VOL. XXXVII NO. 10
`
`THE JOURNAL OF ANTIBIOTICS
`
`1231
`
`ACTIVITY OF RAPAMYCIN (AY-22,989) AGAINST
`TRANSPLANTED TUMORS
`
`C. P. E:-;0 1, S. N. SEHGALt and CLAUDE VEzlNAt~*
`
`Deparlment of Microbiology, Ayerst Research Laboratories,
`P.O. Box 6115, Montreal, Quebec, Canada H3C 3JI
`
`(Received for publication June 18, 1984)
`
`Rapamycin exhibits activity against several ascites and solid transplantable tumors: it is
`slightly acti\·e to inacfo·e against leukemias. On a weight basis, rapamycin was less acti\'e
`than 5-fluorouracil, cyclophosphamide and adriamycin, but rapamycin's maximal acth·itr
`against Colon 38 tumor was similar to that of 5-fluorouracil and cyclophosph:unidc.
`Its ac(cid:173)
`tivity was such that it significantly inhibited tumor growth at any stage of development
`In
`the active dose range, rapamycin appeared less 1oxic than 1he 01her drugs.
`In the Colon 38
`tumor model. rapamycin at a given dose exhibited the same activity when administered ip, iv,
`im and sc; upon oral administration, its activity was reduced but not abolished. Rapamycin
`was compatible with 5-11uorouracil and cyclophosphamide. The sequential treatment 5-
`Huorouracil-rapamycin-cyclophosphamidc was superior to the scqucm:c 5-fluorouracil-adril·
`mycin-cyclophosphamide in protecting Colon 38 tumor-bearing mice.
`29-Demethoxyrapamycin excricd only marginal activity against P388 lymphocytic
`leukemia; it was inactive against Bl6 melanocarcinoma and Colon 38 solid tumor.
`
`Rapamycin is a triene antibiotic produced by Streptomyccs hygroscopicus 1 ·~>. Structure elucida(cid:173)
`tion revealed the presence of a pipecolic acid residue in the macrolide3 •H. Several yeasts, as well as
`yeast-like and filamen1ous fungi, are sensitive to rapamycin; however, the main feature of the antibiotic
`is its high activity against Candida albican.r (MIC 0.02- 0.20 11g/ml)~>. Nucleic acids synthesis inhibi(cid:173)
`tion and degradation in C. albiccms are the primary modes of actionc>. The LD00 ip of rapamycin in
`the mouse is 587 mg/kg. The antibiotic also has immune-suppressant activity~>: it is half as potent as
`cyclophosphamide in inhibiting experimental allergic encephalomyelitis and is as potent as this standard
`reference drug in preventing adjuvant-induced arthritis. The mode of action and the pharmacological
`effects warranted the evaluation of rapamycin in experimental tumor models. The National Cancer
`Institute {NCI, Division of Cancer Treatment) conducted the initial studies and reported modest ac(cid:173)
`tivity against P38B iymphocytic ieukemia (increased iife span (iLS)=30-407~ ai. i.25 mg/kg) and no
`activity against Ll210 lymphoid leukemia and Lewis lung carcinomas>, Activity was reported against
`Bl6 melanocarcinoma (ILS 80% at 100 mg/kg), Colon 26 tumor (ILS 105% at 6.25 mg/kg) and EM
`cpendymoblastoma (ILS 85% at 50 mg/kg and ILS 100% at 200 mg/kg). Rapamycin was also active
`against the solid tumors, CDSFI mammary tumor (80~-~ tumor weight inhibition at 25 mg/kg) and
`Colon 38 tumor (85% tumor weight inhibition at 25 mg/kg). Subrenal capsule CX-1 colon adeno(cid:173)
`carcinoma xenograft and spontaneous colon adenocarcinoma 11/A were sensitive to rnpamycin.
`We report here a more detailed evaluation of the efficacy of rapamycin in transplantable tumor
`models. The effects of dosage, regimen and route of administration were studied. Rapamycin was
`compared to other antitumor agents; the antineoplastic effects of rapamycin in combination were also
`evaluated.
