(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`I lllll llllllll II llllll lllll llll I II Ill lllll lllll 111111111111111111111111111111111
`
`(43) International Publication Date
`19 July 2001 (19.07.2001)
`
`PCT
`
`(10) International Publication Number
`WO 01/51049 Al
`
`(51) International Patent Classification7:
`31/445
`
`A61K 31/44,
`
`(US). SHAW, Leslie, M. [US/US]; 705 Sunnyside Avenue,
`Audubon, PA 19403 (US).
`
`(21) International Application Number: PCT/USOl/01537
`
`(22) International Filing Date: 12 January 2001 (12.01.2001)
`
`(74) Agents: COLBY, Gary, D. et al.; Akin, Gump, Strauss,
`Hauer & Feld, L.L.P., One Commerce Square, 22nd floor,
`2005 Market Street, Philadelphia, PA 19103-7986 (US).
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/176,086
`
`14 January 2000 (14.01.2000) US
`
`(71) Applicant (for all designated States except US): THE
`TRUSTEES OF THE UNIVERSITY OF PENN(cid:173)
`SYLVANIA [US/US]; Suite 300, 3700 Market Street,
`Philadephia, PA 19104-3147 (US).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): WASIK, Mariusz,
`A. [US/US]; 210 Cedarbrook Road, Ardmore, PA 19003
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ,
`DE, DK, DM, DZ, EE, ES, Fl, GB, GD, GE, GH, GM, HR,
`HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR,
`LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ,
`NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM,
`TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW.
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
`patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
`patent (AT, BE, CH, CY, DE, DK, ES, Fl, FR, GB, GR, IE,
`IT, LU, MC, NL, PT, SE, TR), OAPI patent (BF, BJ, CF,
`CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).
`
`[Continued on next page]
`
`;;;;;;;;;;;;;;; ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
`;;;;;;;;;;;;;;;
`;;;;;;;;;;;;;;;
`(54) Title: 0-METHYLATED RAPAMYCIN DERNATNES FOR ALLEVIATION AND INHIBITION OF LYMPHOPROLIF(cid:173)
`ERATIVE DISORDERS
`
`---
`!!!!!!!! ---
`-;;;;;;;;;;;;;;;
`--;;;;;;;;;;;;;;;
`;;;;;;;;;;;;;;; ----
`
`o--
`
`ln
`..........
`,....i
`
`°" ~ = ,....i
`= (57) Abstract: The present invention relates to methods of alleviating and inhibiting a lymphoproliferative disorder in a mammal, the
`
`0 method comprising administering one or more rapamycin derivatives (including rapamycin) to the mammal (see figure 8). Further,
`> the invention provides a method for identifying agents which are useful for alleviating and inhibiting lymphoproliferative disorders,
`~ as well as a method for identifying agents which are capable of inhibiting metastasis of lymphatic tumors in a mammal.
`
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`Page 001
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`

`WO 01/51049 Al
`
`I lllll llllllll II llllll lllll llll I II Ill lllll lllll 111111111111111111111111111111111
`
`Published:
`with international search report
`before the expiration of the time limit for amending the
`claims and to be republished in the event of receipt of
`amendments
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
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`

`WO 01/51049
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`PCT/USOl/01537
`
`TITLE
`
`0-Methylated Rapamycin Derivatives for
`
`5
`
`Alleviation and Inhibition of Lymphoproliferative Disorders
`
`BACKGROUND OF THE INVENTION
`
`Post-transplant lymphoproliferative disorders (PTLDs) which
`
`usually involve expansion ofB lymphocytes infected with the Epstein-Barr virus
`
`10
`
`(EBV), are a life-threatening complication of the immunosupressive therapy
`
`necessary to inhibit graft rejection (Morrison et al., 1994, Am. J. Med. 97:14-24;
`
`Warnke et al., 1995, AFIP Fascicle 14:531-535). PTLDs comprise a whole
`
`spectrum of lymphoproliferative disorders ranging from a polyclonal atypical
`
`lymphoid hyperplasia to a monoclonal, overtly malignant B-cell lymphoma
`
`15
`
`(Morrison et al., 1994, Am. J. Med. 97:14-24; Warnke et al., 1995, AFIP Fascicle
`
`14:531-535; Curtis et al., 1999, Blood 94:2208-2216; Harris et al., 1997, Semin.
