•
`
`•
`
`- 20 -
`
`Case 100-7932
`
`The combined organic layers are washed with lN aqueous sodium
`bicarbonate and saturated brine, dried over anhydrous sodium sulfate,
`filtered and concentrated. The resulting green oil is purified by
`column chromatography on silica gel {60:40-hexane-AcOEt) to afford
`40-0-(2-t-butyldimethylsilyloxy)ethyl-rapamycin as a light brown,
`amorphous solid.
`
`To a stirred, cooled {0°C) solution of 786 mg (0.73 mmol) of
`40-0-(2-t-butyldimethylsilyloxy)ethyl-rapamycin in 20 ml of
`acetonitrile is added 2 ml of HF-pyridine complex. This mixture is
`stirred at 0°C for 1 hour and quenched with lN aqueous sodium
`bicarbonate. The aqueous solution is extracted three times with
`AcOEt. The resulting organic phase is washed with aqueous lN sodium
`bicarbonate, cold lN HCl and saturated brine, dried over sodium
`sulfate, filtered and concentrated. The brown residue is purified by
`column chromatography on silica gel {10:90 hexane-AcOEt) to afford
`40-0-hydroxyethyl-rapamycin as a colorless, amorphous solid, having
`the following characteristic spectroscopic data:
`
`1 H NHR {CDC13) ~ 3.07 {lH,m,H-39), 3.12 {3H,s,C16-0CH3), 3.16
`{lH,m,H-16), 3.32 {4H,s,C27-0CH3 and H-31), 3.43 {4H,s,C39-0CH3 and
`H-6 ax), 3.56 {2H,m,1H of OCH£CH20 and H-6 eq), 3.66 {3H,m,2H of
`OCH 2 CH£0 and H-40), 3.73 {2H,m,1H of OCH£CH20 and H-27), 3.84
`{lH,m,H-14); HS {FAB) m/z 980 {[H+Na]+), 926 {[H-OCH3]+), 908
`{[H-{H20+0CH3}+]).
`
`Hacrophilin binding - rel. IC 50 = 0.9
`IL-6 mediated proliferation - rel. IC 50
`
`0.5
`
`
`
`
`
`•
`
`- 20 -
`
`Case 100-7932
`
`The combined organic layers are washed with lN aqueous sodium
`bicarbonate and saturated brine, dried over anhydrous sodium sulfate,
`filtered and concentrated. The resulting green oil is purified by
`column chromatography on silica gel {60:40-hexane-AcOEt) to afford
`40-0-(2-t-butyldimethylsilyloxy)ethyl-rapamycin as a light brown,
`amorphous solid.
`
`To a stirred, cooled {0°C) solution of 786 mg (0.73 mmol) of
`40-0-(2-t-butyldimethylsilyloxy)ethyl-rapamycin in 20 ml of
`acetonitrile is added 2 ml of HF-pyridine complex. This mixture is
`stirred at 0°C for 1 hour and quenched with lN aqueous sodium
`bicarbonate. The aqueous solution is extracted three times with
`AcOEt. The resulting organic phase is washed with aqueous lN sodium
`bicarbonate, cold lN HCl and saturated brine, dried over sodium
`sulfate, filtered and concentrated. The brown residue is purified by
`column chromatography on silica gel {10:90 hexane-AcOEt) to afford
`40-0-hydroxyethyl-rapamycin as a colorless, amorphous solid, having
`the following characteristic spectroscopic data:
`
`1 H NHR {CDC13) ~ 3.07 {lH,m,H-39), 3.12 {3H,s,C16-0CH3), 3.16
`{lH,m,H-16), 3.32 {4H,s,C27-0CH3 and H-31), 3.43 {4H,s,C39-0CH3 and
`H-6 ax), 3.56 {2H,m,1H of OCH£CH20 and H-6 eq), 3.66 {3H,m,2H of
`OCH 2 CH£0 and H-40), 3.73 {2H,m,1H of OCH£CH20 and H-27), 3.84
`{lH,m,H-14); HS {FAB) m/z 980 {[H+Na]+), 926 {[H-OCH3]+), 908
`{[H-{H20+0CH3}+]).
