Cobalamin and Survival of Tumor-Bearing Mice
`
`Pathobiology 1993:6 1: I 04-1 08
`
`Constance S. Tsao
`Koichi Myashita
`
`Linus Pauling Institute of Science and
`Medicine, Palo Alto. Calif .. USA
`
`Influence of Cobalamin
`on the Survival of Mice
`Bearing Ascites Tumor
`
`KeyWords
`Cobalamin
`Ascites tumor
`Mice survival
`
`Abstract
`The effect of cobalamin (vitami n B12) on the survival time of mice bearing
`P 388 leukemia has been examined. Among the three cobalamins studied, the
`enzymatically active derivatives, methylcobalamin and 5'-deoxyadenosylco(cid:173)
`balamin, were able to significantly increase the survival time of mice
`implanted intraperitoneally with the tumor cells. The pharmaceutical form,
`cyanocobalamin, was not active. The antitumor activity of these cobalamins
`may be associated with their functions in metabolism.
`
`Evidence has accumulated in the recent past that v.ita(cid:173)
`min B12 is associated wi th tumorigenesis. Patients with
`many types of malignant neoplasia and leukemia had ele(cid:173)
`vated serum levels of vitamin B12 [I , 2] and vitamin B12-
`specific binding proteins [3-5]. Certain cancer-bearing
`mice synthesized vitamin B12 and stored more vitamin
`B12 than normal controls did [I , 6]; but severaJ sponta(cid:173)
`neous mammary tumors were shown to destroy this vita(cid:173)
`min [6]. Studies with mouse spleen cells have shown that
`the addition of methylcobalamin to culture medium en(cid:173)
`hanced the production of antibody and suppressor T cells
`[7].
`· Vitamin B12 had been used with apparent benefit in
`the treatment of young children with neuroblastoma [8.
`9], but the results of two survey studies using data from
`several. hospitals failed to confirm that vitamin B12 ther(cid:173)
`apy was effective either when it was administered alone or
`in conjunction with X-ray or chemotherapeutic agents
`[10, 11].
`Experiments with laboratory animals also showed con(cid:173)
`flicting results. The administration of vitamin B12 inhib-
`
`ited the growth of ce1tain tumors implanted in mice [ 12,
`13]. Mice treated with vitamin B12 survived longer than
`did untreated controls. Vitamin 8 12 also inhibited the
`growth of liver tumors induced by p-dimethylaminoazo(cid:173)
`benzene in rats (14) . A mixture of vitamin B12 and vita(cid:173)
`min C was able to inhibit the growth of certain mouse
`ascites or solid tumors and to increase the survival rate of
`tumor bearing mice [ 15- 17). However, in another study
`with Fischer CDF rats, a mixture of vitam ins B12 and C
`had no effects on the growth of~ gliosarcoma, and no
`differences in survival time between treatment and con(cid:173)
`trol group were observed [ 18]. Furthermore, in some
`cases, vitamin B12 enhanced the growth of fibrosarcoma
`in rats and of Rous sarcoma in chickens [ 19]. Vitamin B 12
`was also reported to be procarcinogenic in rats and ham(cid:173)
`sters [20. 21]. In addition. a deficiency of vitamin B12
`decreased the potency of certain carcinogens in rats [22,
`23].
`A recent in vitro study with cultured cells indicated
`that cobalamins were able to inhibit the growth of several
`malignant cell lines [24). In these experiments. the meta-
`
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`te 1993 S. Karger AG. llascl
`I 015-2008193/0612-0 I 04
`$2. 75/()
`
`Rccetvcd:
`August 1.1. 199 1
`Accepted.
`May 11. 19'12
`
`C.S. rsao. PhD
`l.ious Puuling lostitutc
`ofSct~ncc and Medicine
`4~0 Page Mill Road
`Palo Alto. CA 94306 (USA)
`
`Teva – Fresenius
`Exhibit 1007-00001
`
`

`
`bolically active forms, methylcobalamin and 5'-deoxyad(cid:173)
`enosylcobalamin, were found to be effective but the meta(cid:173)
`bolically inactive cyanocobalamin had practically no ef(cid:173)
`fect [24]. In the present study, in vivo experiments were
`carried out to examine the effects of three different forms
`of cobalamin on the survival of mice bearing ascites
`tumors.
`
`tion, the ascites cells were harvested and washed. Then, a homoge(cid:173)
`neous representation of the cells was stained with erythrosin B. The
`numbers of viable cells and dead cells were counted wi th a hemocy(cid:173)
`tometer. The value of ED90 was calculated by comparing viable cell
`numbers in treated and control animals. The survival inocculum
`curves from tJ1e studies of survival time described above were also
`used for the calculation of ED9o. A good agreement was obtained
`between the two methods of calculation.
