4-(Nitrophenylsulfonyl)piperazines mitigate radiation damage to multiple tissues

Abstract

Our ability to use ionizing radiation as an energy source, as a therapeutic agent, and, unfortunately, as a weapon, has evolved tremendously over the past 120 years, yet our tool box to handle the consequences of accidental and unwanted radiation exposure remains very limited. We have identified a novel group of small molecule compounds with a 4-nitrophenylsulfonamide (NPS) backbone in common that dramatically decrease mortality from the hematopoietic acute radiation syndrome (hARS). The group emerged from an in vitro high throughput screen (HTS) for inhibitors of radiation-induced apoptosis. The lead compound also mitigates against death after local abdominal irradiation and after local thoracic irradiation (LTI) in models of subacute radiation pneumonitis and late radiation fibrosis. Mitigation of hARS is through activation of radiation-induced CD11b+Ly6G+Ly6C+ immature myeloid cells. This is consistent with the notion that myeloerythroid-restricted progenitors protect against WBI-induced lethality and extends the possible involvement of the myeloid lineage in radiation effects. The lead compound was active if given to mice before or after WBI and had some anti-tumor action, suggesting that these compounds may find broader applications to cancer radiation therapy.

Fig 1. 4-Nitrophenylsulfonamides effectively mitigate radiation damage in vitro and in vivo.

NPSP (#1–8) and NPS (#9–10) chemical structures with ChemBridge nomenclature arranged by maximal common substructuring. The data underneath each compound refers to % viability of TIL1 lymphocytic cells at 24 hrs, compounds being added at 10μM to TIL1 cells 1 hr after 2Gy irradiation. Viability was assessed by ATPLite production at 24 hrs and is shown relative to 100% of irradiated controls, with >130% (bold) being taken as a significant increase (>3S.D. above control mean). There were no significant toxic or stimulatory effects when added to non-irradiated cells. All except #1 and #8 were tested in vivo (bottom graphs). They were injected in 1% Cremophor s.c. into C3H male mice (8 per group) starting 24 hrs after 7.725Gy WBI (LD70/30 estimate), daily for 5 days. Survival to the day 30 endpoint is expressed using a Kaplan-Meier plot with log rank statistics.

Fig 2. Efficacy of compounds in different conditions.

Compounds #5 (a, b) and #3 (c, d) in C3H male (a, c) and C57Bl/6 female (b, d) mice with 5mg/kg given s.c. on day 1 and daily for 5 days. This dose is generally superior to 10 and 1mg/kg (this figure), and 75 and 25mg/kg (Fig 1) for mitigating hARS after WBI (LD70/30 estimated doses used for the different strains). (e) Male C3H mice treated s.c. for 5dys with 5mg/kg compound #5 or 75mg/kg compound #10 have increased resistance to hARS. LD50/30 control = 7.5Gy (95% c.l. = 7.34–7.67); # 5 LD50/30 = 8.0Gy (95% c.l. = 7.9–8.2); #10 LD50/30 = 8.2Gy (95% c.l. = 8.07–8.58). Compound #5 given by gavage at 5mg/kg for 5dys mitigates hARS (LD70/30) in C3H (f) and C57Bl/6 (g) male mice whereas compound #3 does not (h, i). A single s.c. injection of 5mg/kg of compound #5given 24hrs after WBI (LD70/30) mitigates against hARS in C3H mice (j). (k) Compound #5 given 18hrs before WBI (LD70/30 estimated dose of 7.725Gy) as a single 5mg/kg s.c. injection radioprotects C3H male mice from hARS. (*p<0.05, **p<0.01, ***p<0.001).

Fig 3. Pharmacokinetics.

Pharmacokinetic analysis of compound #5 (triangles) in serum by LC/MS following a single 5 mg/kg s.c. dose showed a drug profile with a C_max (obs) of 0.12 μg/mL, a T_max of 2h, a half-life of 9h, AUC (area) 0.41 μg-hr/mL and CL (expo) 0.37ml/hr. Persistence was superior to #7 (circles) and #11 (diamonds) (which was synthesized specially, suggesting the phenyl group improves pharmacokinetic availability. Note: This lack of persistence of #7 might explain its variable performance in vivo as a mitigator.

Fig 4. Intestinal and lung damage following radiation exposure can be significantly improved with 4-nitrophenylsulfonamides.

(a) Compound #5 (5mg/kg daily x5 s.c.) mitigates lethality from intestinal ARS following 18Gy local abdominal irradiation. (b) Compound #5 (5mg/kg daily x5 s.c.) mitigates lethality from radiation-induced pneumonitis in C3H mice after 18 and 14Gy LTI. (c) Compound #5 (5mg/kg daily x5 s.c.) mitigates lethality from radiation-induced fibrosis in C57Bl/6 mice following 18Gy LTI. Note the differences in time to lethality for these endpoints. Kaplan-Meier with log rank statistics. (d) Picro Sirius Red staining of lungs of C57Bl/6 mice 156 days after LTI with (e) flow cytometric analysis of inflammatory infiltrate. (*p<0.05)

Fig 5. Successfully mitigated mice show favorable immune reconstitution and inflammatory rebalancing.

(a) mRNA levels of various cytokines assessed by RT-PCR in bone marrow derived macrophages treated with 10μM #3 or #5 1 hr after 100ng/ml LPS and assayed at 6hrs. All except IL-6 were significantly decreased (P<0.05). (b) TNF-α production at 24hrs by inflammatory peritoneal exudate cells treated in vitro with 100ng/ml LPS followed at 1hr by 1 or 10 μM #3 or #5 or diluent. (c) Bone marrow cells were removed from C3H mice 30hrs after WBI (LD70/30–7.725Gy) or sham irradiated, with compound #5 or diluent given 6 hrs before harvest. After overnight culture, supernatants were tested for cytokines by multi-arrays. The spider plots show changes after WBI alone (left) compared to control (value = 1 in grey) and WBI plus compound #5 (right) compared to WBI alone (value = 1 in grey). (d) Compound #5 (5mg/kg s.c. at 24 hrs after WBI) increases the number of endogenous CFU-S per spleen at day 10 after the WBI. (*p<0.05)

Fig 6. Irradiated mice have a systemic surge in immature myeloid cells that is essential for mitigation.

(a) The emergence of a CD11b⁺Ly6G⁺Ly6C⁺ population of immature myeloid cells (middle) in the spleens of mice that are easily distinguishable by forward and side scatter (left) in flow cytometry. Proportional increases as a % of all cells are dose dependent (right), which is in part due to loss of other cells and in part mobilization as few of these cells are present in peripheral organs (see control). (b) The same population appears in the blood (left and middle) and bone marrow (right), where it normally represents 20% of all cells. In blood, where it is normally absent, it reaches levels of 20% of all white cells 30hrs after WBI. (c) Treatment with compound #5 (5mg/kg once) at 24hrs after WBI of C3H or C57Bl/6 mice (LD70/30 estimated dose) increases the CD11b⁺Ly6G⁺Ly6C⁺ representation in the spleen (shown) and other organs (not shown). (d) Treatment of mice with anti-Ly6G removes the CD11b⁺Ly6G⁺Ly6C⁺ population (left) and abolishes activity of mitigator #5 (right).

Fig 7. Lung tumors are not protected from radiation damage by the NSPS mitigator #5.

Mice were injected with 5x10⁴ Lewis Lung carcinoma (LLC) cells i.v., treated with compound #5 (20mg/kg s.c.) or diluent on days 4–8 and given 0 or 4Gy LTI on days 5–7. Lungs were harvested on day 14 and the lung tumor nodules counted after staining in Bouin’s solution.