`
`Diagn. Path. 14:8-14). Less advanced forms of PTLDs respond to a less aggressive
`
`course ofimmunosuppressive therapy (Morrison et al., 1994, Am. J. Med. 97:14-
`
`24; Sigal et al., 1992, Ann. Rev. Immunol. 10:519-60). However, lowering the dose
`
`20
`
`of standard immunosuppressive drugs, which nullify the body's ability to reject and
`
`destroy foreign tissue, can jeopardize the survival of a graft. Moreover, this
`
`modification in treatment with conventional agents is not effective against
`
`malignant, lymphoma-type PTLDs which are usually fatal for the graft recipient.
`
`Lymphoma causes significant morbidity and mortality, accounting
`
`25
`
`for more than 50,000 new diagnoses annually in the United States alone. Many
`
`lymphomas are either Hodgkin's or non-Hodgkin's lymphomas, which can be
`
`derived from peripheral, mature B, T, or NK lymphomas. Based on their natural
`
`course, non-Hodgkin's lymphomas are classified into low, intermediate, and high
`
`grades. Low grade lymphomas are usually slowly progressive, but are essentially
`
`30
`
`non-curable. The current 5-year disease-free, post-therapy survival rate for the
`
`intermediate and high grade lymphomas is approximately 60%. These aggressive
`
`types of lymphoma result in a rapid demise of the patients who do not respond to
`
`therapy. Prognosis of lymphomas occurring in patients who are
`- 1 -
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`irnmunocompromised such as AIDS and post-transplant patients, is particularly
`
`poor. Therefore, new treatment modalities are needed to improve cure rate of
`
`lymphoma.
`
`SDZ RAD (40-0-{2-hydroxyethyl}-rapamycin) is one of a class of
`
`5
`
`rapamycin derivatives which exhibit irnmunosuppressive activities (PCT
`
`application WO 94/09010; Schuurman et al., 1997, Transplantation 64:32-5;
`
`Schuler et al. 1997, Transplantation 64:36-42; Sedrani, et al., 1998, Transplant.
`
`Proc. 30:2192-2194; Schuurman et al., 1998, Transplant Proc. 30:2198-2199;
`
`Hausen et al., 1999, J. Heart Lung Transplant 18:150-159). Compounds of this
`
`10
`
`class, including rapamycin, have several points of action in normal T lymphocytes.
`
`They inhibit primarily down-stream signaling events mediated by the IL-2 receptor
`
`(Seghal, 1998, Clin. Biochem. 31 :335-340) and other cytokine receptors (Sakata et
`
`al., 1999, Immunology Letters 68:301-309), but also affect cell-cycle progression at
`
`the early G1 phase (Terada et al., 1993, J. Cell Physiol. 154:7-15; Flanagan et al.,
`
`15
`
`1993, Ann. N.Y. Acad. Sci. 696:31-37). The multi-faceted immunosuppressive
`
`activities exhibited by SDZ RAD and other 0-alkylated rapamycin derivatives
`
`compounds make these compounds versatile irnmunosuppressive agents.
`
`There is a significant need for more effective therapeutic and
`
`prophylactic methods for limiting the severity and frequency of lymphoproliferative
`
`20
`
`disorders such as lymphomas and PTLDs. The present invention satisfies this need.
`
`BRIEF SUMMARY OF THE INVENTION
`
`The invention includes a method of alleviating a lymphoproliferative
`
`disorder in a human patient. The method comprises administering to the patient, in
`
`25
`
`an amount sufficient to alleviating the disorder, a rapamycin derivative having the
`
`chemical structure shown in Formula I in Figure 8. In a preferred embodiment, the
`
`rapamycin derivative is 40-0-(2-hydroxy)ethyl-rapamycin. Numerous other useful
`
`rapamycin derivatives (including rapamycin itself) are described in this disclosure.
`
`Lymphoproliferative disorders that can be alleviated using this method include, for
`
`30
`
`example, PTLDs and lymphatic cancers such as lymphomas. The method can also
`
`be used to alleviate lymphoproliferative disorders caused or associated with
`
`- 2 -
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`treatment of the patient by immunosuppressive therapy (e.g., immunosuppressive
`
`therapy associated with tissue transplantation).
