Radioprotectant Activity of Dicopper(II) Tetrakis(3,5- Diisopropylsalicylate) and Manganese(II) bis(3,5- Diisopropylsalicylate) Alone and in Combination

Dicopper(ll) tetrakis(3,5-diisopropylsalicylate), (Cu(II)2(3,5-DIPS)4, manganese(II) bis(3,5-diisopropylsalicylate), Mn(II)3,5-DIPS)2 or combinations of them were used to treat gamma-irradIated mice in examining the possibility that combination treatments might be more effective in increasing survival than treatment with either complex alone. Doses of 0, 10, 20, or 40 μmol of each complex per kilogram of body mass were administered subcutaneously in a factorial design before 9 Gy gamna irradiation, an LD90 dose of irradiation. Doses of 0, 10, 20, or 40 μmol Cu(II)2(3, 5-DIPS)4 per kg of body mass produced 12, 28, 28, or 36 % survival, respectively, while doses of 0, 10, 20, or 40 μmol (II)3, 5-DIPS)2 per kg of body mass prduced 12, 36, 20, or 24 % survival, respectively. However, the combination of 20 μmol Cu(II)2(3, 5-DIPS)4 and 10 μmol Mn(II)(3, 5−DIPS)2 produced the greatest survival, 48 %, which was 300 % greater than vehicle-treated mice (P=0.01). It is concluded that specific combination treatments can be used to maximize survival of lethally irradiated mice.


Introduction
Both Cu(II)2(3,5-DIPS)4 and Mn(II)(3,5-DIPS)2 have been found to be effective radioprotectants in LDs0/30 and LD00/30 radiation paradigms [1 and references therein]. Other essential metalloelement compounds including chelates of zinc and iron have also been found to have radioprotectant activity. It is suggested that the radioprotectant and radiorecovery activity of various essential metalloelement compounds is based upon roles of these essential metalloelement-dependent enzymes in repair of radiation injury [1].
Since essential metalloelement-dependent enzymes require a specific metalloelement(s) for optimal activity, treatment with a single metalloelement will facilitate the role of that class of metalloelement-dependent enzymes in responding to radiation injury and increasing survival. A priori the response to overcome radiation injury by facilitating a single metalloelement class of enzymes is limited. However, facilitating the response to radiation injury by enhancing the activity of more than one class of essential metalloelement-dependent enzymes should increase the effectiveness of treating radiation injury and increase survival. In using either a single or multiple essential metalloelement treatment approach to overcoming radiation injury there is plausible risk with regard to the incorporation of an essential metalloelement into the wrong apoenzyme to yield a less active or inactive metalloelement-dependent enzyme. Consequently, maximal effectiveness of this approach to treatment may depend upon the use of a somewhat narrow range of combination doses.
We are reporting the radioprotectant activity (survival)of lethally irradiated mice treated with Cu(II)2(3,5-DIPS)4, Mn(II)(3,5-DIPS)2, or combinations of these complexes. The 900 Gy dose selected for these studies was chosen to increase radiation injury as a more rigorous evaluation of combination treatlnents in providing radioprotectant activity.

Materials and Methods
The synthesis Cu(II)2(3,5-DIPS)4 and Mn(II)(3,5-DIPS) have been described in detail [2]. Both compounds were first wetted with a volume of Propy-lene Glycol (Sigma) sufficient to yield a final solution or suspension containing 4 % Propylene Glycol l'a 1. 4  Three hundred and twenty l, tmol (341.1 mg) of Cu(II)(3,5-DIPS)4(H20)3, Mr 1,066 Daltons, was wetted with 4 ml of Propylene Glycol in a Potter-Elvehjem homogenizer while cooling in an ice bath to prevent the increase in viscosity associated with homogenization without cooling. The homogenate was then transferred to a 200 ml graduated cylnder with sufficient incomplete vehicle to make 100 ml. The cylinder was covered with Parafilm (American National Can) and vortex stirred to insure homogeneity. This suspension containing 1.6 ]amol Cu(II)z(3,5-DIPS)4 per 0.5 ml was vortex stirred before all subsequent usage.
Fifty ml of the 1.6 lamol/0.5 ml stirred suspension was diluted with 50 ml of complete vehicle and the diluted 100 ml suspension vortex stirred. This suspension contained 0.8 mol Cu(II)z(3,5-DIPS)4 per 0.5 ml. Treatment of a 20 g mouse with 0.5 ml of this suspension provides 40 ktmol/kg of body mass. Daltons, was wetted with 4 ml of Propylene Glycol in a Potter-Elvehjem homogenizer while cooling in an ice bath to prevent the increase in viscosity associated with homogenization without cooling. The homogenate was then transferred to a 200 ml graduated cylinder with sufficient incomplete vehicle to make 100 ml. The cylinder was covered wth Parafilm (American National Can) and vortex stirred to insure homogeneity of what appeared to be a true solution. This apparent solution was still vortex stirred to guarantee homogeneity before all subsequent dilutions. This solution contained 1.6 lamol Mn(II)(3,5-DIPS)z per 0.5 ml. These solutions were used to provide factorial design treatments shown in Table I. Sixteen groups of 25 randomized 10 week old, young adult, 20 to 22 g female C57BL/6 mice housed 5 per cage, and fed mouse chow pellets and water ad libitum, with 12 hr light (6:00 am to 6:00 pm) and dark cycles for 3 weeks prior to treatment. Each group of 25 mice was treated subcutaneously in the dorsal nape of the neck beginning at 9:00 am with one of the sixteen treatments: either vehicle or one of the single or combination treatments, thr7 to eight hours before gamma-irradiation (900 cGy, 124.5 cGy/min) in a Shepard Mark Cs irradiator beginning at 12:00 pm. All 25 mice in each treatment group were irradiated over a 29 minute period and survival determined over a 30 day post-treatment and irradiation period.
The 900 cGy dose was selected to increase radiation injury beyond the LD50/30 dose of800 c Gy (1) to more rigorously examine these combination therapies for radioprotectant activity.
Statistical comparisons of the proportion surviving among treatment groups were made using Fisher's Exact Test. The software used was StatXact-3 (Cytel Software Corporation).
All animal experiments were approved by the Institute's Animal Care and Use Committee which has an Animal Welfare Assurance on file with the Office of Protection from Research Risks. Table I, (Table II).  Treatment with 10 lamol, 20 lamol, or 40 lamol Mn(II)(3,5-DIPS)z gave survivals of 36%, 20%, 24%, respectively, as shown in Figure 2, which represent 200%, 167%, or 100% increases in survival compared to vehicle-treated mice (Table II).   However, increasing the dose of Mn(II)(3,5-DIPS)z beyond 10 gmol/kg of body mass seems to have decreased survival as shown in Tables I and II and    per kg of body mass in 900 cGy irradiated mice.