`
`Hacrophilin binding - rel. IC 50 = 0.9
`IL-6 mediated proliferation - rel. IC 50
`
`0.5
`
`
`
`
`
`•
`
`•
`
`..,. 21 -
`
`Case 100-7932
`
`CLAIMS
`
`1. A compound of Formula I
`
`41
`
`(I)
`
`y
`
`24
`
`12
`
`wherein
`
`X is (H,H) or O;
`
`18
`
`20
`
`'//
`19
`
`Y is (H,H), (H,OH), or O; and
`
`Rl and R2 are independently selected from .
`H, alkyl, thioalkyl, arylalkyl, hydroxyalkyl,
`dihydroxyalkyl, alkoxyalkyl, acyloxyalkyl,
`carbalkoxyalkyl, amino, alkylamino, aminoalkyl,(cid:173)
`alkylaminoalkyl, allyl and R3
`3 Si where each R3 is
`independently selected from H, methyl, ethyl, isopropyl,
`_!-butyl, and phenyl; wherein "alk-" or "alkyl" refers to
`C1 _ 6 branched or linear alkyl in which the carbon chain may
`be optionally interrupted by an ether (-0-) linkage; .
`
`
`
`
`
`•
`
`•
`
`- 22 -
`
`Case 100-7932
`
`then either Y is other than
`provided that where X is O,
`or R2 is other than H; and
`
`provided that where R1 or R1 and R2 are R3
`both o.
`
`3 Si, X and Y are not
`
`2.
`
`A compound according to claim 1 wherein
`
`a) X is (H,H), Y is (H,H), (H,OH) or O, and R1 and R2 are
`independently selected from H, alkyl, allyl, arylalkyl,
`hydroxyalkyl and carbalkoxyalkyl; or
`
`and R1 and R2 are
`b) X is O or (H,H), Y is. (H,OH),
`independently selected from H, alkyl, allyl, arylalkyl,
`hydroxyalkyl and carbalkoxyalkyl; or
`
`c) X is O or (H,H), Y is (H,H), (H,OH) or O, and R1 and R2 are
`independently selected from alkyl, allyl, arylalkyl,
`hydroxyalkyl and carbalkoxyalkyl.
`
`3. A compound ac~ording to claim 2 selected from:
`
`9-Deoxorapamycin,
`26-Dihydro-rapamycin,
`9-Deoxo-26-dihydro-rapamycin,
`40-0-Carbethoxymethyl-rapamycin,
`40-0-Carbethoxymethyl-9-deoxorapamycin,
`40-0-Benzyl-rapamycin,
`40-0-Allyl-rapamycin, and
`40-0-(2-Hydroxyethyl)-rapamycin.
`
`4. A compound according to any one ~f claims 1-3 for use as a
`pharmaceutical.
`
`
`
`
`
`•
`
`- 23 -
`
`•
`
`Case 100-7932
`
`5. A pharmaceutical composition comprising a compound according to
`any one of claims 1-3 together -wi th-,a-'pharmaceutically acceptable
`diluent or carrier.
`
`6. Use of a compound according to claims 1-3 in the manufacture of a
`medicament for treating or preventing any .. of the following
`conditions:
`
`-- - - .
`--
`autoimmune disease,
`(i)
`(ii) allograf t rejection,
`(iii) graft vs. host disease,
`(iv) asthma,
`(v) multidrug resistance, -
`tumors or hyperproliferative disorders, or
`(vi)
`(vii) fungal infections.
`
`7. Novel products, processes, and utilities substantially as
`described herein.
`
`
`
`
`
`•
`
`Figure . to accompany abstract
`
`- 24 -
`
`Case 100-7932
`
`ABSTRAcr
`
`Novel derivatives of rapamycin, particularly 9-deoxorapamycins,
`26-dihydro-rapamycins, and 40-0-substituted and 28,40-0,0-di(cid:173)
`substituted rapamycins, are found to have pharmaceutical utility,
`particularly as an immunosuppressants.
