- Email: [email protected]
Enhancement of melphalan cytotoxicity in vivo and in vitro by inhibitors of poly (ADP-ribose) polymerase
Ini J Radmron Oncologv F’nnted an the U.S.A All
Lltnl n@i
Phvs trxrwd
Vol
IO.
pi
1665-1668 Copyright
0360-3016184 $03.00 + .OO G 1984 Pcrgamon Pxs Ltd.
??Session V
ENHANCEMENT OF MELPHALAN CYTOTOXICITY BY INHIBITORS OF POLY (ADP-RIBOSE) D. M. BROWN, PH.D., M. R. HORSMAN, PH.D., D. G. HIRST, Division of Radiobiology,
Department
IN VZVO AND IN VITRO POLYMERASE PH.D.
AND J. M. BROWN, PH.D.
of Radiology, Stanford University School of Medicine, Stanford, CA 94305
In these preliminary experiments, we have found enhanced cell killing by the bifunctional nlkylating agent Lphenylalanine mustard (L-PAM) in the presence of inhibitors of poly (ADP-ribose) polymerase (ADPRP)in vitro. In viva enhancement of the tumoricidal effects of L-PAM was observed with the ADPRP inhibitor nicotinamide (1000 mg/kg), although enhanced myelosuppression was also demonstrated. Nicotinamide also increased the plasma elimination half-life of L-PAM by a factor of at least 2. This alteration of L-PAM pharmacokinetics makes it difficult to assess the role that ADPRP inhibition plays in the enhancement of L-PAM tumor cell killing in viva
MelphaIan cytotoxicity,
Poly (ADP-ribose)
polymerase
inhibitors.
INTRODUCTION
In vitro studies The techniques for this assay have been described elsewhere.5 Briefly, Chinese hamster ovary cells designated HA- 1 from exponentially growing cultures were preplated in 60 mm plastic petri dishes in concentrations necessary to achieve approximately 30 colonies post drug treatment. The cells were exposed to either L-PAM alone or L-PAM and ADPRP inhibitors under aerobic conditions at 37°C. After the drug exposure, the cells were washed twice with Hanks’ balanced salt solution followed by 5 ml of MEM plus 15% FCS. Cell survival was assessed by colony formation after 10 days. Plating efficiency for these cells was routinely >90%.
It is now well established that the chromosomal enzyme poly (ADP-ribose) polymerase (ADPRP) is involved in the repair of DNA damage caused by X rays and monofunctional alkylating agents.3 However, the mechanisms are not well understood. Recent evidence suggests that ADPRP is involved in excision repair and that inhibitors of this enzyme retard DNA strand rejoining3 The chemical classes of ADPRP inhibitors include thymidine,’ nicotinamides,” benzamides’ and methylxanthines.2 A previous study showed enhanced cell killing in vitro by nitrogen mustard, a bifunctional alkylator, in the presence of caffeine, a methylxanthine.’ The purpose of this study was to investigate whether ADPRP inhibitors enhance the cytotoxicity ofthe bifunctional alkylating agent L-phenylalanine mustard (L-PAM) in vitro and in vivo. METHODS
AND
Tumor studies The RIF-1 tumor was used in all experiments. Solid tumors were produced in 3-4 month old female C3H/ Km mice by innoculating 2 X 10’ cells into the gastrocnemius muscle. The experimental techniques have previously been described in detaiL6 All drug treatments were carried out when the tumor size was 300-600 mg. For the regrowth delay assay, the time for the tumors to regrow to four times the treatment size was determined. Four to five mice were used in each group. Survival of RIF-1 tumor cells was measured by excising tumors 24 hours after injecting L-PAM. Three tumors were combined for each data point. They were subsequently minced, enzymatically disaggegated and single cell suspensions produced. The colony-forming ability of these cells was then
MATERIALS
Test compounds The ADPRP inhibitors nicotinamide, 3-aminobenzamide and caffeine* were dissolved immediately prior to use in either Minimal Eagle’s Medium (MEM)t plus 15% fetal calf serum (FCS) for the in vitro experiments or in saline for intraperitoneal (i.p.) injection in C3H mice. LPAM* was dissolved at 10 mg/ml in acidic ethanol (5% HCl in 95% ethanol) with further dilutions to 1 mg/ml made in 60% propylene glycol. This investigation
* Sigma Chemical Co., St. Louis, MO. t Gibco, Santa Clara, CA. $ Burroughs-Wellcome, Research Triangle, NC.
was supported by PHS Grants CA15201
and CA25990 awarded by the National Cancer Institute. Reprint requests to: D. M. Brown, Ph.D. Accepted for publication
24 April 1984. 1665
1666
Radiation Oncology 0 Biology0 Physics
1
September 1984, Volume 10, Number 9
2
5 L-PA
Cd.