`
`In another method included in the invention, a lymphoproliferative
`
`disorder is inhibited or prevented in a patient at risk for developing such a disorder
`
`5
`
`(e.g., an immunocompromised patient or a patient undergoing immunosuppressive
`
`therapy).
`
`In these methods, the rapamycin derivative can be co-administered
`
`(in a single composition or in discretely-administered compositions) with a second
`
`pharmacologically active agent, such as an immunosuppressive agent.
`
`10
`
`Immunosuppressive agents are known for use in methods of inhibiting graft
`
`rejection, and those known methods can be improved by administering both the
`
`immunosuppressive agent and a rapamycin derivative disclosed herein to a patient
`
`who has received a graft.
`
`The invention also includes a method of inhibiting metastasis of a
`
`15
`
`lymphatic tumor in a human patient afflicted with a lymphatic cancer. This method
`
`comprising administering to the patient, in an amount sufficient to inhibit
`
`lymphocyte proliferation, a rapamycin derivative having the chemical structure
`
`shown in Formula I.
`
`In another aspect, the invention includes a method of assessing
`
`20 whether an agent is useful for alleviating or inhibiting a lymphoproliferative
`
`disorder in a human patient. This method comprising transforming a B lymphocyte
`
`with an Epstein-Barr virus, injecting the lymphocyte into a mouse having a severe
`
`combined immunodeficiency, administering the agent to the mouse, and monitoring
`
`tumor growth in the mouse for at least about 21 days. If one or more of tumor
`
`25
`
`regression, tumor eradication, and absence of a second tumor is observed in the
`
`mouse, then this is an indication that the agent is useful for alleviating or inhibiting
`
`a post-transplant lymphoproliferative disorder in a mammal.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`30
`
`Figure 1 is a graph which illustrates SDZ RAD-mediated inhibition
`
`of in vitro proliferation of PTLD-like EBV+ B cells. BC-1 is an EBV+/HSV8+ B-
`
`- 3 -
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`cell line derived from primary effusion lymphoma (PEL). The other cell lines are in
`
`vitro EBY-transformed B-cell lines derived from one of a patient with low-grade B(cid:173)
`
`cell lymphoma (15A or 20A), a patient with T-cell lymphoma (LCL) or a healthy
`
`individual (Al or A2D6). The HTLY-I+ malignant T-cell lines, HUT-102, ClOMJ,
`
`5
`
`and ATL"'.'2, were included as control cell lines.
`
`Figure 2 is a graph which illustrates SDZ RAD-mediated inhibition
`
`of cell cycle progression in PTLD-like B cells. Four EBY+ B-cell lines were
`
`cultured for 48 hours in the presence of 0-10 nanomolar SDZ RAD, labeled with
`
`propidium iodine, and analyzed by flow cytometry.
`
`10
`
`Figure 3 is a graph which illustrates SDZ RAD-mediated increase in
`
`apoptotic rate of PTLD-like B cells. Three EBY+ B-cell lines were cultured for 24
`
`hours in the presence of 0-10 nanomolar SDZ RAD, labeled with propidium iodine
`
`and anti-Annexin Y antibody, and analyzed by flow cytometry.
`
`Figure 4, comprising Figures 4A-4D, is a group of images of
`
`15
`
`photomicrographs which depict the morphology and phenotype of 20A tumors
`
`xenotransplanted into SCID mouse. In Figure 4A, hematoxylin-eosin stain shows
`
`large cell lymphoma with high mitotic rate. In Figure 4B, immunoperoxidase stain
`
`using an antibody which binds specifically with human CD20 (i.e., a B-cell antigen)
`
`shows cell-membrane staining in all lymphoma cells. In Figure 4C,
`
`20
`
`immunoperoxidase stain using an antibody which binds specifically with Ki67 (i.e.,
`
`a cell proliferation-related antigen) shows nuclear staining in 50-80% of cells. In
`
`Figure 4D, in-situ hybridization for EBY-encoded EBER-1 RNA shows nuclear
`
`positivity in all lymphoma cells.
