Enhanced Antitumor Activity for the Thymidylate Synthase
`Inhibitor 1843U89 through Decreased Host Toxicity with Oral
`olic Acid
`ary K. Smith, Herbert Amyx, Christine M. Boytos, et al.
`
`(cid:160)F
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` 1995;55:6117-6125. Published online December 1, 1995.Cancer Res
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`
`[CANCER RESEARCH 55. 6| l7—6i25. December I5. I995]
`
`Enhanced Antitumor Activity for the Thymidylate Synthase Inhibitor 1843U89
`
`through Decreased Host Toxicity with Oral Folic Acid
`
`Gary K. Smith," Herbert Amyx, Christine M. Boytos,' David S. Duch, Robert Ferone, and H. Robert Wilson
`Divisions of Cell Biology [(7. K. 5.. D. 5. DJ, Bioch:nti.\‘Iry (C. M. B.. R. F.. H. R. W. I. and Toxicology [H. A.]. The Wellcome Research Labs. Burroughs Wellcome. Research
`Triangle Park. North Carolina 27709
`
`ABSTRACT
`
`The purpose of this investigation was to determine whether antiturnor
`selectivity of the third generation thymidylate synthase inhibitor l843U89
`could be enhanced by a combination of the drug with folic acid. The effects
`of folic acid on toxicity of IMJUS9 to the dog and mouse and on antitumor
`efficacy of l843U89 in the mouse were studied. These data were compared
`to the effect of folic acid on the in vitro cell culture antitumor activity of
`l843U89. The sensitivity of eight cancer cell lines (three ovarian, one
`colon, one ileocecal, one epiderrnoid, one osteosarcoma. and one breast
`line) to l843U89 was tested in vitro in the presence and absence of folic
`acid. Folic acid concentrations greater than 100 pM were required to
`decreme l843U89 activity in seven of the cell lina. Only the activity in
`HCT-8, the ileocecal line, was reversed at folic acid concentrations below
`100 pm. Oral folic acid given 30 min prior to an i.v. dose of 1843089
`increased the maximally tolerated dose and the lethal dose of l843U89,
`both in dogs and in thymidine-depleted mice. in mice, oral folic acid
`produced little or no effect upon the antitumor efficacy of l843U89 in two
`of three tumor cell lines in viva. HCT-8, the line that was setive to folate
`reversal in vitro, was also sensitive in vivo. The rwults show that an oral
`dose of folic acid 30 min prior to i.v. l843U89 can block mouse and dog
`intestinal toxicity without decreming efficacy of l843U89 in two of three
`human tumor lines in the nude mouse. Thus, the data reported here
`indicate that the antitumor selectivity of l843U89 may be enhanced
`through a combination of l843U89 with oral folic acid.
`
`INTRODUCTION
`
`lates (9, 12, 19, 20), l843U89 is resistant to lcucovorin reversal (3).
`Leucovorin can influence antifolate activity by direct competition for
`transport or by elevation of intracellular reduced folate levels (3, 21,
`22). In turn. elevated intracellular folates compete directly with com-
`petitive and mixed inhibitors for binding to their respective target
`enzymes. which decreases inhibition of the target enzyme (1, 9,
`15-18). The cellular folates also compete with the antifolatcs for
`polyglutamation, which decreases the extent of antifolate polygluta-
`mation and cellular retention (9, 15, 23). Since the monoglutamates of
`most antifolates are poorer inhibitors than the polyglutarnates. de-
`creased polyglutamation directly decreases target enzyme inhibition
`(1. 9, 12. 14, 15, 17-20). The effects all decrease the cfficacy of the
`antifolates in the presence of leucovorin. Since the mono- and diglu-
`tamate forms of l843U89 are equipotent noncompetitive inhibitors of
`TS, the reduced folates do not compete with l843U89 for binding to
`TS. and the decreased polyglutamation of l843U89 has less influence.
`The main influence of lcucovorin on l843U89 efficacy then is
`through competition for transport on the reduced folate transporter (3).
`‘These striking differences of 1843U89 from a variety of other
`antifolates led us to seek a means to use the differences to increase
`antitumor selectivity. in the present studies, we have investigated the
`potential of combination chemotherapy with 1843U89 plus folic acid
`to provide the additional selectivity. We report here the use of oral
`folic acid to block the dose-limiting gut toxicity of l843U89 but not
`its antitumor effects. A similar strategy was reported previously for
`the competitive inhibitor DDATHF (24. 25). Differences between the
`two strategies are discussed. The combination of folic acid with
`l843U89 may provide a mechanism for enhanced clinical antitumor
`selectivity.