`
`1 Ayerst Laboratories, 567 Ridge Road, Princeton, NJ 08852, USA.
`Institut Armand-Frappier, 531, Boulevard des Prairies, Ville de Laval, Que, Canada H7V 1B7.
`tt
`
`ROX_EV_00148639
`
`Novartis v. Breckenridge,
`Roxane, Par, C.A. Nos.
`14-1043, 14-1196, 14-1289
`
`JTX 167
`
`Ex. 1113-0001
`
`

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`THE JOURNAL OF ANTIBIOTICS
`
`OCT. 1984
`
`Materials and Methods
`
`Drugs
`Pure crystalline rapamycin and 29-demcthoxyrapamycin (A Y-24,668) were prepared as described
`previously:.r-i. 5-Fluorouracil (5-FU, 99 ~~pure) was obtained from Aldrich Chemical Co., Milwaukee,
`WI, and cyclophosphamide (CYP, 99% pure) from Polyscience Inc., Warrington, PA. Adriamycin
`(ADR) was purchased from a local drug store; each vial contained 10 mg doxorubicin· HCI and 50 mg
`lactose. For injection, rapamycin was dissolved in absolute ethanol containing I .0 mg butylated
`hydroxyanisole/ml, and one volume of this solution was mixed with nine volumes of 10% Cremophor
`EL (BASF, Aktiengesellschaft, West Germany) in water. Sterile physiological saline was used as a
`vehicle for the three reference drugs. All drug solutions were prepared just before use.
`
`Tumor Models and Animals
`P33S lymphocytic leukemia, BI 6 melanocarcinoma and Colon 38 tumor lines were obtained from
`the ;\lason Institute, Worchester, MASS., a tumor bank for the NCI. The tumors were serially trans(cid:173)
`planted in appropriate strains of inbred mice: PJ88 in DBA/2, Bl6 in C57/Bl and Colon 38 in BDF1
`mice. Male BDF1 mice (18-20g) were used in the antitumor tests. All mice were purchased from
`Jackson Laboratories, Bar Harbor, ME., and were or specific-pathogen-free (SPF) grade.
`Antitumor Tests
`The experimental procedures were those recommended by the Developmental Therapeutic Program,
`Drug Evaluation Branch, of the National Cancer Institute10·u>. They are summarized as follows:
`P388 Lymphocytic Leukemia: Ascites tumor cells were aseptically withdrawn from a tumor(cid:173)
`bearing mouse. After one washing with saline, the tumor cells were enumerated with a hcmocytomcter
`and suspended in saline at a concentration of 5.0 :< 106 cells/ml. On Day 0, each test mouse received ip
`I :-: 101 viable tumor cells suspended in 0.2 ml of saline. Drug treatment was given ip once daily from
`Day I to Day 9. Six mice were used in each test group.
`Bl 6 Mclanocarcinoma: Tumor nodules from tumor-bearing mice were excised, gently homo(cid:173)
`genized in a hand-operated tissue grinder and suspended in sterile saline which was then diluted I: 10
`(w/w). On Day 0, each mouse received ip 0.5 ml or the tumor suspension. Treatment was given ip
`once daily from Day 1 to Day 9. Ten mice were used in each test group.
`Colon 38: On Day 0, tumor nodules from tumor-bearing mice were excised and cut into 2- 3
`mm3 fragments. One tumor fragment was placed sc in the back of each test mouse through a trocar.
`Unless otherwise specified, treatment was given ip once on Days 2 and 9 in the 2 x treatment schedule,
`or once on Days 2, 5 and 9 in the 3 ~< treatment schedule. On Day 20, the tumor nodules were excised
`and weighed indi\'idually.
`
`Evaluation
`The effects of the drugs in the P388 and B 16 test systems were evaluated on the basis of the median
`survival time (MST, in days). The results arc expressed as T/C ( %).