`
`Methods and M aterials
`
`Surviral Time
`Drug test was perfom1ed using a murine model. under the aus(cid:173)
`pices of the NCI Division of Cancer Treatment. for screening chemi(cid:173)
`cal agents and natural products against animal tumors. model3PS31.
`In Vivo Cancer Models. US Department of Health and Human Ser·
`vices. National Institutes of Health Publication o. 84-2635 (1984).
`Female DBA and CDF1 mice ( 18-20 g) were purchased from
`Simonsen Laboratories, Inc. (Gilroy, Calif.). They were fed a non pu(cid:173)
`rified diet (Purina Certified Rodent Diet, No. 500 I. Ralston Purina
`Co., St. Louis, Mo.) throughout the experiment. Feed and water were
`offered ad libitum. Mouse lymphoid neoplasm cells P388 were prop(cid:173)
`agated in DBA mice. The ascites cells were harvested on the 7th day
`after tumor implantation. The CDF1 mice were injected intraperito(cid:173)
`neally with I Q6 washed cells. suspended in 0.1 ml phosphate-buffered
`saline. Mice were then randomized into various test groups of I 0
`animals each. Starting 24 h after implantation, test mice were
`injected intraperitoneally with various doses of vitamin B11 in 0.2 ml
`of saline solution daily for I 0 days. The control animals received an
`equal volume of saline. The mice were weighed twice a week and
`were killed 30 days after tumor implantation and the result evaluat(cid:173)
`ed. When 50% or more of the animals in a test group survived to the
`30-day time period after tumor implantation, the experiment was
`extended to a total of 60 days. The number of days that each mouse
`lived after the transplantation of tumor cells was recorded as the sur(cid:173)
`vival time.
`
`Swlislical Calcula1ions
`The Student's two tailed t test was used to detenninc statistical
`differences between the control and experimental groups. The me(cid:173)
`dian survival time of each group was also used as an inde~ of compar(cid:173)
`ison of the test animals to their corresponding controls. The median
`survival time of the untreated control groups of these experiments
`was 16 ± 4 days. A test-to-control ratio of survival time of 1.3 for a
`test agent was considered to demonstrate activity, whereas a ratio of
`I. 75 or greater was considered significant activity.
`
`Ca/culariont~(Titerapelllh' Index
`The therapeutic index. referring to the dose ratio between toxic
`and therapeutic effect, is expressed as the ratio LD5o!ED9o. where
`LD5o is the dosage lethal to 50% oft he untreated animals and ED<>o is
`the dosage that give a 90% cell kill in the experimental mice [25].
`Values of ED9u were measured using CDF1 mice. The mice were
`injected intraperitoneally with 106 cells, suspended in 0.1 ml phos(cid:173)
`phate-buffered saline. The mice were then randomized into various
`groups of I 0 animals each. Starting 24 h after implantation. test mice
`were injected intraperitoneally with various doses or the test agents
`in 0.2 ml of saline solution. On the tentl1 day after tumor implanta-
`
`Chemicals
`Cyano-, methyl- and 5'-deoxyadenosyl cobalamin were pur(cid:173)
`chased from Sigma Chemical Co .. St. Louis, Mo. The cobalamin
`derivatives were prepared by introducing the dry crystals into small
`sterile tubes. A sufficient number of tubes were prepared and stored
`at -20 o C. T m mediately before injection, sterile saline solution was
`added to the tubes to dissolve the compounds.
`
`Results and Discussion
`
`We have tested the antitumor activity of three cobal(cid:173)
`amin derivatives that were available to us, at three differ(cid:173)
`ent dosages: 25.50 and 100 mg/kg body weight/injection.
`The mice were able to tolerate the injection of cobalamin
`at a daily dose of 100 mg!kg body weight without any
`apparent adverse effect. Table I shows the survival times
`of test and control mice bearing P388 leukemia after
`treatment with cobalamins. The data indicate a statisti(cid:173)
`call y significant increase in the survival of mice treated
`with methylcobalamin or adenosylcobalamin when com(cid:173)
`pared with the controls. Adenosylcobalamin was appar(cid:173)
`ently more effective than methylcobalamin. However.
`cyanocobalamin has practically no effect on tumor
`growth. These observations were in good agreement with
`results in previous experiments in which in vitro cell cul(cid:173)
`ture technique was used [24]. The ratios of med.ian sur(cid:173)
`vival time of a test group to median survival time of con(cid:173)
`trol for these mice are shown in parentheses in table I.