`
`6300/TH/RT 5818
`06.0ct.1992 Tue 10:10
`
`
`
`
`
`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`Case No. 100-7932/PCT
`Patent
`
`In re Application of
`COTTENS, et al.
`
`Serial No. ~8/416,673
`
`Art Unit 1202
`
`Filed: April 7, 1995
`
`Examiner: R. Bond
`
`For: 0-ALKYLATED RAPAMYCIN
`DERIVATIVES ... AS
`IMMUNOSUPPRESSANTS
`
`CLAIM OF PRIORITY
`
`Honorable Commissioner of Patents
`and Trademarks
`Washington, D.C.
`
`20231
`
`Dear Sir:
`
`In accordance with 35 USC 119 and the International Convention,
`the priority and benefit of the filing date of each of the
`following foreign patent applications mentioned in the
`declaration of this application is hereby claimed:
`
`GREAT BRITAIN APPLICATION NO. 9221220.8, filed October 9, 1992
`
`[x]A certified copy of each of the above-mentioned foreign patent
`applications is appended.
`
`[ ]A certified copy of each of the above-mentioned foreign patent
`applications appears in the file of
`application Serial No.
`, filed
`, now
`, having been filed therein
`
`on
`
`Respectfully submitted,
`
`~a?OJ?J~
`Thomas 0. McGovern
`Registration No. 25,741
`Agent/Attorney for Applicants
`(201) 503-8480
`
`SANDOZ CORPORATION
`59 Route 10
`East Hanover, NJ 07936
`
`Date: October 10,1996
`TOM:mjl
`
`
`
`
`
`• Ur... The
`
`rdtent
`Otf1Ce
`
`.-------------------------------...-~·-··="'
`~
`PRIORITY DOCUiViCN·r· l
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`{) ¥ { c/l&Lal.5
`PCT!ZP 9 3 / 0 2 6 0 4
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`•
`
`The Patent Off ice
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`2 5 0 CT 1993
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`
`•
`
`ORGANIC COMPOUNDS
`
`This invention comprises novel derivatives of rapamycin having
`pharmaceutical utility, especially as immunosuppressants.
`
`Rapamycin is a known macrolide antibiotic produced by
`Streptomyces hygroscopicus, having the structure depicted in Formula
`A:
`
`41
`
`(A)
`
`24
`
`See, e.g., HcAlpine, J.B., et al., J. Antibiotics (1991) 44: 688;
`Schreiber, S.L., et al., J. Am. Chem. Soc. (1991) 113: 7433; US
`
`
`
`
`
`•
`
`•
`
`Case 100-7932
`
`- 2 ...;.
`
`Patent No. 3 929 992. Rapamycin is an extremely potent
`immunosuppressant and has also been shown to have antitumor and
`antifungal activity. Its utility as a pharmaceutical, however, is
`restricted by its very low and variable bioavailabilicy _~..§. well as
`its high toxicity.
`
`It has now surprisingly been discovered that certain novel
`derivatives of rapamycin (the Novel Compounds) have an improved
`pharmacologic profile over rapamycin and exhibit greater stability
`and bioavailability. The Novel Compounds are compounds having the
`structure of Formula I:
`
`41
`
`36
`
`33
`: 34
`
`.
`
`3
`
`2
`
`6
`
`7
`- N
`
`5 ~· 4 ,,,,~ ... · 35
`
`1 6
`·x~ o
`0
`9
`~~OH·
`°
`11
`0---
`
`(I)
`
`y
`
`24
`
`12
`
`18
`
`20
`
`'/"'
`19
`
`wherein
`
`X is (H,H) or O;
`
`Y is (~,H), (H,OH), or O; and
`
`R1 and R2 are independently selected from
`B, alkyl, thioalkyl, arylalkyl, hydroxyalkyl,
`dihydroxyalkyl, alkoxyalkyl, acyloxyalkyl, carbalkoxyalkyl,
`
`
`
`
`
`•
`
`Case 100-7932
`
`- 3 -
`
`3 Si where each R3
`aminoalkyl, alkylaminoalkyl, allyl and R3
`is independently selected from -H, methyl, .. ethyl, .isopropyl,
`!-butyl, and phenyl; wherein "alk-" or "alkyl" refers to
`c1 _5 alkyl, branched or linear, preferably C1 =3 Jllkyl, in
`which the carbon chain may be optionally interrupted by an
`ether (-0-) linkage; and
`
`provided that where X is O, then either Y is other than O, or R1
`or R2 is other than H; and
`
`provided that where R1 or R1 and R2 are R3
`both o.