`
`MATERIALS AND METHODS
`
`l843U89 is a third generation TS’ inhibitor under clinical devel-
`opment as an anticancer agent. The compound is cytocidal against a
`variety of cancer cell types in culture (I-4) and is active in several
`antitumor models in mice (3. 5-7). l843U89 displays several attrac-
`tive features compared to other antifolates. In human cells, the major
`metabolite of l843U89 is the diglutamate (8). Both l843U89 itself
`and the diglutamate metabolite bind to TS with noncompetitive ki-
`netics (I). The affinities of l843U89 and the diglutamate for TS are
`virtually identical (l ). In comparison. the major cellular metabolites
`for a variety of other antifolatcs. including MTX. DDATHF. 5-dea-
`zaacyclotetrahydrofolate, CB37l7, and Tomudex. are the m'- to hexa-
`glutamates (9-15). Furthermore. these polyglutamates are competi-
`tive or mixed inhibitors (with strong competitive components) of their
`respective enzymes (1. 9. 15-18). Lastly, the affinities of these other
`antifolate polyglutamates are greater for their respective enzymes than
`are those of the parent compounds (1. 9. 12. 15-18).
`These properties of l843U89 described above participate to pro-
`duce another unusual feature of l843U89. Compared to other antifo-
`
`Received 7/l9l95: accepted I0/l6I95.
`The costs of publication or this article were defrayed in pan by the payment of page
`charges. This article must therefore be hereby maritcd advertisement in accordance with
`I8 U.S.C. Section I734 solely to indicate this fact.
`' Present address: Department of Cancer Biology, Glaxowellcome. 5 Moore Drive.
`Research Triangle Park. NC 27709.
`’To whom requests for reprints should be addressed. at Department of Cancer
`Biology. Glaxowcllcomc. S Moore Drive. Research Triangle Park, NC 27709. Phone:
`(919) 315-4179: Fax: (919) 315-332].
`thymidylate synthase: MTX. methotrcxate:
`‘ The abbreviations used are: TS.
`DDATHF. 5.10-didcazatetrahydrofolate. lomotrexol; diglutamate of l843U89. I843U89
`with one additional glutamate; PEG-TPase. polyethylene glycol-thymidine phosphoryl-
`ase; RTCB. residual tumor cell burden: MTD. maximum tolerated dose.
`
`l843U89 was synthesized at the Wellcome Research Labora-
`Chemicak.
`tories as described (2). it was solubilized with approximately 2 molar equiv-
`alents of NaOH in endotoxin-tested Dulbecco's PBS without calcium or
`magnesium (Sigma Chemical Co.. St. Louis, MO). adjusted to pH 6.8-7.2. and
`was sterile filtered prior to use. Folic acid (Sigma Chemical Co.) for oral
`administration was dissolved in water by adjusting the pH to 7.2 to 7.4 with
`NaOH.
`Cell Culture. Human tumor cell lines were obtained from the following
`sources: breast adenocarcinoma MCF7.
`ileocecal adcnocarcinoma HCT-8.
`ovarian carcinomas A2780. OVCAR-3 and Sl(~OV-3 and osteosarcoma 143 B
`TK' (American Type Culture Collection, Rockville, MD); colon carcinoma
`GC3TK‘ (J. Houghton. St. Jude Children's Research Hospital. Memphis. TN):
`ileocecal adenocarcinoma HCT-8/TK‘ (Y. Rustum. Roswell Park Cancer
`Institute. Buffalo. NY); and human epidermoid carcinoma KB3—l
`(Dr.
`Michael Gottesman. National Cancer institute, Bethesda, MD). Cells were
`propagated as monolayer cultures as described (3). The basal medium for all
`cell lines was folate-free RPMI 1640 (GIBCO-BRL) supplemented with 10 nM
`calcium leucovorin and l0% dialyzed FCS [except A2780 and OVCAR-3.
`which contained l0% heat-inactivated (30 min at 56°C) FCS that had been
`depleted of thymidine by an incubation for 15 min at 37°C with l unit/ml
`Escherichia cali thymidine phosphorylase]. MCF7. A2780. and OVCAR-3
`media contained 10 ttg/ml insulin (Sigma l-I882): the plates for these three
`cell lines were coated with l0 ug/ml PepTitc-2000 (Tclios Pharmaceutical
`Research Products) for l h to aid cell attachment.