`These ratios are in accordance with tile corresponding
`p values.
`The therapeutic index is the ratio between toxic and
`therapeutic effect. or the ratio of LD50 and ED9o [25]. The
`values of ED9o for methyl- and 5'-deoxyadenosylcobal(cid:173)
`amin were 120 and 100 mg!kg body weight. respectively,
`whereas cyanocobalamin did not affect cell growth at a
`daily dose as high as I ,000 mg!kg body weight. 1 t has been
`shown that the addition of vitamin B11 to food in
`amounts far in excess of need or absorbability appears to
`be without hazard. Cyanocobalamin has caused no toxic(cid:173)
`ity in ani mats at levels several thousand times their nutri(cid:173)
`tional requirements [29]. Toxicity tests in our laboratory
`indicated that these three cobalimin derivatives were
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`105
`
`Teva – Fresenius
`Exhibit 1007-00002
`
`

`
`Table 1. Effect of cobalamin derivatives
`on the survival of mice bearing ascites
`tumor
`
`Test agent
`
`Dosage, mglkg body weight/day
`so
`
`25
`
`0
`
`Cyanocobalamin
`
`17.8 :t 2.9
`
`19.6 :t 3.5
`
`12.6 :t 7.5 L9.3 :t 4.2
`(0. 97)
`( 1.03)
`
`17.1 :t 3.5 20.S :t 2.7
`(0.94)
`(I. I 7)
`
`18.1 :t 3.6 17.9 :t 3.4
`(0.97)
`
`18.3 :t 4.5
`( 1.00)
`
`Therapeutic
`Index
`
`100
`
`18.7± 1.5
`( 1.06)
`
`20.5 :t 1.4
`(I. I 7)
`
`18.5±4.2
`( 1.03)
`
`Methylcobalamtn
`
`18.0 ± 6.0
`
`17.5 ± 2.8
`( 1.00)
`
`17.3± 0.5
`(1.00)
`
`38.0 ± 1 8.4"
`(2.12)
`
`I 5
`
`17.0 ± 3.2
`
`16.7±0.9
`
`-
`
`-
`
`18.9 ± 0.3
`( 1.06)
`
`17.3±6.1
`(0.91)
`
`22.7 ± 9.7
`( 1.61)
`
`32.7 ± 10.6°
`(2.18)
`
`Adenosylcobalamin
`
`9.4±2.4
`
`9. 1±1.4
`( 1.18)
`
`8.9±2.3
`(I. 18)
`
`29.1 ± 1.9°
`(3.53)
`
`20
`
`I 5.5±0.9 16.7 ± 4.5 21.2± 6.2b
`(I. II )
`( 1.43)
`
`34.3± 10.3C
`(2.86)
`
`15.0±2.1
`
`28.4± 13.7b 40.7±2S.2b
`( 1.33)
`(3.83)
`
`Values are means ± SD of survival times (days) for n = I 0. Significance of the difference
`between control and experimental values: • p < 0.05; b p < 0.01: c p < 0.001. Values in
`parentheses are ratios of median survival time of test group to median survival time of con(cid:173)
`trol group. A value of I. 75 or greater for a test agent was considered to demonstrate signifi(cid:173)
`cant activity.
`Therapeutic index is expressed as the ratio LD5o/ED90, where LD50 is the dose lethal to
`SO% of a population and ED90 is the dose that gives a 90% cell kill.
`
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`remarkably nontoxic when administered intraperitone(cid:173)
`ally. The LD50 for metbylcobalamin was I ,800 mg/kg
`body weight. which was 15 times the effective dose. The
`LD50 for 5'-deoxyadenosylcobalamin was higher than
`that for rnetbylcobalamjn. However, when a dose of2,000
`mg/kg body weight was injected into these mice, they
`started to Jose weight. Thus, the value of 2,000 mg/kg
`body weight was used and the therapeutic index for 5'(cid:173)
`deoxyadenosylcobalamin was 20. Although very large
`doses of cobalamins injected intraperitoneally were non(cid:173)
`toxic. it became lethal when a much smaller dose was
`injected intravenously into the mouse tail vein. This may
`be the effect of the large volume of fluid entering the
`mouse bloodstream during a relatively short period of
`injection time.