`
`T/C~'=~~-~!~~atment group)>: 100
`0 MST (control group)
`The effects of the drugs in the Colon 38 tumor model were assessed on the basis of median tumor
`weight (MTW, in mg) on Day 20, unless otherwise specified. The results are expressed as T/C~·~.
`
`T/C%
`
`MTW(treatment group) ;.:
`MTW (control group)
`
`100
`
`or percent tumor inhibilion (100-T/C~·~).
`For the evaluation of the effects of rapamycin on established and advanced Colon 38 tumors (Table
`3), the width (a) and the length (b) of individual tumors were measured (in mm) ai various time intervals.
`The tumor weight was calculated by the formula:
`Tumor weight (mg)= J /2 ab~ (mm)
`
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`VOL. xxxvn NO. to
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`THE JOURNAL OF ANTIBIOTICS
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`1233
`
`Results
`
`Activity against P388 Lymphocytic Leukemia, Bl 6 Melanocarcinoma
`and Colon 38 Tumor
`As shown in Table 1, rapamycin was slightly active against P388 leukemia: in two separate experi(cid:173)
`ments, T/C values of 160 and 137% were obtained after the ip administration of JOO mg/kg once daily
`for 9 days. By comparison, a T/C value of 191 % was obtained when 5-FU was given ip at 10 mg/kg/
`day for the same period. The antibiotic was active against Bl6 melanocarcinoma; T/C values of 141
`and 179% were attained with 100 mg/kg/ip injection in the 9 x treatment schedule. This effect is com(cid:173)
`parable to that obser\'ed with 20 mg/kg/ip injection of 5-FU (T/C 143 %). Rapamycin also showed
`anti tumor activity against the Colon 38 tumor. Relative to the tumor growth observed in the untreated
`controls, 400 mg/kg/ip injection (2 >~ treatment schedule) of rapamycin (T/C 7.0 and 3.7 %) inhibited
`tumor weight by 93 and 96.3 ~{. Thus, rapamycin was more ac1ive than 5-FU (T/C 23.8%) adminis(cid:173)
`tered ip at a dose of 70 mg/kg on Days 2 and 9. When given according to the treatment schedules des(cid:173)
`cribed in Table l, rapamycin did not cause any early deaths, indicating that it was devoid of acute toxicity
`at the doses used.
`In these experiments, clcmethoxyrapamycin was compared to rapamycin and found completely
`inactive against Bl6 melanocarcinoma and Colon 38 solid tumor; it exhibited slight activity (T/C%
`135) against P388 lymphocytic leukemia. Therefore, the absence of the methoxy group in position 29
`almost completely abolishes the anti tumor activity of rapamycin~>.
`
`Anti-Colon 38 Activity of Rapamycin Administered by Various Routes
`In the present experiment five routes of administration were compared with respect to their effects
`on 1he antilumor activity of rapamycin (400 mg/kg, 2 x treatment schedules). As shown in Table 2,
`the im and sc (adjacent to the 1umor) routes were as effective as the ip route. These three routes afforded
`over 90 % tumor inhibition. When the sc injection was given on the other side of 1he back, distant from
`the tumor, the antitumor activity (84.8% tumor inhibition) decreased slightly. Oral administration
`produced significantly less antitumor activity (64.8% tumor inhibition) than the other routes.
`
`Table I. Effects of rapamycin and 5-FU against P388 lymphocytic leukemia, Bl 6 melanocarcinoma and
`Colon 38 tumor (expressed as T/C%)".