`
`Figure 5, comprising Figures 5A-5C, is a trio of graphs which
`
`25
`
`illustrates SDZ RAD-mediated inhibition of in vivo growth of PTLD-like B-cells;
`
`treatment of established tumors. Fragments of tumors derived from EBY+ B-cell
`
`lines 15A (Fig. 5C), 20A (Fig. 5B), and A2D6 (Fig. 5A) were implanted into
`
`recipient SCID mice. The number of mice per group is indicated in parentheses.
`
`Treatment with 5 milligrams per kilogram body weight per day of the drug was
`
`30
`
`started when the tumors reached 0.4- 0.5 cm in diameter.
`
`-4-
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`Figure 6, comprising Figures 6A-6C, is a trio of graphs which
`
`illustrates SDZ RAD-mediated inhibition of in vivo growth of a malignant PTLD(cid:173)
`
`like B-cells (i.e., inhibition of tumor growth). Fragments of tumors derived from
`
`EBV+ B-cell lines 15A (Fig. 6C), 20A (Fig. 6B), and A2D6 (Fig. 6A) were
`
`5
`
`implanted into recipient SCID mice. The number of mice per group is indicated in
`
`parentheses. The treatment with 5 milligrams per kilogram body weight per day of
`
`the drug was started 3 days prior to the tumor implantation.
`
`Figure 7 depicts the chemical structure of SDZ RAD.
`
`Figure 8 depicts the chemical formula for 0-methylated rapamycin
`
`10
`
`derivatives which can be used in the methods described herein (i.e., Formula I).
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`The present invention relates to methods of alleviating or inhibiting
`
`lymphoproliferative disorders such as lymphomas and post-transplant
`
`15
`
`lymphoproliferative disorders (PTLDs) in a mammal such as a human. The method
`
`comprises administering to the mammal an 0-alkylated rapamycin derivative such
`
`as SDZ RAD in amount sufficient to inhibit proliferation of lymphocytes in the
`
`mammal.
`
`0-alkylated rapamycin derivatives are known to inhibit graft
`
`20
`
`rejection. Thus, when the rapamycin derivative is administered to a mammal which
`
`has undergone a tissue or organ graft (i.e., a xenograft), administration of one of
`
`these derivatives can both inhibit graft rejection and alleviate or prevent a PTLD.
`
`For treatment or inhibition of a PTLD, the amount administered can be sufficient to
`
`inhibit both graft rejection and proliferation of lymphocytes.
`
`25
`
`Not previously recognized by others is the fact that 0-alkylated
`
`rapamycin derivatives such as those described herein can be used to alleviate or
`
`prevent lymphoma. 0-alkylated rapamycin derivative such as SDZ RAD can also
`
`be used to inhibit proliferation of lymphocytes, as occurs in a variety of
`
`lymphoproliferative disorders other than lymphoma (e.g., chronic lymphocytic
`
`30
`
`leukemia). 0-alkylated rapamycin derivatives such as SDZ RAD can be used to
`
`- 5 -
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`induce in a cell, either in vitro or in vivo, cell cycle arrest or apoptosis, and to
`
`inhibit lymphocyte growth and proliferation.
`
`The 0-alkylated rapamycin derivatives that are useful in the methods
`
`described herein have been described by others, as has methods for making them.
`
`5
`
`In this regard, the PCT patent application having international publication number
`
`WO 94/09010 is incorporated herein by reference.
`
`0-alkylated rapamycin derivatives that are useful in the methods
`
`described in this disclosure include those having the chemical structure shown in
`
`Formula I in Figure 8, wherein
`
`10
`
`X is (H, H) or O;
`
`Y is (H, OH) or O;
`1
`2
`and R
`is independently selected from the group consisting of -H,
`each ofR
`
`alkyl, thioalkyl, arylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylarylalkyl,
`
`dihydroxyalkylarylalkyl, alkoxyalkyl, acyloxyalkyl, aminoalkyl, alkylaminoalkyl,
`
`alkoxycarbonylaminoalkyl, acylaminoalkyl, arylsulfonamidoalkyl, ally!,
`3
`dihydroxyalkylallyl, dioxolanylallyl, carbalkoxyalkyl, and (R
`)} Si;
`3
`is independently selected from the group consisting of -H, methyl,
`
`each R
`
`15
`
`20
`
`25
`
`30
`
`either R
`
`ethyl, isopropyl, tert-butyl, and phenyl; and
`1
`4
`4
`together form a C2-s alkylene moiety.