`6ll7
`
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`
`FOLIC ACID INTESTINAL PRO'l'E(TlON FOR l843U89
`
`In Virro Protection Studies. Cell growth inhibition and reversal experi-
`ments were done as described (3). The ability of folic acid or leucovorin to
`block the cytotoxicity of l843U89 in several human tumor cell lines was also
`assessed by a clonogenic assay. Log phase cells were allowed to attach
`overnight to 60-mm plates before a 24-h exposure to l843U89 t folic acid or
`leucovorin (0.0l—l(X) um). After the 24-h drug exposure. drug was removed.
`the plates were washed with 3 ml of sterile Dulbecco‘s PBS without calcium
`or magnesium. and 3 ml of fresh media containing the appropriate levels of
`folic acid or leucovorin (but not
`l843U89) were added. The plates were
`incubated I0-I4 days at 37°C in 95% air/5% CO2. the media was removed.
`and the plates were stained with a solution of IO mglml crystal violet in l0%
`fonnaldehyde. 5% acetic acid, and 60% methanol (5-min stain, water rinse,
`and air dry). Colonies of >0.1 mm were counted on an image analyzer (Artck
`Systems Corp.: Model 9823). All determinations were nm in triplicate. The
`inoculum density was adjusted to obtain 200-300 colonies on the control
`plates. and the l843U89 concentration was chosen from a previous titration to
`yield -80% inhibition of colony formation.
`Animals. Male CD-I and female BALB/c athymic mice mice were pur-
`chased from Charles River Laboratories (Wilmington. DE) and used for all
`studies. Mice were housed in polycarbonate. filter-capped micro-isolator cages
`and given sterilized food and water ad Iibirum. Female purebred beagles at
`least l0 months of age were purchased from Marshall Farms. USA. Inc. (North
`Rose. NY) and used for all studies. Beagles were housed individually in cages
`and given Prolab Canine I600 Certified Diet (Agway. Inc.) and municipal tap
`water ad Iibitum. All animals were maintained in a temperature (22°C t 1°C)-.
`humidity (50 : 10%)-. and photoperiod (12-h light/12-h dark)-controlled room
`with 10 to l5 air changes/h.
`l843U89 was administered i.v. by slow bolus
`Dog Protection Studies.
`infusion via the cephalic vein once daily for five consecutive days. From a 20
`mg/ml solution of l843U89. 0.3, 0.6 and 0.9 ml/kg were dosed to obtain 6. 12.
`and 18 mg/kg, respectively. Oral folic acid was given 30 to 40 min prior to
`l843U89. To facilitate rapid ingestion of the entire dose. folic acid was given
`by gavage by #l2 gelatin capsules as a 50 mg,/ml solution. Animals were
`monitored during the dosing period and for 30 days after the dose.
`Mouse Protection Studios. The ability of folic acid to block weight loss
`and death was detennined in mice given l843U89. The high plasma thyrnidine
`levels in mice. which prevent the toxicity of thymidylate synthase inhibitors.
`was reduced by treating the mice with a conjugate of PEG-'l'Pase. PEG-TPase
`(Wellcome Research Laboratories, Research Triangle Park. NC) was admin-
`istered at 2500 units/kg, i.p.. on days I and 4. 1.5 h prior to l843U89, which
`was dosed at 200 and 400 mg/kg. i.p.. bid X 7 days. Folic acid was dosed p.o.
`at 300 mg/kg 30 min prior to each l843U89 dose. in all cases. five mice were
`used per group. The mice were maintained as described above and monitored
`for weight change and death.
`Subrenal Capsule Tumor Growth Assay. Prior to their use in viva. all
`cell
`lines were verified to be free of Mycaplasma bacteria and adventitial
`murine viral pathogens by mouse antibody production test (Charles River
`Biotechnical Laboratories). Cell preparation. implantation. and evaluation pro-
`cedures of cell
`lines and tumors in in vivo studies have been described
`elsewhere (3. 26). Briefly. cells were implanted as fragments under the renal
`capsule of l8—22 g male CD-l athymic mice by published procedures (26).
`
`Length and width of implanted fragments were measured at the time of surgery
`and again at autopsy. l843U89 was administered to mice by the i.p. route in
`volumes of either 10 or 20 ml/kg. At each dose interval. mice were weighed.