6
7
(pg/ml)
Fig. 1. Survival of aerobic HA-I cells exposed for 1 hr at 37°C to I and 4 Icg/ml L-PAM alone (0) or in the presence of 5 mM concentrations of ADPRP inhibitors nicotinamide (B), 3-aminobenzamide (A) and caffeine Iv). The cells were then incubated for an additional 2 hours in 5 mM concentrations of the inhibitors without L-PAM.
determined. Survival was expressed as surviving fraction/ g tumor. This is the product of the plating efficiency and cell yield/g of treated tumor relative to that for untreated tumors.
Normal tissue studies White blood cell counts were determined four days after injecting drug by taking blood samples (5 ~1) from the tails of tumor-bearing mice and adding to 95 ~1 of 2% glacial acetic acid to lyse the erythrocytes. The resulting suspension of leucocytes was counted using a hemacytometer. Six mice were used for each treatment group.
Plasma L-PAM concentration assay The plasma pharmacokinetics of L-PAM was determined by high pressure liquid chromatography (HPLC)* according to methods previously described.4 RESULTS Preplated HA-l cells were initially exposed to L-PAM (1 and 4 &ml) with 5 mM concentrations of nicotinamide, 3-aminobenzamide or caffeine for 1 hr under * Waters Associates, Milford, MA.
aerobic conditions at 37’C. This was followed by an additional 2 hour post-incubation with the inhibitors alone. The survival results are shown in Figure 1. While not cytotoxic to the cells themselves, all of the inhibitors studied enhanced the cell killing by L-PAM. The order of effectiveness was caffeine > 3-aminobenzamide > nicotinamide. The effect of these ADPRP inhibitors on the cytotoxicity of L-PAM to RIF- 1 tumor cells was screened in vivo by injecting i.p. -2/3 LDso of caffeine (200 mg/kg) or nicotinamide (1000 mg/kg) simultaneously with 8 mg/ kg L-PAM. Because of solubility limitations, 3-aminobenzamide was injected i.p. below its 2/3 LD50 at a dose of 100 mg/kg with LPAM. An enhancement of L-PAM cell killing by all three inhibitors was observed (Fig. 2). In the in vivosituation nicotinamide was the most effective followed by caffeine and 3-aminobenzamide. Because of the solubility limitations of 3-aminobenzamide and the other known complicating biochemical effects caused by caffeine” further experiments with nicotinamide alone were pursued. Preliminary time course studies showed that the simultaneous injection of nico-
1667
Melphalan cytotoxicity and ADP-I&SC inhibiton 0 D. M. BROWN er al.