`
`3 Si, X and Y are not
`
`Among the pref erred groups of Novel Compounds are
`
`a) 9-deoxorapamycins where X. is (H,H), Y is (H,H), (H,OH) or 0,
`and R1 and R2 are independently selected from H, alkyl, _allyl,
`arylalkyl, hydroxyalkyl and carbalkoxyalkyl;
`
`b) 26-dihydro-rapcµnycins where X is 0 or (H,H), Y is (H,OH),
`and R1 and R2 are independently selected from H, alkyl, allyl,
`arylalkyl, hydroxyalkyl and carbalkoxyalkyl; and
`
`c) 40-0-substituted and 28,40-0,0-disubstituted rapamycins where
`X is 0 or (H,H), Y is (H,H), (H,OH) or O, and R1 and R2 are
`independently selected from alkyl, allyl, arylalkyl, hydroxyalkyl and
`carbalkoxyalkyl.
`
`The most preferred Novel Compounds are
`
`1. 9-Deoxorapamycin (X=H,H; Y=O; R1 =R2 =H).
`2. 26-Dihydro-rapamycin (X=O; Y=H,OH; R1 =R2 =H).
`3. 9-Deoxo-26-dihydro-rapamycin (X=H,H; Y=H,OH; R1 =R 2 =H).
`4. 40-0-Carbethoxymethyl-rapamycin (X=Y=O; R1 =CH 2 COOCH 2 CH 3 ; R2 =H).
`
`
`
`
`
`•
`
`- 4 -
`
`Case 100-7932
`
`5. 40-0-Carbethoxymethyl-9-deoxorapamycin (X=H,H; Y=O,
`R1 =CH2COOCH2CH3, R2=H).
`6. 40-0-Benzyl-rapamycin (X=Y=O; R1 =CH2C6 H5 ; R2=H).
`7. 40-0-Allyl-rapamycin (X=Y=O; R1 =CH2CHCH2; R2=H).
`8. 40-0-(2-Hydroxyethyl)-rapamycin (X•Y=O; R1 =CH2CH20H, R2=H).
`
`The 9-deoxorapamycin compounds are produced by reducing a
`rapamycin using hydrogen sulfide-, e.g. -as- described in Example 1,
`by reacting rapamycin with dipbenyldiselenide and tributylphosphine
`or by other suitable reduction reaction.
`
`The 26-dihydro-rapamycins are produced by reducing rapamycins or
`9-deoxorapamycins -from-keto to hydroxy at C26 by a mild reduction
`reaction, such as a borohydride reduction reaction, -e.g., as
`described in Example 2.
`
`0-substitutions at C40 are accomplished by reacting the compound
`with a radical attached to a leaving group under acidic or neu~ral
`conditions, e.g., as described in Example 3. Further modi~ications
`are possible. For -exa~ple, where the substituent at C40 is allyl,
`the isolated, monosubstituted double bond of the allyl moiety is
`highly amenable to further modification. 0-substitutions at C28 are
`accomplished in the same manner, but with prior protection at .C40.
`
`The Novel Compounds are particularly useful for the following
`conditions:
`
`a) Treatment and prevention of organ or tissue transplant
`rejection, e.g. for the treatment of recipients of e.g. heart, lung,
`combined heart-lung, liver, kidney, pancreatic, skin or corneal
`transplants. They are also indicated for the prevention of
`graft-versus-host disease, such as following bone marrow
`transplantation.
`
`
`
`
`
`,.