Discussion
None of the single or combination doses selected for this study caused acute toxicity, death in a treatment group prior to death in the vehicle-treated group. As shown in Figures to 5, there were no deaths in any group prior to day 5 after treatment.
Data presented in Tables and II and Figure show that treatment with Cu(II)z(3,5-DIPS)4 caused an increase in survival in 900 cGy irradiated mice as the dose was increased from 10 lamol/kg to 40 lamol/kg with no change in survival when the dose was increased from 10 tmol/kg to 20 tmol/kg. Treatment with 10 lamol Mn(II)(3,5-DIPS)z/kg caused an increase in survival compared with vehicle-treated mice. However, treatment with the higher doses, 20 lamol and 40 lamol/kg, did not cause a dose-related increase in survival which decreased with increasing dose of Mn(II)(3,5-DIPS)z above 10 tmol/kg (Tables and II and Figures 2, 3, 4, and 5). This decrease in survival may be due to a Mn-induced interference wherein Mn may be inappropriately incorporated into an apoenzyme that requires some other essential metalloelement to maximally fulfill its role in repair of a 900 cGy radiation injury. This possible interference, which may arise with larger doses of irradiation, is offered as a rationale in explaining why Mn(II)(3,5-DIPS) given to 800 cGy irradiated mice gave 100 % survival [3]. The greater radiation injury caused by 900 cGy may have caused an increased essential metalloelement-dependent response wherein there is an increased physiological response involving other essential metalloelement-dependent enzymes that may be less active as a result of the inappropriate incorporation of Mn.
This concept that treatment with a combination of metalloelement chelates provides a synergistic increase in survival of lethally irradiated mice. It may also be biologically significant that the ratio of Cu Mn in this combination treatment is 2 1, 40 lamol Cu and 20 tmol Mn since the copper complex is binuclear and contains 2 atoms of copper while the manganese complex is mononuclear and contains atom of manganese.
Acute toxicities for both the binulcear Cu(II)2(3,5-DIPS)4 and mononuclear Mn(II)(3,5-DIPS)2 complexes have been reported to be 261 + 36 lamol/kg or 91 + 13 lamol/kg and 842 + 92 or 706 + 64 lamol/kg respectively for female or male C57BL/6 mice [1, 2,9]. Both complexes have been shown to increase survival in LDs0 mice at a dose of 49 to 80 lamol/kg of body mass in female and male C57BL/6 mice when given subcutaneously or orally either up to 24 hrs before irradiation or up to 24 hrs after irradiation. The dose of irradiation for this experiment was selected to approach the LD00 dose in order to more rigorously examine combination therapy with regard to its potential in facilitating survival of mice experiencing greater radiation injury. Results of these studies do support the possibility that combination therapy does offer the potential for recovery from increased radiation injury.
In this study the treatment and irradiation intervals ranged from three to eight hours starting with the control group and moving from left to right for the treatment groups shown in Table   and ending with the group treated with 40 lamol/kg Cu(II)2(3,5-DIPS)4 and Mn(II)(3,5-DIPS)2/kg. This is not likely to have influenced the outcome of this experiment 67 67 since, as we have shown [7,8], Cu administered as Cu labeled Cu(II)2(3,5-DIPS)4 was conserved and remained in blood, liver, kidney, intestine, muscle, lung, thymus, femur, spleen, and brain throughout the 5 day duration of our study. These complexes have also been found to increase survival of LD0 irradiated mice when administered up to twenty-four hours before irradiation. Data presented in Table II do not reveal an influence of time of treatment before irradiation on survival. There were no statistically significant differences due to time of irradiation following treatment. However, this point remains to be addressed in future experiments when time before irradiation is held constant for all treatment groups.