`and doses were adjusted according to individual body weight to the nearest
`gram. In all cases. five mice were used per group. Antitumor activity was
`assessed in three ways. Differences between various treatments were deter-
`mined by Kruskal-Wallis analysis (SAS. Cary. NC) of relative tumor volumes
`of individual mice by group; values of P < 0.05 were used to indicate statistical
`significance. The percentage of growth inhibition was calculated fmm group
`median doublings of treated and control tumors. treatment initiation to sacri-
`fice. Histological evaluation of tumors in early studies indicated that l843U89
`might effect lysis of tumor cells with varying degrees of replacement with scar
`tissue. Accordingly. the RTCB of each lesion was also assessed and scored as
`follows by an individual unfamiliar with the particular study: histological
`score = 0. no effect of therapy evident from histological evaluation of lesion
`(RTCB > 60%); histological score = l. equivocal to minimal effect (RTCB 40
`to 60%); histological score = 2. moderate to strong effect (RTCB 5% to 40%):
`and histological score = 3. strong effect (RTCB < 5%).
`
`RESULTS
`
`In vitro Effects of Folic Acid or Leucovorin on 1843U89. Based
`upon three—way mutual competition for transport among l843U89.
`leucovorin. and MTX but not
`folic acid, we have reported that
`l843U89 appears to enter MOLT-4 human T—cell leukemia on the
`reduced folate carrier (3). We have now also observed similar mutual
`transport competition among l843U89, MTX. and leucovorin but not
`folic acid in MCF7 breast adenocarcinoma and SW480 and WiDr
`
`colon carcinoma cell lines, which suggests broader tumor use of this
`transporter for l843U89 (data not shown). Since folic acid does not
`compete for this l843U89 tumor cell transport and the products of
`cellular metabolism of folic acid. the reduced folates. do not compete
`for l843U89 binding to T8 (1). folic acid should not effectively
`reverse l843U89 cytotoxicity in tumor cells. Table 1 shows the ability
`of folic acid and leucovorin to reverse the inhibition of clonogenic
`growth by l843U89 in eight separate human tumor lines. HCT-8 was
`the most sensitive line to folic acid reversal, requiring 28 um for 50%
`reversal of l843U89 activity; all others required over 100 |.LM for 50%
`reversal. in contrast to folic acid, leucovorin. which does compete for
`l843U89 transport in the p.M range. reversed l843U89 cytotoxicity
`more efficiently (Table 1). To further investigate this resistance of
`l843U89 to folic acid or leucovorin reversal. reversal of l843U89 and
`another TS inhibitor. Tomudex. by the two agents were compared in
`three cell lines (Table 2). Since Tomudex has been reported to be
`efficiently reversed by leucovorin (3). we expected Tomudex activity
`to be more sensitive to both folic acid and leucovorin. As can be seen
`
`in Table 2. both 100 and 10 um leucovorin very efficiently reversed
`the activity of Tomudex but only weakly reversed the activity of
`
`Table l Reversal of l843U89 cytotoxicity by folic acid or leucovorin in cell: in culture
`Concentration required for 50%
`reversal of IB43U89 cytotoxicity
`(rm)
`
`% reversal of l843U89 cytotoxicity with l or 10 pm
`reversal agent“
`
`Folic acid
`
`Cell line
`SK~OV-3
`A2780
`OVCAR-3
`
`HCT-8
`
`Tm
`Ovarian
`Ovarian
`Ovarian
`
`lleocecal
`
`>100
`>100
`
`28
`
`0.6
`5
`I0
`
`0.07
`
`KB3~l
`Epidermoid
`Osteosarcoma
`143B TK-
`Colon
`OC3/1'K"
`Breast
`MCF7
`" Reversal of 80% inhibition by the drug; thus. 50% reversal leads to 40% inhibition remaining. Partial (80%) inhibition was chosen to be able to detect small reversal effects.
`61 18
`
`I2
`2|
`
`53
`25
`
`>l00
`>100
`> I (0
`>l(l)
`
`3.7
`5.0
`8
`32
`
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`
`FOLIC ACID INTESTINAL PROTECTION FDR l843U89
`
`Table 2 Eflea offalic acid or leucovorin on cell growth inhibition by Tomudex and l843U89
`Reversal agent
`I0 um leucovorin
`Fold
`increase
`
`l00 p.M leucovorin
`Fold
`increase
`
`lC50
`um)
`
`tc,.,
`(nu)
`
`SW480
`GC3TK'
`HCT-8
`
`SW480
`GC3TK'
`HCT-8
`" From Duch er al. (3).