`
`Dose
`(mg/kglip
`injec1ion)
`
`P388 lymphocytic
`leukemia~
`
`Bl6
`mclanocarcinoma•
`
`Colon 38
`tumor**
`
`Expt I
`
`Expt II
`
`Exptl
`
`Expt JI
`
`Expt I
`
`Expt 1I
`
`160
`145
`145
`145
`
`137
`141
`141
`141
`191
`
`5-FUb
`
`141
`134
`124
`129
`
`179
`171
`150
`131
`
`143
`
`Rapamycin 400
`200
`100
`50
`25
`12.5
`JO
`20
`70
`Treatment schedule " 9 ;,;(Days 1-9), ** 2 :-:(Days 2 and 9)
`• Drug considered active when T/C% :a: 130 (P388), :a: 125 (B 16) and :<:;;42 (Colon 38}.
`b Posith·e control.
`
`7.0
`13.4
`38.9
`133.4
`
`3.7
`7.4
`24.2
`39.3
`
`23.8
`
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`123.i
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`THE JOUR.i"'l(AL OF Ail\TlBIOTICS
`
`OCT. 198.i
`
`Table 2. Ami·Colon 38 effect of rapamycin administered by various routes (treatment on Days 2 and 9).
`
`Route of
`administration
`
`Median tumor weight (mg)
`
`Vehicle
`(0.4 ml ip)
`
`Rapamycin
`(400 mg/kg)
`
`ip
`im
`SC
`
`1.096
`1,061
`
`a) Adjacent to tumor
`b) Distant to tumor
`po
`
`567
`885
`838
`~ Drug considered active when T/C~,~ 5:42.
`
`70
`70
`
`52
`135
`262
`
`T/C~~·
`
`6.4
`6.6
`
`9.1
`15.2
`3.5.2
`
`Tumor
`inhibition
`(%>
`
`93.6
`93.4
`
`90.9
`84.S
`64.8
`
`Effects of Rapamycin on Established and Advanced Colon 38 Tumors
`In the standard test, tumor inoculation is conducted on Day 0 and treaur:ent given on Days 2 and
`In this experiment, rapamycin treatment was delayed until a) Days 6 and 13; b) Days 13 and 20;
`9.
`and c) Days 20 and 27. Tumors were excised and weighed on Day 29 or 30. All treated groups re(cid:173)
`ceived rapamycin ip at 400 mg/kg/injection, and the corresponding control groups were given the vehicle
`ip. As shown in Table 3, the tumor weights in the treated and control groups were similar up to the
`first day of treatment. However, there was a great reduction of tumor growth in all the rapamycin(cid:173)
`treated groups in comparison to the vehicle-treated controls. When the tumors were measured on
`Day 29 or 30, 81.1, i6.I and 54.8% inhibition occurred in groups (a), (b) and (c), respectively. Thus,
`there was a direct correlation between the time of treatment and the maximum tumor inhibition attained.
`
`Table 3. Activity of mpamycin against established and advanced Colon 38 tumors.
`
`Days after tumor
`inoculation
`
`Average tumor weight (mg)
`
`Vehicle
`(0.4 ml, ip)
`
`Rapamycin. ip
`(400 mg/kg)
`
`Tumor
`inhibition
`(%)"
`
`a) Treatment on Days 6 and 13
`
`b) Treatment on Days 13 and 20
`
`6
`9
`13
`16
`20
`23
`26
`30
`
`13
`16
`19
`23
`26
`29
`
`39
`66
`213
`306
`564
`908
`896
`1,121
`
`97
`139
`244
`415
`646
`844
`
`c) Treatment on Days 20 and 27
`
`594
`1.159
`1,915
`2,112
`~ Drug considered active when tumor inhibition ;;:: 58 ~~ (TfC ~~ 5: 42l.
`
`20
`23
`27
`29
`
`38
`43
`50
`70
`103
`i40
`194
`212
`
`128
`220
`139
`194
`190
`202
`
`602
`792
`845
`955
`
`2.6
`34.9
`76.6
`77.2
`81.8
`84.6
`78.4
`81.1
`
`-31.9
`-58.2
`40.4
`53.3
`70.6
`76.1
`
`-1.3
`31. 7
`57.3
`54.8
`
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`VOL. XXXVII NO. 10
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`THE JOURNAL OF ANTIBIOTICS
`
`1235
`
`Table 4. Comparati\'e an1i-Colon 38 activity of 5-FU, CYP, ADR and rapamycin (treatment on Days 2
`and 9).