`
`•
`
`- 5 -
`
`•
`
`case 100-7932
`
`b) Treatment and prevention of autoimmune disease and of
`inflammatory conditions, in particular inflammatory conditions with
`an aetiology including an autoimmune component such as arthr1tis (for
`example rheumatoid arthritis, arthritis chronica prog~~~}~nte and
`arthritis deformans) and rheumatic diseases. Specific autoimmune
`diseases for which the compounds of -the invention .may be employed
`include, autoimmune haematological disorders (including e.g. haemo(cid:173)
`lytic anaemia, aplastic anaemia, pure red cell anaemia and idiopathic
`thrombocytopaenia), systemic lupus erythematosus, polychondritis,
`sclerodoma, Vegener granulamatosis, dermatomyositis, chronic active
`hepatitis, myasthenia gravis, psoriasis, Steven-Johnson syndrome,
`idiopathic sprue, autoimmune inf~ammatory bowel disease (including
`e.g. ulcerative coli tis and Cr.ohn.' s disease) endocrine
`ophthalmopathy, Graves disease, sarcoidosis, multiple sclerosis,
`primary billiary cirrhosis, juvenile diabetes (diabetes mellitus type
`I), uveitfs (anterior and posterior), keratoconjunctivitis sicca and
`vernal keratoconjunctivitis, interstitial lung fibrosis, psoriatic
`arthritis, glomerulonephritis (with and without nephrotic syndrome,
`e.g. including idiopathic nephrotic syndrome or minimal change
`nephropathy) and juven~le dermatomyositis.
`
`c) Treatment and prevention of asthma.
`
`d) Treatment of multi-drug resistance (MDR). The Novel
`Compounds suppress P-glycoproteins (Pgp), which are the membrane
`transport molecules associated with MDR. MDR is particulary
`problemafic in cancer patients and AIDS patients who will not respond
`to conventional chemotherapy because the medication is pumped out of
`the cells by Pgp. The Novel Compounds are therefore useful for
`enhancing the efficacy of other chemotherapeutic agents in the
`treatment and control of multidrug resistant conditions such as
`multidrug resistant cancer c;>r multidrug resistant AIDS.
`
`
`
`
`
`•
`
`•
`
`Case 100-7932
`
`- 6 -
`
`The Novel Compounds are also useful in treating proliferative
`disorders, e.g. tumors, hyperproli-f.erative skin disorder and the
`like, and in treating fungal injections.
`
`The pharmacological activity of the Novel Compounds are
`demonstrated in, e.g., the following tests:
`
`-· _...,..
`
`1. Mixed lymphocyte reaction (HLR)
`
`The Mixed Lymphocyte Reaction was originally developed in
`connection with allografts, to assess the tissue compatability
`between potential organ donors and recipients, and is one of the best
`established models of immune reaction in vitro. A murine model HLR,
`e.g., as described by T.Meo in "Immunological Methods", L. Lefkovits
`and B. Peris, Eds., Academic Press, N.Y. pp. 227-239 (1979), is used
`to demonstrate the immunosupressive effect of the Novel Compounds.
`Spleen cells (0.5 x 106 )
`from Balb/c mice (female, 8-10 weeks) are
`co-incubated for 5 days with 0.5 x 106 irradiated (2000 rads) or
`mitomycin C treated spleen cells from CBA mice (female, 8-10 weeks).
`The irradiated allogene_ic cells induce a proliferative response in
`the Balb/c spleen cells which can be measured by labeled precursor
`incorporation into the DNA. Since the stimulator cells are irradiated
`(or mitomycin C treated) they do not respond to the Balb/c cells with
`proliferation but do retain their antigenicity. The
`antiproliferative effect of the Novel Compounds on the Balb/c cells
`is measured at various dilutions and the concentration resulting in
`50% inhibition of cell proliferation (IC 50 ) is calculated. The
`inhibitory capacity of the test sample may be compared to rapamycin
`and expressed as a relative IC 50 (i.e. IC 50 test sample/IC50
`rapamycin).
`
`
`
`
`
`I"'
`
`•
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`- 7 -
`
`•
`
`Case 100-7932
`
`2.