`is methyl or R
`andR
`1
`4
`, "alk-" or "alkyl" refers to a c 1_6 alkyl
`In the descriptions of R
`-R
`moiety, the moiety being branched or linear, and preferably being a c 1_3 alkyl
`moiety, in which the carbon chain can, optionally, be interrupted by an ether (-0-)
`1
`4
`linkage (e.g., -CH2-CH2-0-CH2-). In the descriptions ofR
`-R
`, "ar-" or "aryl"
`refers to a c5_g aryl moiety, the aryl moiety optionally having one or two nitrogen
`1
`4
`-R
`, "allyl" means -CH2-
`atoms in place of a carbon atom. In the descriptions of R
`1
`4
`-R
`
`CH=CH2. In the descriptions ofR
`
`, "acyl" refers to a C1-6alkanoyl moiety
`
`(i.e., an alkyl moiety having a carbonyl {i.e., -CO-} moiety in place of a methylene
`4
`1
`,
`moiety {i.e., -CH2-} moiety of the alkyl moiety). In the descriptions of R
`-R
`"alkylene" refers to a c 1_6 alkylene moiety, the moiety being branched or linear.
`Examples of 0-alkylated rapamycin derivatives that are suitable for
`
`use in the methods described in this disclosure include
`
`- 6 -
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`• 40-0-benzyl-rapamycin,
`
`• 40-0-( 4'-hydroxymethyl)benzyl-rapamycin,
`
`• 40-0-[4'-(l,2 dihydroxyethyl)]benzyl-rapamycin,
`
`• 40-0-Allyl-rapamycin,
`
`5
`
`• 40-0-[3'-(2,2-dimethyl-1,3-dioxolan-4(S)-yl)-prop-2'-en-1 '-yl]-rapamycin,
`
`•
`
`(2'E, 4'S)-40-0-( 4',5'-dihydroxypent-2'-en-1 '-yl)-rapamycin,
`
`• 40-0-(2-hydroxy)ethoxycarbonylmethyl-rapamycin,
`
`• 40-0-(2-hydroxy)ethyl-rapamycin,
`
`• 40-0-(3-hydroxy)propyl-rapamycin,
`
`10
`
`• 40-0-(6-hydroxy)hexyl-rapamycin,
`
`• 40-0-[2-(2hydroxy)ethoxy ]ethyl-rapamycin,
`
`• 40-0-[(3S)-2,2-dimethyldioxolan-3-yl]methyl-rapamycin,
`
`• 40-0-[(2S)-2,3-dihydroxyprop-1-yl]-rapamycin,
`
`• 40-0-(2-acetoxy)ethyl-rapamycin,
`
`15
`
`• 40-0-(2-nicotinoyloxy)ethyl-rapamycin,
`
`• 40-0-[2-(N-morpholino )acetoxy ]ethyl-rapamycin,
`
`• 40-0-(2-N-imidazolylacetoxy)ethyl-rapamycin,
`
`• 40-0-[2-(N-methyl-N'-piperazinyl)acetoxy]ethyl-rapamycin,
`
`• 39-0-desmethyl-39,40-0,0-ethylene-rapamycin,
`
`20
`
`•
`
`(26R)-26-dihydro-40-0-(2-hydroxy )ethy 1-rapamycin,
`
`• 28-0-methyl-rapamycin,
`
`• 40-0-(2-aminoethyl)-rapamycin,
`
`• 40-0-(2-acetaminoethyl)-rapamycin,
`
`• 40-0-(2-nicotinamidoethyl)-rapamycin,
`
`25
`
`• 40-0-(2-(N-methyl-imidazo-2'-ylcarbethoxamido )ethyl)-rapamycin,
`
`• 40-0-(2-ethoxycarbonylaminoethyl)-rapamycin,
`
`• 40-0-(2-tolylsulfonamidoethyl)-rapamycin, and
`
`• 40-0-[2-( 4'5'-dicarboethoxy-1 ',2',3'-triazol-l'-yl)-ethyl]-rapamycin.