`
`5-FU
`
`CYP
`
`ADR
`
`Dose ip
`(mg/kg/injection)
`200
`100
`50
`200
`100
`50
`20
`JO
`5
`
`Rapamycin
`
`400
`200
`100
`~ Drug considered active when TIC% =:;;42.
`
`Early
`dea1hs
`4/10
`0:10
`0/10
`O/IO
`0/10
`O/IO
`8/10
`3/10
`0/IO
`0/10
`0/10
`O/IO
`
`T/C% 3
`
`Toxic
`5.8
`54.4
`0.8
`7.9
`59.0
`Toxic
`26.8
`89.7
`6.8
`9.6
`12.3
`
`Tumor inhibition
`(%)
`Toxic
`94.2
`45.6
`99.2
`92.1
`41.0
`Toxic
`73.2
`J0.3
`93.2
`90.4
`87.7
`
`Comparison of the Anti-Colon 38 Activity of Rapamycin and Other
`Antitumor Drugs
`In this study, Colon 38 tumor-bearing mice were treated ip on Days 2 and 9 with 5-FU, CYP, ADR
`or rapamycin. Three dose levels of each drug were tested. As shown in Table 4, the intermediate
`doses of the reference drugs had very high anti-Colon 38 activity; 5-FU ( 100 mg/kg), CYP (100 mg/kg)
`and ADR (10 mg/kg) inhibited tumor growth by 94.2, 92.1 and 73.2 %, respectively. The highest
`closes of 5-FU and ADR were toxic to the tumor-bearing mice, and the lowest doses of 5-FU, CYP
`and ADR did not exert significant anti-Colon 38 activity. Rapamycin exhibited significant anti-Colon
`38 activity at all three doses; tumor growth was inhibited by 93.2, 90.4 and 87.7 % at 400, 200 and
`JOO mg/kg/injection, respectively. No early deaths occurred at any of three doses, an indication of
`low acute toxicity. Therefore, rapamycin exerted the same maximal activity as 5-FU and CYP against
`Colon 38, and the activity of rapamycin was observed for a wider range of concentrations.
`
`Combination of Rapamycin, 5-FU and CYP for the Treatment of
`Colon 38 Tumor-bearing Mice
`The purpose of this study was to compare the anti-Coion 38 activiiy of two regimens: FRC (5·
`FU, mpamycin and CYP given in sequence) and FAC (5-FU, ADR and CYP given in sequence). Single
`drug treatments were also included for comparison. Treatment was administered on Days 2, 5 and 9.
`As shown in Table 5, 5-FU was toxic at 50 and I 00 mg/kg/injection and ADR at 5 and l 0 mg/kg/injec(cid:173)
`tion; CYP and rapamycin exhibited no toxicity at the doses tested.
`In the FAC regimen, the optimal
`dose was 50 mg/kg of 5-FU on Day:?., 5 mg/kg of ADR on Day 5, and 50 mg/kg of CYP on Day 9;
`no early deaths occurred and tumor inhibition was 87.4%.
`The FAC regimen was more effective than either 5-FU or ADR given alone, but only the inter(cid:173)
`In the FRC regimen, activity was obtained at all
`mediate dose level exhibited high tumor inhibition.
`three dose levels. These results indicate that rapamycin can be combined with 5-FU and CYP to
`provide a wide range of doses effective against Colon 38 tumor.
`
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`

`
`1236
`
`THE JOURNAL OF ANTIBIOTICS
`
`OCT. 1984
`
`Table 5. Anti-Colon 3S acti\'ity or 5-FU, CYP, ADR, rapamycin and combinations FAC and FRC
`(treatment on Days 2, 5 and 9).