`
`IL-6 mediated proliferation
`
`The capacity of the Novel Compounds to interfere with growth
`factor associated signalling pathways is assessed us~!!_g_.~~
`interleukin-6 (IL-6)-dependent mouse hybridoma cell line. The assay
`is performed in 96-well microtiter plates. 5000 cells/well are
`cultivated in serum-free medium (as described by M. H. Schreier and
`R. Tees in Immunological Methods, I. Lefkovits and B. Pernis, eds.,
`Academic Press 1981, Vol. II, pp. 263-275), supplemented with 1 ng
`recombinant IL-6/ml. Following a 66 hour incubation in the absence
`or presence of a test sample, cells are pulsed with 1 µCi
`(3-B)-thymidine/well for another 6·hours, harvested and counted by
`liquid scintillation.
`(3-B)-thymidine incorporation into DNA
`correlates with the increase in cell number and is thus a measure of
`cell proliferation. A dilution series of the test sample allows the
`calculation of the concentration resulting in 50% inhibition of cell
`proliferation (IC50 ). The inhibitory capacity of the test sample may
`be compared to rapamycin and expressed as a·relative IC 50 (i.e. IC 50
`test sample/IC50 rapamycin).
`
`3. Macrophilin binding assay
`
`Rapamycin and the structurally related immunosuppressant,
`FK-506, are both known to bind in vivo to macrophilin-12 (also known
`as FK-506 binding protein or FKBP-12), and this binding is thought to
`be related to the immunosuppressive activity of these compounds.
`The Novel Compounds also bind strongly to macrophilin-12, as is
`demonstrated in a competitive binding assay.
`
`In this assay, FK-506 coupled to BSA is used to coat microtiter
`wells. Biotinylated recombinant human macrophilin-12 (biot-MAP) is
`allowed to bind in the presence or absence of a test sample to the
`immobilized FK-506. After washing (to remove non-specifically bound
`macrophilin), bound biot-MAP is assessed by incubation with a
`
`
`
`
`
`•
`
`•
`
`Case 100-7932
`
`- 8 -
`
`streptavidin-alkaline phosphatase conjugate, followed by washing and
`subsequent addition of p-nitrophenyl phosphate as a substrate. The
`read.-out is the OD at 405nm. Binding of a test sample to biot-MAP
`results in a decrease in the amount of biot-MAP bound -~o., . .!..he FK-506
`and thus in a decrease in the 00405. A dilution series of the test
`sample allows determination of the concentration resulting in 50%
`inhibition of the biot-MAP binding to the immobilized FK-506 (IC 50 }.
`The inhibitory capacity of a test sample is compared to the IC 50 of
`free FK-506 as a standard and expressed as a relative IC50 (i.e.,
`IC 50 -test sample/ IC 50 -free FK-506).
`
`4.
`
`Localised Graft-Versus-Host (GvB)-Reaction
`
`In vivo efficacy of the Novel Compounds is proved in a suitable
`animal model, as described, e.g., in Ford et al, TRANSPLANTATION 10
`(1970} 258. Spleen cells (1 x 107 } from 6 week old female
`Vistar/Furth (VF} rats are injected subcutaneously on da~ 0 into the
`left hind-paw of female (F344 x VF)F 1 rats weighing about lOOg.
`Animals are treated for 4 consecutive days and the popliteal lymph
`nodes are removed and weighed on day 7. The difference in weight
`between the two lymph nodes is taken as the parameter for evaluating
`the reaction.
`
`5.
`
`Kidney Allograft Reaction in Rat
`
`One kidney from a female fisher 344 rat is transplanted onto the
`renal vessel of a unilaterally (left side} nephrectomised VF
`recipient rat using an en.d-to-end anastomosis. Ureteric anastamosis
`is also end-to-end. Treatment commences on the day of
`transplantation and is continued for 14 days. A contralateral
`nephrectomy is done seven days after transplantation, leaving the
`recipient relying on the performance of the donor kidney. Survival
`of the graft recipient is taken as the parameter for a functional
`graft.