`
`0.4
`2.l
`I .4
`
`I
`0.34
`0.82
`
`>55.000
`> I .0(X)
`
`30
`
`>l l0.0tl)"
`>5(X)
`
`I 0.000
`I .000
`
`30"
`34
`
`25.(X)0°
`500
`
`l"
`
`The ability of folic acid to protect against the lethal toxicity of
`l843U89 was tested in beagle dogs given a lethal dose of drug with
`or without prior treatment with oral folic acid. On a 5-day dosing
`schedule. 6 mg/kg/day l843U89 i.v. was a 100% lethal dose in dogs
`(Table 3). By contrast, when 50 mg/kg oral folic acid was given 30
`ntin prior to the i.v. doses of l843U89 (Table 4), all of the animals
`survived both this dose and twice this dose (6 and 12 mg/kg/day
`l843U89. respectively). However, even with the oral 50 mg/kg folic
`acid doses, five daily doses of 18 mg/kg/day l843U89 i.v. were fatal
`to all dogs treated. importantly, dose-limiting toxicity at 18 mg/kg
`l843U89 with 50 mglkg folic acid was intestinal and comparable to
`that seen with 6 mg/kg l843U89 alone. Thus. oral administration of
`folic acid protects against the lethal toxicity of i.v. l843U89. thereby
`raising the MTD for the drug more than 2-fold and potentially greater
`than 6-fold [MTD for l843U89 alone is between 2 and 6 mg/kg
`(Table 3); MTD for l843U89 plus folic acid is between 12 and l8
`mg/kg].
`In other experiments (data not shown). it was found that five daily
`doses of up to 100, 200. or 500 mg/kg/day of folic acid were well
`tolerated and effective in protection against a 10 mg/kg/day dose of
`l843U89. The minimal dose of folic acid to provide protection to all
`dogs was between 10 and 50 mg/kg/day since a dose of IO mg/kg/day
`of folic acid provided protection to three of four dogs receiving the i0
`mg/kg/day l843U89. However. the use of multiple daily doses of folic
`acid did pennit the reliable use of lower individual doses; thus. 16.7
`or I0 mg/kg folic acid three times daily (30 min before and 3 and 6
`h after the l843U89 dose) each protected four of four dogs receiving
`10 mglkyday l843U89.
`Leucovorin at 10 to 200 mglkg/day could replace folic acid and
`protect all dogs receiving 10 mg/ltglday l843U89. As will be shown
`below. leucovorin more effectively reversed l843U89 antitumor ef-
`ficacy and was, therefore, not pursued further.
`Effect of Oral Folic Acid on Weight Loss and Death of Mice
`Administered l843U89. In contrast to human cells. l843U89 is not
`efficiently transported into mouse cells (3). in addition, high circulat-
`ing thymidine levels in rrtice decrease the efficacy and toxicity of all
`TS inhibitors in mice (3. 4. 27. 28). As such. l843U89 has very little
`toxicity in mice (data not shown; see also Refs. 3 and 4). Nonetheless.
`if mice are first depleted of thyntidine by prior administration of a
`polyethylene glycol conjugate of thymidine phosphorylase (PEG-
`TPase). high doses of l843U89 can produce weight loss and death
`(Fig. 1). This effect is presumably due to depletion. by PEG-TPase. of
`circulating thymidine available for salvage since circulating t.ltyn1i-
`dine levels drop from 0.7-1.5 rm to <0.03 p.M.‘ The toxicity produced
`by this regimen appears to be gut toxicity, as shown by bloody
`diarrhea and histopathology (data not shown). To test whether folic
`
`l843U89 in either GC3'l‘K‘ or SW480 cells. Furthermore. 25 p.M
`folic acid increased the IC,o of Tomudex approximately 20-fold in all
`three of SW480. GC3TK', and HCT-8 cells. while these conditions
`only increased the l843U89 IC,o 1- to 3-fold. Thus. the efficacy of
`l843U89 in cell culture is less sensitive than Tomudex to reversal by
`either leucovorin or folic acid. and folic acid is the less effective
`reversing agent.
`Effect of Oral Folic Acid on Toxicity of l843U89 in the Beagle
`0 Dog. As shown in Table 3. the MTD for i.v.