`
`Dose
`(mg/kgl
`
`Number of
`injection
`
`Early
`deaths
`
`T/C~~
`
`Tumor
`inhibition
`(%>
`Toxic
`9/10
`Toxic
`Toxic
`Toxic
`6/10
`6S.S
`31.5
`O/IO
`------··· ·~-- _+ _______ J-.. ·-----------
`6.2
`0/10
`93.8
`85.3
`0/10
`14.7
`O/IO
`109.8
`-9.8
`----~-- - - - - - -
`Toxic
`Toxic
`Toxic
`Toxic
`22.5
`77.5
`4.5
`95.5
`86.5
`13.5
`11.6
`88.4
`Toxic
`Toxic
`12.6
`87.4
`29.4
`70.6
`---
`99.3
`0.7
`2.7
`97.3
`89.2
`10.8
`
`8/IO
`6}10
`O/JO
`0/10
`O/IO
`OilO
`
`9/IO
`0/10
`O/IO
`2/IO
`0/10
`0/10
`
`5-FU
`
`3x
`100
`3·.·
`50
`3·.·
`25
`----- . --------- - - - - - - -
`3 ·.·
`100
`CYP
`3..-
`50
`25
`3 :-:
`3"
`10
`3 ,.
`5
`3-·
`2.5
`400
`200
`100
`lOO(F)+ lO(AH- IOO(C)
`50(F)+5(A)+50(C)
`25(F) +2. S(A) +25(C)
`100(F)-!-400(R)+50(C)
`SO{F) +200(R) +50(C)
`25(F)-i- IOO(R)+25(C)
`
`ADR
`
`RAPA"'
`
`FAC
`
`FRC
`
`* Rapam~·cin.
`
`Discussion
`
`The screening strategy of the National Cancer Institute consists of testing natural products such
`as antibiotics against the murine transplantable tumor, P388 lymphocytic leukemia!ll; compounds with
`ILS ~30 are then evaluated in a tumor panel which includes ascites as well as solid tumors. Rapamycin
`shows only slight activity against P388 (maximal ILS ranges from 30 to 40), and its activity could easily
`be missed if an activity of ILS ;;:::30 were mandatory for further evaluation.
`Rapamycin exhibited little or no activity in the leukemia models. However, it was active against
`Bl6 melanocarcinoma, EM ependymoblastoma, CDSFl mammary and Colon 38 tumors. The re(cid:173)
`sistant tumor P388 and the sensitive Bl6 and Colon 38 tumors were used in the present study. 5-FU
`served as the control drug and exhibited the? expected activity in all of the experirnents reported. Rapa(cid:173)
`mycin \\as less active than 5-FU on a weight basis, but the antibiotic's maximal activity at ~25 mg/kg
`against 816 and Colon 38 tumors was higher than that obtained with 5-FU (Table I). In the Colon
`38 tumor model, CYP surpassed rapamycin on the basis of weight and maximal activity attainable;
`ADR was less active (Table 4).
`Rapamycin exhibited the same activity whether administered ip, i\· (not shown in Table 2), im or
`sc; upon oral administration, its activity was reduced but not abolished. The dose-response observed
`In the active dose
`with rapamycin was not as sharp as with the other drugs tested (Tables 4 and 5).
`range, rapamycin appeared less toxic than the other drugs. For example, tumor-bearing mice treated
`with a single ip injection of 400 mg/kg on Day 1 had an average weight loss of less than S % by Day S
`(data not shown).
`In the same experiment, 5-FU, CYP and ADR administered at optimal doses caused
`a 10 to 15% weight loss. Also. rapamycin was capable of inhibiting the growth of Colon 38 tumors
`at any stage of development (Table 3). If this clfect can be reproduced in humans, rapamycin may
`prove useful as a pre-operative treatment.
`Another feature of rapamycin is its compatibility with 5-:FU and CYP in the treatment of Colon 38
`tumor-bearing mice (Table 5). This sequential combination afforded better protection than any of the
`
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`
`VOL. XXXVII NO. 10
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`THE JOUlli~AL OF ANTIBIOTICS
`
`1237
`
`It was also superior to the sequence 5-FU, ADR and CYP, a combination that
`drugs given alone.