1.0 f
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10-l
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1o-2
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1o-5 I
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1
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6
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12
DOSE OF L-PAM (mg/kg) Fig. 2. The effect of ADPRP inhibitors on L-PAM cytotoxicity in RIF-1 tumors measured by tumor excision assay 24 hours after drug administration. Results are means from 2 separate experiments for L-PAM alone (8 mg/kg) @). 3-aminobenzamide alone ( 100 mg/kg) (O), 3-aminobenzamide + L-PAM (O), caffeine alone (266 m&/kg) (A), caffeine + L-PAM (A), nicotinamide alone (1060 mg/kg) (Cl), and nicotinamide + L-PAM (m). Dashed line represents L-PAM dose response curve. I
I
I
I
tinamide with L-PAM was the most effective (data not shown). The effect of nicotinamide on the L-PAM dose response curve, as measured by both tumor regrowth delay and white blood cell counts are shown in Figure 3. Regrowth delay is expressed as time required for tumors to grow to four times their initial treatment volume. in this figure, a slight delay in tumor growth was observed with nicotinamide alone (1000 mg/kg). However, this concentration of nicotinamide gave rise to a dose modification of approximately 2 at each L-PAM concentration. Myelosuppression is the dose-limiting normal tissue for L-PAM. Therefore, to determine whether a thempeutic gain could result from this treatment combination, the effect of nicotinamide ( 1000 mg/kg) with L-PAM on the white blood cell count of treated mice was studied. An enhanced depression of the white blood cell count four days post-treatment was observed for the nicotinamide and L-PAM combination (Fig. 3). It was observed that nicotinamide alone ( 1000 mg/kg) caused a pronounced drop in mouse body temperature of up to 4°C for -2 hours (data not shown). This depression of body temperature by nicotinamide may result in alteration of LPAM pharmacokinetics. Figure 4 is a plot of the plasma L-PAM concentration as a function of time after injection of L-PAM (8 mg/kg). A very marked pharmacokinetic effect by nicotinamide ( 1000 mg/kg) on LPAM plasma concentration was found. An increase in the area under the curve for plasma L-PAM concentration by a factor of approximately greater than two for the nicotinamide treated animals resulted. DISCUSSION It has been well demonstrated in vim that the cytotoxic effects of X ray and monofunctional alkylating agents 1
1
I
1
1 , 1 i 2 4 6 8 6 8 2 4 DOSE OF L-PAM (mg/kg) DOSE OF L-PAM (mg/kg) Fig. 3. The effect of nicotinamide plus L-PAM on both the RIF-I tumor regrowth delay (left panel) and white blood cell counts (right panel). Data points represent the mean f 1 S.E. for 4-6 data points for saline + L-PAM (8 mg/kg) (0 A) and nicotinamide ( 1006 mg/kg) + L-PAM (0 A). The different symbols are duplicate experiments. I
Radiation Oncology 0 Biology0 Physics
10.0 r
1.00 F
0.10 r 1
2
3
4
TIME AFTER L-PAM (hours) Fig. 4. The effect of nicotinamide on L-PAM pharmacokinetics. Plasma L-PAM measured by HPLC at various times post drug injection. Individual data points are shown for saline + LPAM (8 mg/kg) (0) and nicotinamide (1000 mg/kg) + L-PAM (0).
like dimethyl by inhibitors
sulfate and methylnitrosourea
are enhanced
of ADPRP (for review see 8). Furthermore, caffeine enhances the cytotoxicity in vitro of nitrogen mustard, a bifunctional alkylator.’ In our preliminary studies, we have shown an enhancement of cell killing by the bifunctional alkylator L-
September 1984,Volume IO, Number 9
PAM can be achieved by ADPRP inhibitors in vitro and also in vivo. In vitro, greater enhancement of L-PAM cell killing was obtained with caffeine than with 3-aminobenzamide or nicotinamide (Fig. 1). However, caffeine is a weak inhibitor of ADPRP (Ki = -250 PM for permeabilized L 12 IO cell~).~ Thus other biochemical effects caused by caffeine” may have been responsible for its effectiveness in vitro and in vivo (Fig. 2). The most effective inhibitor of ADPRP studied was 3aminobenzamide (Ki = 4.4 f 1.1 PM)*; like caffeine it also enhanced in vitro L-PAM cytotoxicity, but because of poor water solubility a limited dose ( 100 mg/kg) was used in vivo (Fig. 2). Only marginal enhancement of L PAM tumor cell kill was observed. Currently more water soluble benzamide analogs are being studied both in vitro and in vivo (Horsman et al., unpublished data, 1984) both in vitro and in vivo. Nicotinamide was the least effective compound studied in the in vitro experiments, but it is intermediate in its ability to inhibit ADPRP (Ki = 13 + 2 PM).* However, because of its good water solubility and low acute toxicity in the mouse (approximate LD50 = 1.5 g/kg), it was examined in more detail in vivo (Fig. 3). Unfortunately enhanced myelosuppression was observed at the large nicotinamide dose studied with LPAM. Preliminary data suggest that nicotinamide doses can be reduced to a level where no effect on the white blood cell count occurs, yet enhanced L-PAM tumor cell killing is observed (Horsman, unpublished data, Dec., 1983). However, since nicotinamide also enhances the plasma half-life of LPAM (Fig. 4), it is difficult from our results to assess the role that nicotinamide plays as an inhibitor of poly (ADPribose) polymerase in the enhanced L-PAM tumor cell killing observed in vivo.