`
`Vitamin B12 and folate are involved in the process of
`one-carbon-unit metaboJjsm and methylcobalamin is a
`source of one-carbon functional groups [26, 27]. Although
`the mechanism of the antitumor activity is not known, it
`is quite evident that the roles of vitarrun 8 12 in carcino(cid:173)
`genesis may be associated with its functions in normal
`metabolism. particularly in the one-carbon-unit metabo(cid:173)
`lism and in the positive and negati ve control of DNA syn(cid:173)
`thesis by normal and malignant ceUs [26, 27]. Vitamin 8 12
`is expected to correct defective DNA-thymine synthesis
`in vitamin B1r defective marrow [28], because thymidy(cid:173)
`late synthase requires N>,N 10-methylenetetrahydrofolate
`as methyl donor. For the same reason, in patients with
`folate and vitamin 8 12 deficiency, the addition of these
`vitamins to marrow and lymphocyte cultures enhanced
`the incorporation of3H-deoxyuridine into DNA [29].
`
`106
`
`Tsao/Myashita
`
`Cobalamin and Ascites Tumor
`
`Teva – Fresenius
`Exhibit 1007-00003
`
`

`
`Previous in vitro findings have indicated that the met(cid:173)
`abolically active cobalamins were able to inhibit malig(cid:173)
`nant cell growth, while the metabolically inactive forms
`had practically no effect (24]. Folic acid and vitamin B12
`are intimately related to the synthesis of DNA and RNA;
`lack of either damages DNA synthesis. The primary dam(cid:173)
`age is to de novo DNA synthesis, with the result that there
`may be a secondary increment in salvage DNA synthesis
`[27, 29]. In those tumors in which synthesis of DNA by
`the salvage pathway is relatively greater than in normal
`cells, as compared to the de novo pathway of DNA syn(cid:173)
`thesis, it is theoretically possible that folate and vitamin
`8 12, by enhancing de novo DNA synthesis, could be rela(cid:173)
`tively more helpful to normal than to tumor cells and rela(cid:173)
`tively more harmful to certain tumor cells [27, 29].lt has
`been suggested that these vitamins and their antagonists
`could be involved in the control of normal gene expres(cid:173)
`sion and that deficiency of folate or vitamin B12 or any
`cause of failure to methylate DNA or RNA can activate
`malignancy by hypomethylating oncogenes, leading to
`such gene expression or gene amplication, and that meth(cid:173)
`ylating oncogenes can inhibit malignancy by making them
`dormant. Furthermore. these vitamins can be useful in
`controlling tumors that grow more rapidly as more of
`them arc supplied, because the tumor cells can be stimu(cid:173)
`lated into the DNA synthesis phase in which a number of
`cancer chemotherapy agents exert their deadly effects.
`These agents can be used in a sequence right after folate or
`vitam in 8 12 [27, 30]. Large doses of folic acid and vitamin
`B12 were able to potentiate cytotoxici ty of fluoropyrimid(cid:173)
`i ne by stabilizing the ternary complex and bet ween fluoro(cid:173)
`deoxyuridylate and thymidylate synthase [30]. The find-
`
`ing that adenosylcobalamin was more effective than
`methylcobalamin indicated that the mechanism of cobal(cid:173)
`ami n in carcinogenesis involved not only methylation but
`also other metabolic pathways of vitamin B12 metabo(cid:173)
`lism.
`Most of the cobalamin in animals exists as the two
`coenzymatically active forms, methylcobalamin and ade(cid:173)
`nosylcobalamin (26]. Methylcobalamin constitutes 60-
`80% of the total plasma cobalamin. Adenosylcobalamin
`is the major cobalamin in cellular tissues. The stable phar(cid:173)
`maceutical form, cyanocobalamin, is not nutritionally
`active. Although animals have the biochemical machin(cid:173)
`ery to convert cyanocobalamin and other cobalamins into
`the two metabotically active cobalamins, the difference in
`activity of the three cobalamins indic.ated that the rate of
`the conversion was probably very low and insufficient in
`view of the rather high required dosage of the active
`cobalamins.
`In summary, these studies indicate that methyl- and
`5'-deoxyadenosylcobalamin were able to significantly in(cid:173)
`crease the survival time of mice implanted with the P388
`tumor cells. Because there has been criticism of the use of
`P388 as a tumor system for drug discovery and develop(cid:173)
`ment, other tumor systems are being studied in our labo(cid:173)
`ratory for further evaluation.
`
`Acknowledgements
`
`This work was supponed in pan by the Foundation for utri(cid:173)
`tional Advancement. We thank M. Prender, V. Andrews and D.
`Jiang for technical assistance.
`
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`
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`107
`
`Teva – Fresenius
`Exhibit 1007-00004
`
`

`
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`108
`
`Tsao/Myashita
`
`Cobalamin and Ascites Tumor
`
`Teva – Fresenius
`Exhibit 1007-00005