`
`0-substituted rapamycins where X and Y are both 0, R
`
`Preferred compounds for use in the methods described herein are 40-
`2
`4
`1
`is H, R
`is methyl, and R
`
`30
`
`is selected from hydroxyalkyl, hydroxyalkoxyalkyl, acylaminoalkyl, and
`
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`aminoalkyl. Examples of preferred compounds include 40-0-(2-hydroxy)ethyl(cid:173)
`
`rapamycin (i.e., SDZ RAD, the structure of which is shown in Fig. 7), 40-0-(3-
`
`hydroxy)propyl-rapamycin, 40-0-[2-(2-hydroxy)ethoxy ]ethyl-rapamycin, and 40-
`
`0-(2-acetaminoethyl)-rapamycin. Rapamycin itself (i.e., the compound having the
`2
`4
`1
`structure of Formula I, wherein R
`and R
`are each -H, R
`
`is methyl, and each ofX
`
`5
`
`and Y are 0) can also be used in the methods described herein.
`
`Definitions
`
`As used herein, each of the following terms has the meaning
`
`10
`
`associated with it in this section.
`
`The articles "a" and "an" are used herein to refer to one or to more
`
`than one (i.e., to at least one) of the grammatical object of the article. By way of
`
`example, "an element" means one element or more than one element.
`
`A process in a cell, such as cell growth, cell cycle progression,
`
`15
`
`proliferation, or tumorigenesis, is "inhibited" by a composition or method of
`
`treatment if, upon administering the composition to the cell or employing the
`
`method of treatment on the cell, the process is altered relative to the same process in
`
`a cell to which the composition was not administered or on which the method of
`
`treatment was not employed. For example, ifthe level of proliferation in a cell is
`
`20
`
`decreased following administration of a composition comprising SDZ RAD, as
`
`compared with the level of proliferation in a cell to which the composition is not
`
`administered, then the composition inhibits proliferation in the cell.
`
`A process in a mammal, such as tumor growth, establishment of a
`
`tumor, a lymphoproliferative response, or graft rejection, is "inhibited" by a
`
`25
`
`composition or method of treatment if, upon administering the composition to the
`
`mammal or employing the method of treatment on the mammal, the process is
`
`decreased in rate or magnitude relative to the same process in a mammal to which
`
`the composition was not administered or on which the method of treatment was not
`
`employed. For example, ifthe level of proliferation of lymphocytes in a mammal is
`
`30
`
`decreased following administration of a composition comprising SDZ RAD, as
`
`compared with the level of proliferation of lymphocytes in a mammal to which the
`
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`composition is not administered, then the composition inhibits a proliferation of
`
`lymphocytes in the mammal.
`
`A process in a cell, such as apoptosis or cell cycle arrest, is
`
`"induced" by a composition or method of treatment if, upon administering the
`
`5
`
`composition to the cell or employing the method of treatment on the cell, the level
`
`of the process in the cell is increased relative to the process in a cell to which the
`
`composition was not administered or on which the method of treatment was not
`
`employed. For example, if the degree, level, or likelihood of apoptosis in a cell is
`
`increased following administration of a composition comprising SDZ RAD, as
`
`10
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`compared with the degree, level, or likelihood of apoptosis in a cell to which the
`
`composition is not administered, then the composition induces apoptosis in the cell.
`
`As used herein, the term "pharmaceutically acceptable carrier"
`
`means a chemical composition with which an 0-alkylated rapamycin derivative can
`
`be combined and which, following the combination, can be used to administer the
`
`15
`
`rapamycin derivative to a subject.
`
`As used herein, the term "physiologically acceptable" ester or salt
`
`means an ester or salt form of an 0-alkylated rapamycin derivative which is
`
`compatible with any other ingredients of the pharmaceutical composition and which
`
`is not deleterious to the subject to which the composition is to be administered.
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`20
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`As used herein, "parenteral administration" of a pharmaceutical
`
`composition includes any route of administration characterized by physical
`
`breaching of a tissue of a subject and administration of the pharmaceutical
`
`composition through the breach in the tissue. Parenteral administration thus
`
`includes, but is not limited to, administration of a pharmaceutical composition by
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`25
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`injection of the composition, by application of the composition through a surgical
`
`incision, by application of the composition through a tissue-penetrating non(cid:173)
`
`surgical wound, and the like. In particular, parenteral administration can include,
`
`but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrastemal
`
`injection, and kidney dialytic infusion techniques.