REFERENCES 1. Das, S.K., Lau, C.G., Pardee, A.B.: Abolition by cyclohexamide of caffeine-enhanced lethality of alkylating agents in hamster cells. Cancer Res. 42: 4499-4504, 1982. 2. Davies, M.I., Shall, S., Skidmore, C.J.: Poly (Adenosine disphosphate ribose) polymerase and deoxyribonucleic acid damage. Biochem. Sot. Trans. 5: 949-950, 1978. 3. Durkacz, B.W., Omidiji, O., Gray, D.A., Shah, S.: (ADPribose)n participates in DNA excision repair. Nature, u(3: 593-596, 1980. 4. Fumer, R.L., Mellett, L.B., Brown, R.K., Duncan, G.: A method for the measurement of L-phenylalanine mustard in the mouse and dog by high-pressure liquid chromatography. Drug Metab. Dispos. 4(6): 577-583, 1976. 5. Hot-smart, M.R., Brown, J.M., Schelley, S.A.: The effect of misonidazole on the cytotoxicity and repair of potentially lethal damage from alkylating agents in vitro and in vivo. Inl. J. Radial. Oncol. Biol. Phys. 8: 761-765, 1982.
Law, M.P., Hint, D.G., Brown, J.M.: Enhancing effect of misonidazole on the response of the RIF-I tumor to cyclophosphamide. Br. J. Cancer 44: 208-2 18, 198 1. Preiss, J., Schlaeger, R., Hilz, H.: Specific inhibition of poly (ADP-ribose) polymerase by thymidine and nicotinamide in HeLa cells. FEBS Lett. 19: 244-246, 197 1. Shah, S.: ADP-ribose in DNA repair. In ADP-ribosylation Reactions, Biology and Medicine, Hayaishi, O., Veda, K. (Eds.). New York, Academic Press. 1982, pp. 477-520. 9. Shall, S.: Experimental manipulation of the specific activity of poly (ADP-rib) polymera.% J. Biochem. 77: 2p., 1975. Jo*
Shall, S., Goodwin, P., Halldorsson, H., Khan, H., Skidmore, C., Tsopanakis, C.: Post-synthetic modification of nuclear macromolecules. B&hem. Sot. Symp. 42: 103-I 16, 1977.
1 I. Timson, J.: Caffeine. Mutaf. Res. 47: l-52, 1977.
Lltnl n@i
Phvs trxrwd
Vol
IO.
pi
1665-1668 Copyright
0360-3016184 $03.00 + .OO G 1984 Pcrgamon Pxs Ltd.
??Session V
ENHANCEMENT OF MELPHALAN CYTOTOXICITY BY INHIBITORS OF POLY (ADP-RIBOSE) D. M. BROWN, PH.D., M. R. HORSMAN, PH.D., D. G. HIRST, Division of Radiobiology,
Department
IN VZVO AND IN VITRO POLYMERASE PH.D.
AND J. M. BROWN, PH.D.
of Radiology, Stanford University School of Medicine, Stanford, CA 94305
In these preliminary experiments, we have found enhanced cell killing by the bifunctional nlkylating agent Lphenylalanine mustard (L-PAM) in the presence of inhibitors of poly (ADP-ribose) polymerase (ADPRP)in vitro. In viva enhancement of the tumoricidal effects of L-PAM was observed with the ADPRP inhibitor nicotinamide (1000 mg/kg), although enhanced myelosuppression was also demonstrated. Nicotinamide also increased the plasma elimination half-life of L-PAM by a factor of at least 2. This alteration of L-PAM pharmacokinetics makes it difficult to assess the role that ADPRP inhibition plays in the enhancement of L-PAM tumor cell killing in viva
MelphaIan cytotoxicity,
Poly (ADP-ribose)
polymerase
inhibitors.
INTRODUCTION
In vitro studies The techniques for this assay have been described elsewhere.5 Briefly, Chinese hamster ovary cells designated HA- 1 from exponentially growing cultures were preplated in 60 mm plastic petri dishes in concentrations necessary to achieve approximately 30 colonies post drug treatment. The cells were exposed to either L-PAM alone or L-PAM and ADPRP inhibitors under aerobic conditions at 37°C. After the drug exposure, the cells were washed twice with Hanks’ balanced salt solution followed by 5 ml of MEM plus 15% FCS. Cell survival was assessed by colony formation after 10 days. Plating efficiency for these cells was routinely >90%.