`
`
`
`
`
`!"
`
`.J
`
`•
`
`- 9 -
`
`•
`
`Case 100-7932
`
`6.
`
`Experimentally Induced Allergic Encephalomyelitis (EAE) in Rats
`
`Efficacy of the -Novel Compounds in EAE is measur~d,_,__!.g., by the -·
`.+
`procedure described in Levine.& Venlt, AMER J PATH 47 (1965) 61;
`McFarlin et al, J IKMUNOL 113 (1974) 712; Borel, TRANSPLANT. & CLIN.
`IMMUNOL 13 (1981) 3. EAE is a widely accepted model for multiple
`sclerosis. Male Vistar rats are injected in the hind paws with a
`mixture of bovine.spinal cord and complete Freund's adjuvant.
`Symptoms of the disease (paralysis of the tail and both hind legs)
`usually develop within 16 days. The number of diseased animals as
`well as the time of onset of the disease are recorded.
`
`7.
`
`Freund's Adjuvant Arthritis
`
`Efficacy against experimentally induced arthritis is shown using
`the procedure descr~bed, e.g., in Vinte~ & Nuss, ARTHRITIS &
`RHEUMATISM ~ (1966) 394; Billingham & Davies, HANDBOOK OF
`EXPERIMENTAL PHARMACOL (Vane & Ferreira Eds, Springer-Verlag, Berlin)
`50/II (1979) 108-144. OFA and Vistar rats (male or female, lSOg body
`weight) are injected i.e. at the base of the tail or in the hind paw
`with 0.1 ml of mineral oil containing 0.6 mg of lyophilised
`heat-killed Mycobacterium smegmatis.
`In the developing arthritis
`model, treatment is started immediately after the injection of the
`adjuvant (days 1 - 18); in the established arthritis model treatment
`is started on day 14, when the secondary inflammation is well
`developed (days 14-20). At the end of the experiment, the swelling
`of the joints is measured by means of a micro-caliper. ED50 is the
`oral dose in mg/kg which reduces the swelling (primary or secondary)
`to half of that of the controls.
`
`
`
`
`
`•
`
`-·
`
`Case 100-7932
`
`- 10 -
`
`8. Antitumor and HDR activity
`
`The antitumor activity of the No'lel Compounds and their ability
`to enhance the perfomance of anti tumor agents by allev_!.a~.!,_ng
`multidrug resistance is demonstrated, e.g., by administration of an
`anticancer agent, e.g., colchicine or etoposide, to multidrug
`resistant cells and drug sensitive cells in vitro or to animals
`having multidrug resistant or drug sensitive tumors or infections,
`with and without co-administration of the Novel Compounds to be
`tested, and by administration of the Novel Compound alone.
`
`-· _.,f.
`
`Such in vitro testing is performed employing any appropriate
`drug resistant cell line and control (parental) cell line, generated,
`e.g. as described by Ling et al., J. Cell. Physiol. 83, 103-116
`(1974) and Bech-Hansen et al. J. Cell. Physiol. 88, 23-32 (1976).
`Particular clones chosen are the multi-drug resistant (e.g.
`colchicine resistant) line CHR (subclone CSS3.2) and the parental,
`sensitive line AUX Bl (subclone ABl Sll).
`
`In vivo anti-tumor_ and anti-HDR activity is shown, e.g., in mice
`injected with multidrug resistant and drug sensitive cancer cells.
`Ehrlich ascites carcinoma (EA) sub-lines resistant to drug substance
`DR, VC, AH, ET, TE or CC are developed by sequential transfer of EA
`cells to subsequent generations of BALB/c host mice in accordance
`with the methods described by Slater et al., J. Clin. Invest, 70,
`1131 (1982).
`
`Equivalent results may be obtained employing the Novel Compounds
`test models of comparable design, e.g. in vitro, or employing test
`animals infected with drug-resistant and drug sensitive viral
`strains, antibiotic (e.g. penicillin) resistant and sensitive
`bacterial strains, anti-mycotic resistant and sensitive fungal
`strains as well as drug resistant protozoal strains, e.g. Plasmodial
`strains, for example naturally occurring sub-strains of Plasmodium
`
`
`
`
`
`••
`
`- 11 -
`
`•
`
`Case 100-7932
`
`falciparum exhibiting accquired chemotherapeutic, anti-malarial drug
`resistance.