`l843U89 on a daily
`5-day schedule in the beagle dog was between 2 and 6 mg/kg/day; at
`2 mg/kg. toxicity was mild. but 6 mg/kg was letltal. Gastrointestinal
`toxicity. including severe diarrhea and maturation arrest enteritis. was
`dose limiting. Hematological toxicity was observed but was consis-
`tently mild and never dose limiting (data not shown). Thus, a mech-
`anism to decrease the gastrointestinal
`toxicity should result
`in an
`increased MTD for the drug.
`
`Table 3 Toxicity afflvz daily r‘. v. doses of I843U89 in beagle dogs‘
`l843U89 dose
`(ms/ks/day)
`
`M 7
`
`Yes
`
`No
`
`No
`
`Yes
`
`No
`
`Clinical signs: hypothermia. labored breathing.
`tetching. righting reflex slow or absent. pale
`gingiva, prostration. salivation, and
`Iacrimation
`
`Diarrhea. emesis. andlor decreased activity.
`and/or dehydration
`
`Body weight decrease
`
`Food consumption decrease
`
`Gross pathology: dark red discoloration of the
`lining and contents of intestinal tract. andlor
`severe. reddened mucosal lining or brown.
`watery contents in stomach; and/or
`longitudinal dark red streaking of the colon.
`and/or severe thymic hemorrhage; andlor
`minimal to severe injection site hemorrhage
`
`Hislopathology: maturation arrest enteritis of
`intestinal tract: severe thymus atrophy.
`congestion; and/or bile stasis in liver;
`minimal to severe involution of the white
`pulp of spleen: mild to moderate involution
`of cervical and mesenteric lymph nodes
`
`Maturation arrest of myeloid elements in bone
`IHZKTOW
`
`" Four dogs were treated at each dose level of l843U89. two males and two females.
`" M. male; F. female.
`" Deaths.
`
`‘ R. Ferone. unpublished data.
`
`6119
`
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`
`FOUC ACID INTESTINAL PROTEX.'T1ON FOR l843U89
`
`Table 4 Eflecl of 50 mg/kg oral folic acid prerrealntenr on Ihe toxicity of 5-day daily r'.v. I843U89 in beagle dogs“
`l843UB9 dose (mg/kg/day)
`Folic Acid dose (mg/lrglday)
`
`Severe clinical signs: altered stool with blood. frequent emesis. decreased activity. cool to the
`touch. blood and/or stains amund anogenital area. salivation. labored breathing. body uemors.
`and nystagmus
`
`Mild clinical signs: occasional altered stool without blood. occasional emesis. and salivation
`
`Body weight decrease
`
`Food consumption decrease
`
`Histopathology: mild. minimal. or very minimal maturation arrest enteritis of the cecum. colon.
`andlor ileum
`
`Yes
`
`Yes
`
`Yes
`
`Yes
`
`Yes
`
`Yes
`
`Yes
`Yes
`
`Yes
`
`Yes
`
`Yes
`
`No
`
`No
`
`Gross pathological changes noted in the 6- and I2-mg/kg/day group: red streaking of the colon. approximately 5 mm raised reddened areas at the junction of the colon with the
`ileum.
`
`Gross pathological changes noted in the I8-mykglday group only: dark red discoloration and congestion of the mucosa of the duodenum. jejunum. ileum. cecum. and colon.
`minimal white streaking of the renal cortex.
`
`Histopathological changes noted in the I8-mykg/day group only: moderate to severe maturation anest enteritis of the duodenum. jejunum. ileum. cecum. and colon. Maturation
`arrest was noted in the bone marrow smars from two of the three dogs humanely sacrificed. Kidney congestion. minimal to mild dilated tubules. casts. very minimal focal
`tubular epithelial necrosis. and very minimal tubular epithelial regeneration.
`
`Note: Effects in all parameters were reversed or reversing at the end of the postdose recovery period in all surviving animals.
`" Four dogs were treated at each dose level of l843U89. two males and two females.
`{’ Folic acid dose (myltg/day).
`‘ M. male: F. female.
`" Deaths.
`
`acid can protect in this model of l843U89 toxicity. drug was admin-
`istered to TPase-pretreated mice via i.p. injection twice daily for 7
`days with or without oral 300 mg/kg folic acid 30 min prior to
`l843U89. The data in Fig.