It is now well established that the chromosomal enzyme poly (ADP-ribose) polymerase (ADPRP) is involved in the repair of DNA damage caused by X rays and monofunctional alkylating agents.3 However, the mechanisms are not well understood. Recent evidence suggests that ADPRP is involved in excision repair and that inhibitors of this enzyme retard DNA strand rejoining3 The chemical classes of ADPRP inhibitors include thymidine,’ nicotinamides,” benzamides’ and methylxanthines.2 A previous study showed enhanced cell killing in vitro by nitrogen mustard, a bifunctional alkylator, in the presence of caffeine, a methylxanthine.’ The purpose of this study was to investigate whether ADPRP inhibitors enhance the cytotoxicity ofthe bifunctional alkylating agent L-phenylalanine mustard (L-PAM) in vitro and in vivo. METHODS
AND
Tumor studies The RIF-1 tumor was used in all experiments. Solid tumors were produced in 3-4 month old female C3H/ Km mice by innoculating 2 X 10’ cells into the gastrocnemius muscle. The experimental techniques have previously been described in detaiL6 All drug treatments were carried out when the tumor size was 300-600 mg. For the regrowth delay assay, the time for the tumors to regrow to four times the treatment size was determined. Four to five mice were used in each group. Survival of RIF-1 tumor cells was measured by excising tumors 24 hours after injecting L-PAM. Three tumors were combined for each data point. They were subsequently minced, enzymatically disaggegated and single cell suspensions produced. The colony-forming ability of these cells was then
MATERIALS
Test compounds The ADPRP inhibitors nicotinamide, 3-aminobenzamide and caffeine* were dissolved immediately prior to use in either Minimal Eagle’s Medium (MEM)t plus 15% fetal calf serum (FCS) for the in vitro experiments or in saline for intraperitoneal (i.p.) injection in C3H mice. LPAM* was dissolved at 10 mg/ml in acidic ethanol (5% HCl in 95% ethanol) with further dilutions to 1 mg/ml made in 60% propylene glycol. This investigation
* Sigma Chemical Co., St. Louis, MO. t Gibco, Santa Clara, CA. $ Burroughs-Wellcome, Research Triangle, NC.
was supported by PHS Grants CA15201
and CA25990 awarded by the National Cancer Institute. Reprint requests to: D. M. Brown, Ph.D. Accepted for publication
24 April 1984. 1665
1666
Radiation Oncology 0 Biology0 Physics
1
September 1984, Volume 10, Number 9
2
5 L-PA
Cd.
6
7
(pg/ml)
Fig. 1. Survival of aerobic HA-I cells exposed for 1 hr at 37°C to I and 4 Icg/ml L-PAM alone (0) or in the presence of 5 mM concentrations of ADPRP inhibitors nicotinamide (B), 3-aminobenzamide (A) and caffeine Iv). The cells were then incubated for an additional 2 hours in 5 mM concentrations of the inhibitors without L-PAM.
determined. Survival was expressed as surviving fraction/ g tumor. This is the product of the plating efficiency and cell yield/g of treated tumor relative to that for untreated tumors.
Normal tissue studies White blood cell counts were determined four days after injecting drug by taking blood samples (5 ~1) from the tails of tumor-bearing mice and adding to 95 ~1 of 2% glacial acetic acid to lyse the erythrocytes. The resulting suspension of leucocytes was counted using a hemacytometer. Six mice were used for each treatment group.
Plasma L-PAM concentration assay The plasma pharmacokinetics of L-PAM was determined by high pressure liquid chromatography (HPLC)* according to methods previously described.4 RESULTS Preplated HA-l cells were initially exposed to L-PAM (1 and 4 &ml) with 5 mM concentrations of nicotinamide, 3-aminobenzamide or caffeine for 1 hr under * Waters Associates, Milford, MA.
