 5, and to the preferred embodiment depicted therein, in step 265 one can determine the harmonic frequencies that correspond to the resonant frequency

determined in step 264. One may use a first harmonic of such resonant frequency, a second harmonic of such resonant frequency, and, in fact, any harmonic of the resonant frequency. As is known to those skilled in the art, a harmonic is one of a series of sounds, each of which has a frequency that is ain integral multiple of some fundamental frequency.

One may apply the resonant frequency to the stabilized microtubules and/or one of the harmonic frequencies, and/or a second of the harmonic frequencies and/or a third of the harmonic frequencies and/or a fourth of the harmonic frequencies and/or a fifth of the harmonic frequencies, etc. These frequencies may be applied simultanoueously, and/or they may be applied sequentially. One may alternate this application of frequency or frequencies with the administration of one or more stabilizing agents and/or synchronizing agents and/or antimototic agents and/or cytotacitc agents.

In this process, and in step 266 thereof, one may use any of the means for generating and focusing ultrasound energy that are known to those skilled in the art. Thus, e. g. , one may use the ultrasound generator disclosed in United States patent 6, 685, 639, the entire disclosure of which is hereby incorporated by reference into this specification. This patent claims:"A high intensity focused ultrasound system, comprising: a controllable power supply ; a B-mode ultrasound scanner; a therapeutic bed having a through hole; a liquid bag placed in the through hole and having opposite upper and lower portions, the lower portion of the liquid bag being attached to a combined probe, whereby a body portion of a patient lying immediately above the through hole may be scanned and treated by said system; and the combined probe comprising: a therapeutic head coupled to said controllable power supply for generating and focusing a ultrasound beam on a focal region at a temperature greater than 70 degrees centigrade, said therapeutic head comprising a ultrasound lens and piezoelectric ceramics coupled to said controllable power supply and disposed beneath the ultrasound lens, and an imaging probe coupled to said B-mode ultrasound scanner and mounted on a central axis of said therapeutic head so that the focal region of said therapeutic head is fixed at a predetermined location on a scanning plane; wherein said liquid bag contains vacuum degassed water having an acoustic impedance similar to that of human tissue, the upper portion of said liquid bag including an opening exposing said vacuum degassed water, said opening being open to an upper surface of said therapeutic bed so as said vacuum degassed water is adapted to be placed in direct contact with the skin of the patient's body portion; said system further comprising a multi-dimensional motional apparatus, on which the combined probe is mounted and which is moveable along three-dimensional rectangular coordinate axes and rotatable about one or two rotational coordinate axes, for driving said combined probe, said multidimensional motional apparatus

includes a plurality of one-dimensional motional devices each being configured to either translate or rotate said combined probe in a specific direction." By way of yet further illustration, and not limitation, one may use one or more of the ultrasound generators described in United States patents 3,735, 756 (duplex ultrasound generator); 4,718, 421 (ultrasound generator); 4,957, 100 (ultrasound generator and emitter); 4,976, 255 (extracorporeal lithotripsy using shock waves and therapeutic ultrasound) ; 5,102, 534; 5,184, 065 (therapeutic ultrasound generator); 5,443, 069 (therapeutic ultrasound applicator for the urogenital region); 6,270, 342; and the like. The entire disclosure of each of these United States patents is hereby incorporated by reference into this specification.

By way of further illustration, one may also use the ultrasound generator disclosed in an article by article by I. Hrazdira et al. ,"Ultrasonicallly inducted altrations of cultured tumour cells, "European Journal of Ultrasound 8 : 43-49,1998. At page 45 of this article, is it disclosed that:"A laboratory generator operating at a frequency of 0.8 MHz was used as the source of continuous ultrasound." Without wishing to be bound to any particular theory, applicants believe that the resonant freqency will will vary with the square root of the average length of the microtubules in the cells being treated. They also believe that the microtubules in diseased cells do not necessarily have the same length as the microtubules in non-diseased cells. It is believed, e. g., that cancer cells have microtubules that are up to about 10 percent longer than the microtubules of comparable non-cancer cells. Thus, by applying frequencies that are specific for the microtubules in the diseased cells, they preferentially treat the diseased cells with the process of this invention. Moreover, for the ultrasound application to be most effective in reaking up tumor cell microtubules, an apprpriate superposition of frequencies must be applied in correspondence to the lengths and rigidities of microtubules targette.