`has found applications in cancer therapy1:.w.
`The results of these studies demonstrate that the rapamycin treatments afford latitude with respect
`to the dose, the route of administration and the treatment schedule which can be used. This latitude
`is useful to the investigator who can therefore select effective, minimal toxic doses for specific clinical
`situations.
`
`References
`
`I)
`
`7)
`
`11)
`
`I.
`\'Ez1:-:A, C.; A. KuoELSKI & S. N. SEHG,\L: Rapamycin (AY-22,989). a new an1ifungal antibiotic.
`Taxonom>' of the producing strep1omyee1e and isolation of the active principle. J. Antibiotics 28: 721 -
`726, 1975
`2) SEHGAL, S. N.; H. BA1'ER & C. Viz1:-:A: Rapamycin (AY-22,989), a new antifungal antibiotic.
`mentation, isolation and characterization. J. Antibiotics 28: 727-732, 1975
`3) Sw1:-:DELLS, D. C.; P. s. WHITE & J. A. Fll'LAY: The X-ray crystal s1ructure of rapamycin, C;.,H:~NO:,.
`Can. J. Chem. 56: 2491-2492. 1978
`4) FINLAY, J. A. & L. RA01cs: On the chemistry and high field nuclear magnetic resonance spectroscopy of
`rapamycin. Can. J. Chem. 58: 579-590, 1980
`5) BAKER, H.; A. S100ROW1cz, S. N. SEHGAL & C. VEZll'A: Rapamycin (AY-22,989), a new antifungal anti·
`111 1·itro and i11 rfro e\'aluation. J. Antibiotics JI: 539-545, 1978
`Ill.
`biotic.
`6) S1:-;011, K.; S. Sui-; & C. VEZlt'A: Rapamycin (A Y-22,989), a new antifungal antibiotic.
`of action. J. Antibiotics 32: 630,.., 645, 1979
`l\IARTEL, R.R.; J. Kucius & S. GALET:
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`8) Dou1tos, J. & M. SUFF:-:Ess: New antitumor substances of natural origin. Cancer Treat. Rev. 8: 63-Si,
`1981
`9) SEHGAL, S. N.; H. BAKER, C. P. E:-.:a, K. S1:-;GH & C. V£zti-:A: Demethoxyrapamycin (AY-24,668), a new
`antifungal antibiotic. J. Antibiotics 36: 351 - 354, 1983
`JO) GERAX, I.: N. H. GREEXllERG, M. M. MACDOl'ALD, A. M. SCHU.MACHER & B. J. Aonorr: Protocols for
`screening chemical agents and natural products against animal tumors and other biological systems. Cancer
`Chemotherapy Reports 3: I, 1972
`Instruction 271B: Summary or 1he usual characteristics of selected murinc models used under the auspices
`of the NCI Division of Cancer Treatment. Apr. I, 1978
`12) BllZDAR, A. U.; G. R. GLU!>.IEl'SCHEIS, S. s. lEGtlA, G. N. HORTABAGYI, H. Y. YAY, T. L. SMITH & E. M.
`HERSH: Adjuvant chemotherapy with fluorouracil, doxorubicin and cyclophosphamidc (FAC) in stage H
`and III breast cancer 5-year results. Proc. Am. Ass. Cancer Res. 22: 144, 1981
`13) VOGEL, C. L.; J. LEFA~TE. D.R. EAST, B. J. RoGERS & R. V. S:1.tALLEY: Cyclophosphamide, adriamycin
`and 5-fluorouracil (CAF) alternating with cycle-acti\"e regimen in metastatic breast cancer. Proc. Am.
`Ass. Cancer Res. 22: 439, 1981
`
`II. Fer(cid:173)
`
`IV. Mechanism
`
`
`
`ROX_EV_00148645
`
`Ex. 1113-0007