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`30
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`A first pharmacological agent and a second pharmacological agent
`
`are "co-administered" if the two agents are administered sufficiently closely in time
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`that the period during which the pharmacological effect attributable to the first
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`agent is at least half its maximal value overlaps with the period during which the
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`pharmacological effect attributable to the second agent is at least half its maximal
`
`value. Co-administered agents can be administered in a single composition
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`5
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`containing the agents, or they can be administered in discrete compositions.
`
`As used herein, the term "induction of apoptosis" means a process
`
`which causes a cell to begin or accelerate the process of programmed cell death.
`
`A disorder is "alleviated" ifthe severity of the disorder or one of its
`
`symptoms or the frequency with which the disorder or one of its symptoms is
`
`10
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`experienced by a patient is reduced.
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`A disorder is "inhibited" by a treatment if the onset of the disorder or
`
`one of its symptoms is delayed or prevented, relative to its 'onset in the absence of
`
`the treatment.
`
`15 Description
`
`The present invention relates to a method of using an 0-alkylated
`
`rapamycin derivative to alleviate or inhibit a lymphoproliferative disorder in a
`
`mammal. Lymphoproliferative disorders which can be alleviated or inhibited using
`
`the method described herein include post-transplant lymphoproliferative disorders
`
`20
`
`(PTLDs), lymphomas, and other disorders. This method comprises administering
`
`to the mammal a sufficient amount of an 0-alkylated rapamycin derivative to
`
`inhibit the disorder. In patients who are afflicted with both a lymphoproliferative
`
`disorder and graft rejection, an amount of the derivative sufficient to alleviate or
`
`inhibit both the disorder and the graft rejection can be administered. For example,
`
`25
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`both rejection of a graft (i.e., a transplanted tissue or organ) and a PTLD can be
`
`alleviated simultaneously.
`
`PTLDs include a wide spectrum of lymphoproliferative disorders,
`
`ranging from a polyclonal atypical lymphoid hyperplasia to a monoclonal, overtly
`
`malignant B cell lymphoma. Examples of such disorders have been described in
`
`30
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`the art (e.g., Morrison et al., 1994, Am. J. Med. 97:14-24; Warnke et al., 1995,
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`AFIP Fascicle 14:531-535; Curtis et al., 1999, Blood 94:2208-2216; Harris et al.,
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`1997, Semin. Diagn. Path. 14:8-14) and can be identified by the skilled artisan.
`
`One class of lymphoproliferative disorders for which the methods
`
`described herein are useful are those disorders which are characterized by infection
`
`5
`
`of lymphocytes by Epstein-Barr virus (EBV). Proliferation ofEBV-infected
`
`lymphocytes is a frequent complication of immunosuppressive therapy, and thus
`
`sometimes occurs during immunosupression associated with prevention of graft
`
`rejection, with cancer-related chemotherapy, or with other immune-suppressing
`
`medical interventions. Furthermore, proliferation of EBY-infected lymphocytes can
`
`10
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`be accelerated in immunocompromised patients, such as patients afflicted with
`
`AIDS or other immune disorders. The 0-alkylated rapamycin derivatives described
`
`herein can be used before, after, or during treatment of one of these other immune
`
`system-affecting disorders or treatments. For example, a patient who is to undergo
`
`immunosuppressive therapy can first (i.e., minutes, hours, days, or weeks
`
`15
`
`beforehand) be administered one or more 0-alkylated rapamycin derivatives
`
`(including rapamycin) in order to inhibit or prevent proliferation of lymphocytes
`
`during the immunosuppressive therapy. One or more 0-alkylated rapamycin
`
`derivatives (including rapamycin) can be co-administered (i.e., in a single
`
`composition or in discrete compositions administered closely in time or alternately)
`
`20
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`during the course of immunosuppressive therapy in order to decrease or prevent
`
`proliferation oflymphocytes during the therapy. The derivative(s) need not be the
`
`same one(s), if any, administered prior to the therapy. Similarly, one or more 0-
`
`alkylated rapamycin derivatives (including rapamycin) can also (or instead) be
`
`administered to a patient who has undergone immunosuppressive therapy, in order
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`25
`
`to alleviate, inhibit, or prevent post-therapy lymphocyte proliferation and symptoms
`
`associated therewith.