aerobic conditions at 37’C. This was followed by an additional 2 hour post-incubation with the inhibitors alone. The survival results are shown in Figure 1. While not cytotoxic to the cells themselves, all of the inhibitors studied enhanced the cell killing by L-PAM. The order of effectiveness was caffeine > 3-aminobenzamide > nicotinamide. The effect of these ADPRP inhibitors on the cytotoxicity of L-PAM to RIF- 1 tumor cells was screened in vivo by injecting i.p. -2/3 LDso of caffeine (200 mg/kg) or nicotinamide (1000 mg/kg) simultaneously with 8 mg/ kg L-PAM. Because of solubility limitations, 3-aminobenzamide was injected i.p. below its 2/3 LD50 at a dose of 100 mg/kg with LPAM. An enhancement of L-PAM cell killing by all three inhibitors was observed (Fig. 2). In the in vivosituation nicotinamide was the most effective followed by caffeine and 3-aminobenzamide. Because of the solubility limitations of 3-aminobenzamide and the other known complicating biochemical effects caused by caffeine” further experiments with nicotinamide alone were pursued. Preliminary time course studies showed that the simultaneous injection of nico-
1667
Melphalan cytotoxicity and ADP-I&SC inhibiton 0 D. M. BROWN er al.
1.0 f
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10-l
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\
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1o-2
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A 1o-4
a
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6
6
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DOSE OF L-PAM (mg/kg) Fig. 2. The effect of ADPRP inhibitors on L-PAM cytotoxicity in RIF-1 tumors measured by tumor excision assay 24 hours after drug administration. Results are means from 2 separate experiments for L-PAM alone (8 mg/kg) @). 3-aminobenzamide alone ( 100 mg/kg) (O), 3-aminobenzamide + L-PAM (O), caffeine alone (266 m&/kg) (A), caffeine + L-PAM (A), nicotinamide alone (1060 mg/kg) (Cl), and nicotinamide + L-PAM (m). Dashed line represents L-PAM dose response curve. I
I
I
I
tinamide with L-PAM was the most effective (data not shown). The effect of nicotinamide on the L-PAM dose response curve, as measured by both tumor regrowth delay and white blood cell counts are shown in Figure 3. Regrowth delay is expressed as time required for tumors to grow to four times their initial treatment volume. in this figure, a slight delay in tumor growth was observed with nicotinamide alone (1000 mg/kg). However, this concentration of nicotinamide gave rise to a dose modification of approximately 2 at each L-PAM concentration. Myelosuppression is the dose-limiting normal tissue for L-PAM. Therefore, to determine whether a thempeutic gain could result from this treatment combination, the effect of nicotinamide ( 1000 mg/kg) with L-PAM on the white blood cell count of treated mice was studied. An enhanced depression of the white blood cell count four days post-treatment was observed for the nicotinamide and L-PAM combination (Fig. 3). It was observed that nicotinamide alone ( 1000 mg/kg) caused a pronounced drop in mouse body temperature of up to 4°C for -2 hours (data not shown). This depression of body temperature by nicotinamide may result in alteration of LPAM pharmacokinetics. Figure 4 is a plot of the plasma L-PAM concentration as a function of time after injection of L-PAM (8 mg/kg). A very marked pharmacokinetic effect by nicotinamide ( 1000 mg/kg) on LPAM plasma concentration was found. An increase in the area under the curve for plasma L-PAM concentration by a factor of approximately greater than two for the nicotinamide treated animals resulted. DISCUSSION It has been well demonstrated in vim that the cytotoxic effects of X ray and monofunctional alkylating agents 1
1
I
1
1 , 1 i 2 4 6 8 6 8 2 4 DOSE OF L-PAM (mg/kg) DOSE OF L-PAM (mg/kg) Fig. 3. The effect of nicotinamide plus L-PAM on both the RIF-I tumor regrowth delay (left panel) and white blood cell counts (right panel). Data points represent the mean f 1 S.E. for 4-6 data points for saline + L-PAM (8 mg/kg) (0 A) and nicotinamide ( 1006 mg/kg) + L-PAM (0 A). The different symbols are duplicate experiments. I
Radiation Oncology 0 Biology0 Physics
10.0 r
1.00 F
0.10 r 1
2
3
4
TIME AFTER L-PAM (hours) Fig. 4. The effect of nicotinamide on L-PAM pharmacokinetics. Plasma L-PAM measured by HPLC at various times post drug injection. Individual data points are shown for saline + LPAM (8 mg/kg) (0) and nicotinamide (1000 mg/kg) + L-PAM (0).