`I show that folic acid prevented the 20%
`weight
`loss caused by 200 mg/kg l843U89. A higher dose of
`l843U89. 400 mg/kg. was lethal to all mice by day 10 in the absence
`of folic acid, but in the presence of the protectant. 80% of the animals
`survived (Fig. 2). Thus. folic acid can protect against
`the lethal
`toxicity of l843U89 in mice as well as dogs.
`Effect of Folic Acid on the Antitumor Efficacy of 1843U89. To
`determine the effect of folic acid or leucovorin on the in vivo antim-
`mor efficacy of l843U89. three human tumor lines were grown under
`the renal capsule of mice and treated with l843U89 with or without
`prior oral doses of folic acid or leucovorin. These tumor lines
`(GC3Tl(" colon carcinoma. HCT-8/TK‘ ileocecal adenocarcinoma.
`and 1438 TK‘ osteosarcoma) all lack thymidine kinase. preventing
`thymidine salvage.
`GC3Tl(" is the most sensitive of the three cell lines to l843U89.
`Table 5 shows that 3 to I0 mg/kg of l843U89 twice daily for 5 days
`greatly inhibited tumor growth and led to cell kill and regression of
`GC3'I‘K' (all measured on day 10. experiments 1 through 4). In this
`table. the term histological score (measured as described in “Materials
`and Methods") is introduced and used as a measure of the extent of
`cell kill caused by drug; values for histological score of 2 to 3 indicate
`extensive tumor cell kill. As can be seen in Table 5. most treatment
`schedules produced histological scores of 2 to 3. indicating a substan-
`tial antitumor effect. The effect of folic acid upon therapy of GC3Tl(“
`by l843U89 is also shown in Table 5. Addition of 50 to 500 mg/kg
`Fig. I. Effect of oral folic acid on the weight loss of mice given 200 mg/kg l843U89.
`BALBIC female mice were dosed. i.p.. on days I and 4 with 25(1) units of PEG-TPase/ltg.
`folic acid p.o. 30 min prior to l843U89 did not change either growth
`On days 1 through 7. the mice were dosed. i.p.. with 200 mg l843U89Iltg. twice daily. 55
`inhibition or tumor cell kill (Table 5. experiments 1 through 4). Since
`h apart alone (I) or along with oral predoses of folic acid (D). For animals receiving the
`in vitro leucovorin reversed l843U89 activity in GC3TK' only
`oral folic acid protection. 300 mg folic acid/kg were dosed 0.75 to 0.5 h prior to all
`l843U89 doses.
`poorly. although more efficiently than folic acid. leucovorin was also
`6l20
`
`
`
`AverageWeight(9)
`
`Dav
`
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`Copyright © 1995 American Association for Cancer Research
`
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`
`

`
`FOUC ACID INTESTINAL PRCYTKTION FOR 1843089
`
`tested on the activity of l843U89 in this tumor. The addition of oral
`200 mg/kg leucovorin had little effect (Table 5. experiments 2 and 3).
`Since multiple doses of folic acid were also protective in the dog,
`we investigated the effect of multiple daily doses of folic acid on
`therapy in mice. Four separate conditions were tested: (a) l843U89
`alone once daily for 5 days; (b) folic acid 30 min prior to the l843U89
`dose; (c) folic acid 30 min prior to and 3 h after the 1843U89 dose;
`and (d) folic acid 30 min prior to and 3 and 6 h after the 1843U89
`dose. The results are displayed in Table 5, experiment 5. In this
`experiment. 50 mg/kg of 1843U89 alone once daily for 5 days
`inhibited tumor growth and led to cell kill and regression of GC3‘1‘1('.
`Administration of the single or multiple daily oral doses of 366 mg/kg
`folic acid did not change this antitumor efficacy. although multiple
`doses may have produced some decrease in cell kill. as measured by
`the histological score. For this experiment, the effect of the folic acid
`administrations upon tumor outgrowth to day 21 was also measured.
`This is shown in Fig. 3. No significant outgrowth was observed in the
`1843U89 treated tumors, while control tumors increased their volume
`14-fold. Folic acid once or twice daily had no effect on the antitumor
`activity of 1843U89. Thus, folic acid produced no measurable reversal
`of 1843U89 efficacy in GC3TK'.