`
`A particular method included within the scope of the invention
`
`comprises administering SDZ RAD to a human prior to transplantation of a tissue
`
`(i.e., an allograft or a xenograft) into or onto the body of the human. Such
`
`30
`
`administration can occur before, during, or after transplantation, or at any
`
`combination of these. By way of example, SDZ RAD can be administered to a
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`human patient prior to, during, and after receiving an allogeneic kidney transplant.
`
`Other examples of transplants include allogeneic heart, kidney, and liver
`
`transplants, heart valves, vascular grafts, skin grafts, dura mater grafts, pericardium
`
`grafts, cartilage grafts and implants, and xenogeneic transplants.
`
`5
`
`Another type of lymphoproliferative disorder that can be, but often is
`
`not, associated with tissue transplantation is lymphatic cancers, including
`
`lymphomas and lymphocytic leukemias. The lymphocytic leukemias include
`
`disorders such as acute and chronic lymphocytic leukemias. Lymphomas
`
`encompass a wide variety of cancers characterized by lymphocyte proliferation.
`
`10
`
`Examples of lymphomas include AIDS-related lymphomas, Hodgkin's lymphoma
`
`(sometimes designated Hodgkin's disease), non-Hodgkin's lymphoma, Burkitt's
`
`lymphoma, diffuse large cell lymphoma, T-cell lymphoma, and cutaneous T-cell
`
`lymphoma.
`
`Regardless of whether a patient is diagnosed with a disease or
`
`15
`
`exhibits symptoms of a disease, it can be desirable to inhibit lymphocyte
`
`proliferation to a patient who harbors lymphocytes infected with EBV, particularly
`
`ifthe patient is expected to undergo immunosuppressive therapy or if the patient is
`
`at particular risk for developing an immune-compromising disorder. Such patients
`
`can be identified by isolating lymphocytes from the patient and assessing the
`
`20
`
`presence or absence of all or part of the EBV genome in those lymphocytes. If
`
`EBV is detected, administration of rapamycin or an 0-alkylated rapamycin
`
`derivative can be indicated.
`
`The methods described herein for inhibiting or alleviating a
`
`lymphoproliferative disorder comprise administering rapamycin or an 0-alkylated
`
`25
`
`rapamycin derivative to a mammal. The precise form in which the compound is
`
`administered is not critical, numerous pharmaceutical compositions, dosage forms,
`
`and pharmaceutically acceptable carriers and excipients being known in the art. A
`
`formulation administered to a mammal can contain the rapamycin derivative as the
`
`sole active agent, or it can be admixed with one or more other active agents (e.g., an
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`30
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`immunosuppressive agent such as azathioprine, a mycophenolic acid such as
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`mycophenolate mofetil, Rh0(D) immune globulin, cyclosporin, tacrolimus, cisplatin,
`
`a cyclophosphamide, and leflunomide ).
`
`The invention encompasses the preparation and use of medicaments
`
`and pharmaceutical compositions comprising one or more 0-alkylated rapamycin
`
`5
`
`derivatives (including rapamycin) as an active ingredient. Such a pharmaceutical
`
`composition may consist of the active ingredient alone, in a form suitable for
`
`administration to a subject, or the pharmaceutical composition may comprise the
`
`active ingredient and one or more pharmaceutically acceptable carriers, one or more
`
`additional ingredients, or some combination of these. Administration of one of
`
`10
`
`these pharmaceutical compositions to a subject is useful for inhibiting proliferation
`
`of lymphocytes in the subject, as described elsewhere in the present disclosure. The
`
`active ingredient may be present in the pharmaceutical composition in the form of a
`
`physiologically acceptable ester or salt, such as in combination with a
`
`physiologically acceptable cation or anion, as is well known in the art.
`
`15
`
`The formulations of the pharmaceutical compositions described
`
`herein may be prepared by any method known or hereafter developed in the art of
`
`pharmacology. In general, such preparatory methods include the step of bringing
`
`the active ingredient into association with a carrier or one or more other accessory
`
`ingredients, and then, if necessary or desirable, shaping or packaging the product
`
`20
`
`into a desired single- or multi-dose unit.
`
`Although the descriptions of pharmaceutical compositions provided
`
`herein are principally directed to pharmaceut