like dimethyl by inhibitors
sulfate and methylnitrosourea
are enhanced
of ADPRP (for review see 8). Furthermore, caffeine enhances the cytotoxicity in vitro of nitrogen mustard, a bifunctional alkylator.’ In our preliminary studies, we have shown an enhancement of cell killing by the bifunctional alkylator L-
September 1984,Volume IO, Number 9
PAM can be achieved by ADPRP inhibitors in vitro and also in vivo. In vitro, greater enhancement of L-PAM cell killing was obtained with caffeine than with 3-aminobenzamide or nicotinamide (Fig. 1). However, caffeine is a weak inhibitor of ADPRP (Ki = -250 PM for permeabilized L 12 IO cell~).~ Thus other biochemical effects caused by caffeine” may have been responsible for its effectiveness in vitro and in vivo (Fig. 2). The most effective inhibitor of ADPRP studied was 3aminobenzamide (Ki = 4.4 f 1.1 PM)*; like caffeine it also enhanced in vitro L-PAM cytotoxicity, but because of poor water solubility a limited dose ( 100 mg/kg) was used in vivo (Fig. 2). Only marginal enhancement of L PAM tumor cell kill was observed. Currently more water soluble benzamide analogs are being studied both in vitro and in vivo (Horsman et al., unpublished data, 1984) both in vitro and in vivo. Nicotinamide was the least effective compound studied in the in vitro experiments, but it is intermediate in its ability to inhibit ADPRP (Ki = 13 + 2 PM).* However, because of its good water solubility and low acute toxicity in the mouse (approximate LD50 = 1.5 g/kg), it was examined in more detail in vivo (Fig. 3). Unfortunately enhanced myelosuppression was observed at the large nicotinamide dose studied with LPAM. Preliminary data suggest that nicotinamide doses can be reduced to a level where no effect on the white blood cell count occurs, yet enhanced L-PAM tumor cell killing is observed (Horsman, unpublished data, Dec., 1983). However, since nicotinamide also enhances the plasma half-life of LPAM (Fig. 4), it is difficult from our results to assess the role that nicotinamide plays as an inhibitor of poly (ADPribose) polymerase in the enhanced L-PAM tumor cell killing observed in vivo.
REFERENCES 1. Das, S.K., Lau, C.G., Pardee, A.B.: Abolition by cyclohexamide of caffeine-enhanced lethality of alkylating agents in hamster cells. Cancer Res. 42: 4499-4504, 1982. 2. Davies, M.I., Shall, S., Skidmore, C.J.: Poly (Adenosine disphosphate ribose) polymerase and deoxyribonucleic acid damage. Biochem. Sot. Trans. 5: 949-950, 1978. 3. Durkacz, B.W., Omidiji, O., Gray, D.A., Shah, S.: (ADPribose)n participates in DNA excision repair. Nature, u(3: 593-596, 1980. 4. Fumer, R.L., Mellett, L.B., Brown, R.K., Duncan, G.: A method for the measurement of L-phenylalanine mustard in the mouse and dog by high-pressure liquid chromatography. Drug Metab. Dispos. 4(6): 577-583, 1976. 5. Hot-smart, M.R., Brown, J.M., Schelley, S.A.: The effect of misonidazole on the cytotoxicity and repair of potentially lethal damage from alkylating agents in vitro and in vivo. Inl. J. Radial. Oncol. Biol. Phys. 8: 761-765, 1982.
Law, M.P., Hint, D.G., Brown, J.M.: Enhancing effect of misonidazole on the response of the RIF-I tumor to cyclophosphamide. Br. J. Cancer 44: 208-2 18, 198 1. Preiss, J., Schlaeger, R., Hilz, H.: Specific inhibition of poly (ADP-ribose) polymerase by thymidine and nicotinamide in HeLa cells. FEBS Lett. 19: 244-246, 197 1. Shah, S.: ADP-ribose in DNA repair. In ADP-ribosylation Reactions, Biology and Medicine, Hayaishi, O., Veda, K. (Eds.). New York, Academic Press. 1982, pp. 477-520. 9. Shall, S.: Experimental manipulation of the specific activity of poly (ADP-rib) polymera.% J. Biochem. 77: 2p., 1975. Jo*
Shall, S., Goodwin, P., Halldorsson, H., Khan, H., Skidmore, C., Tsopanakis, C.: Post-synthetic modification of nuclear macromolecules. B&hem. Sot. Symp. 42: 103-I 16, 1977.
1 I. Timson, J.: Caffeine. Mutaf. Res. 47: l-52, 1977.