Referring again to Figure 5, and to step 264 thereof, a series of experiments may be preferably conducted with ultrasound waves with a power level of 10 milliwatts per square centimeter and different frequencies, at temperature of 37 degrees Celsius, and atmospheric pressure, and then the breakage of microtubules caused by such exposure is determined. That frequency which breaks the maximum number of microtubules is the resonant frequency. as will be apparent, the results of these experiments may be used to corroborate the estimates made by mathematical means of the resonant frequency of the stabilized microtubules.

Alternatively, they may be used independently to determine the resonant frequency of the microtubules.

One may determine the extent to which any particular ultrasound wave breaks microtubules by conventional means. Thus, e. g. , one may use the means described in the afrorementioned article by I. Hrazdira et al. ("Ultrasonically induced alterations of cultured tumor cells, "European Journal of Ultrasound 8 [1998], 43-49), in section 2.3 thereof. As is disclosed in such article, "For visualization of cytoskeleton components, an indirect immunofluorescence method was used. The cells in the monolayer were washed with phosphate buffer before adding 0. 1 % Triton for stabilization of membrane permeability. The cells were subsequently fixed by means of 3% paraformaldeyde. After fixation, secondary antibodies were added for 45 min... for microtubules.... Between each operation, the cells were washed by PBS. Finally, samples for fluorescene microscopy were prepared.... A total of 20 microphotographs of each controal and experimental sample were evaluated anonymously.... Changes in cytoskeletal structre were evaluated quantitatively...." Referring again to Figure 5, and in step 266 of the process, the stabilized microtubules are then contacted with ultrasound energy.

In one embodiment, the frequency of the ultrasound energy is approximately the resonant frequency, plus or minus about ten percent. In one aspect of this embodiment, the frequency of the ultrasound energy is approximately the resonant frequency, plus or minus about 5 percent. In general, such frequency will often be in the range of from about 100 kilohertz to about 500 kilohertz. and, more preferably, from about 110 to about 200 kilohertz.

In yet antoher embodiment, such frequency is from about 130 to about 170 kilohertz.

The power used for such exposure is preferably from about 1 to about 30 milliwatts per square centimeter and, more preferably, from about 5 to about 15 milliwatts per square centimeters.

At page 46 of the aforementioned Hrazdira et al. article, it was disclosed that"The disassembly of cytoskeleton components was not permanent. According to the time interval between sonication and cell fixation, a partial (at higher intensities) or total (at lower intensitivies) recovery of the cytoskeleton took place. "At page 49 of the Hradzdira et articles, it was disclosed that"We didnot find any changes in the cells that could be entirely attributed to ultrasound action only. From the point of view of cytoskeletal alterations, ultrasound has to be considered as a non-specific stress factor. " To help insure that applicants'process is more effective in causing permanent changes in the cell, an in step 268, the ultrasound excitation of the stabilized microtubules is ceased when the temperature of such microtubules reaches a specified temperature such as, e. g. , a temperature of 70 degrees Celsius.

United States patent 6, 685, 639, the entire disclosure of which is hereby incorporated by reference into this specification, describes and claims"a high intensity focused ultrasound system for scanning and treating tumor"which creates a very high temperature (in excess of 70 degrees Celsisus) in the area of the"focal region. "As is disclosed in column 3 of this patent, "By means of focusing, the sytem causes ultrasonic waves to form a space-point with high energy (focal region); the energy of the region reaches over 1000 W/M and the temperature instaneously rises to greater than 70 degrees centigrade...." Applicants wish to avoid prolonged exposure of the cells of living organisms to a temperature in excess of a specified temperature, such as, e. g. , 42 degrees Celisus. Thus, when the temperature of the microtubules reaches such specified temperature, and in step 268, the process of ultrasound excitation is repeated.

Thereafter, in step 270, step 266 (the contacting of the stabilized microtubules with ultrasound energy) is repeated until the temperature of the microtubules reaches the aforementioned maximum temperature, at which point step 268 is repeated (in step 272). The cycle is continued for as many times as is necessary to induce apoptosis.

In one embodiment, step 266 is conducted for from about 1 to about 5 minutes, the microtubules are allowed to cool, and then such step 266 is repeated again and again.