`This effect of folic acid on 1843U89 therapy of GC3'I'K‘ was
`compared with the effect on therapy with two other anticancer agents
`under clinical development. DDATHF and Tomudex (12. 19). Since
`both of these agents are reversed by folates in cell culture more
`efficiently than is l843U89. we wished to determine whether a similar
`reversal would be observed in vivo. The results of this experiment are
`shown in Fig. 4. Both Tomudex and DDAT1-{F inhibited the growth of
`GC3'l‘K'. but upon addition of folic acid. a modest reversal of the
`efficacy of each drug was observed. As before, no such reversal was
`seen for 18425089. The effect of folic acid upon the action of these
`three agents was further substantiated with measurement of tumor cell
`kill, as determined by the histological score. Folic acid had no effect
`on cell kill by l843U89 but decreased cell kill by both DDATHF and
`
`3
`
`Dav
`
`Fig. 2. Effect of oral folic acid on the lethality of 400 mg/kg l843U89 in mice.
`BALB/C female mice were dosed, i.p.. on days 1 and 4 with 25(X) units of PEG-TPaseIkg.
`On days I through 7. the mice were dosed. i.p.. with 400 mg l843U89/kg. Mice daily. 55
`h apart alone (I) or along with oral predoses of folic acid (El). For animals receiving the
`oral folic acid protection. 300 mg folic acid/lrg were dosed 0.75 to 0.5 h prior to all
`l843U89 doses.
`
`Table 5 Eject offolic acid or leucovorin on the anlinanor activity of 18-43089 against GC3TK' colon nunor.r'
`1B43U89 dose
`
`Experiment no.
`I
`
`Dose
`mg/ltg
`10
`10
`3.2
`3.2
`
`10.0
`|0.0
`10.0
`3.2
`3.2
`3.2
`
`40.0
`40.0
`12.7
`12.7
`4.0
`4.0
`
`10.0
`10.0
`
`50.0
`50.0
`$0.0
`50.0
`
`Schedule
`bid X 5
`bid X 5
`bidx 5
`bid X 5
`
`bid X 5
`bid X 5
`bid X 5
`bid X 5
`bid X 5
`bid X 5
`
`bid X 5
`bid X 5
`bid X 5
`bid X 5
`bid x 5
`bid X 5
`
`bid X 5
`bid X 5
`
`qd X 5
`qt! x 5
`qd X 5
`qd x 5
`
`FA
`FA
`FA
`
`:Therapy was from days 3 to 7 post implant. and tumors were measured on day 10.
`FA, folic acid; LV. leuoovorin; N/A, not applicable.
`‘ 1nhib.. inhibition. Tumors with greater than 100% growth inhibition regressed upon therapy.
`" Histol.. histology. Score - 0. no eflect of therapy evident from histological evaluation of lesion; score - 1, equivocal to minimal efiect; score = 2. modem: to strong effect;
`score = 3. strong effect.
`6121
`
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`
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`
`

`
`FOUC ACID INTESTINAL PROTECTION FOR l843U89
`
`in an outgrowth assay. In this experiment, 50 mg/kg l843U89 alone
`completely inhibited growth measured on day l0. and little outgrowth
`occurred over the 21 days. With the addition of at twice daily oral folic
`acid dose of 366 mg/kg. a substantial delay in outgrowth was still
`apparent on day 10: however. significant reversal was apparent by
`day 21.
`
`DISCUSSION
`
`It is well accepted that a variety of classical folate-based antitumor
`agents in use or currently under development are reversed by folates
`such as folic acid or the reduced folates (5-for-myltetrahydrofolate,
`leucovorin. is the reduced folate commonly used; Refs. 9. l2. l9, and
`20). These folates are often used to “rescue" a patient from severe
`toxicity after therapy with a large dose of MTX (9). However. since
`there is little selectivity between cancer and normal cells with the
`drugs. both tumor cells and normal cells are rescued from the action
`of the antifolate.
`The effect of both folic acid and leucovorin in the rescue of cancer
`cells front these classical antifolates is clearly shown with the glyci-
`namide ribonucleotide transformylase inhibitors DDATHF and 5—dea-
`zaacyclotetrahydrofolatc and the TS inhibitor Tomudex (Table 2;
`Refs. 12. 19, 20, 29. and 30). These compounds and/or their active
`forms, the polyglutamated compounds. are all competitive or mixed
`inhibitors (with respect to the folate substrate) of their respective
`target enzymes (12, 19. 20). Therefore. folic acid and leucovorin.
`which are efficiently metabolized to the reduced