`
`9. Dosage forms
`
`The Novel Compounds are utilized by administration of a
`pharmaceutically effective dose in pharmaceutically acceptable form
`to a subject in need of treatment. Appropriate dosages of the Novel
`Compounds will of course vary, e.g. depending on the condition to be
`treated (for example the disease type or the nature of resistance),
`the effect desired and the mode of administration.
`
`In general however satisfactory results are obtained on
`administration orally at dosages on the order of from 0.05 to 5 or up
`to lOmg/kg/day, -e.g .•. on the.order_ of -from 0.1. to 2·or up to 7.5
`mg/kg/day administered once or, in divided doses 2 to 4x per day, or
`on administration parenterally, e.g. intravenously, for example by
`i.v. drip or infusion, at dosages on the order of .from 0.01 to 2.5 up
`to 5 mg/kg/day, e.g. on the order of from 0.05 or 0.1 up to 1.0
`mg/kg/day.
`
`Suitable daily dosages for patients are thus on the order of
`500 mg p.o., e.g. on the order of from 5 to 100 mg p.o., or on the
`order of from 0.5 to 125 up to 250 mg i.v., e.g. on the order of from
`2.5 to 50 mg i.v ••
`
`Alternatively and even preferably, dosaging is arranged in
`patient specific manner to provide pre-determined trough blood
`levels, e.g. as determined by RIA technique. Thus patient dosaging
`may be adjusted so as to achieve regular on-going trough blood levels
`as measured by RIA on the order of from 50 or 150 up to 500 or
`lOOOng/ml, i.e. analogously to methods of dosaging currently employed
`for Ciclosporin immunosuppressive therapy.
`
`
`
`
`
`•
`
`- 12 -
`
`Case 100-7932
`
`The Novel Compounds .are administered by any conventional route,
`in particular enterally, e.g. orally, for example in the form of
`solutions for drinking, tablets or capsules or parenterally, for
`example in the form of injectible solutions or suspensions. Suitable
`unit dosage forms for oral administration comprise, e.g. from 1 to SO-::;.
`mg of a compound of the invention, usually 1 to 10 mg.
`
`
`
`
`
`•
`
`Case 100-7932
`
`- 13 -
`
`EXAMPLES:
`
`In the following examples, characteristic spectroscopic data is
`given to facilitate identification. Peaks which do no.L.differ
`significantly from rapamycin are not included. Biological data is
`expressed as a relative IC50 , compared to rapamycin in the case of
`the MLR and IL-6 mediated proliferation assays, and to FK-506 in the
`macrophilin binding assay. A higher IC 50 correlates with lower
`binding affinity.
`
`EXAMPLE 1 - 9-deoxorapamycin
`
`A stream of hydrogen sulfide is passed at room temperature
`through a stirred solution of 3.2 g (3.5 mmol) of rapamycin in 50 ml
`pyridine and 2.5 ml DMF. The solution turns from colorless to
`yellow. After two hours, the introduction of hydrogen sulfide is
`stopped and stirring is continued for five days, during yhich time
`the solution turns gradually orange. TLC and HPLC analysis verifies
`complete consumption of the starting material and the presence of a
`single new compound. The solution is purge~ with nitrogen for one
`hour and concentrated under reduced pressure. The residue is taken
`up in ethyl acetate, washed with cold lN HCl solution (3x), saturated
`sodium bicarbonate solution and saturated brine. The organic layer
`is dried over anhydrous sodium sulfate and filtered and concentrated
`under reduced pressure. The residue is taken up in ether and the
`precipitated sulfur is filtered off. Concentration of the ethereal
`solution followed by column chromatography on silica gel (10:4:1
`CH 2 Cl 2 /i-Pr 2 0/MeOH) yields 9-deoxorapamycin as a colorles
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