Regression of glioma tumor growth in F98 and U87 rat glioma models by the Nitrone OKN-007

Abstract

Background: Glioblastoma multiforme, a World Health Organization grade IV glioma, has a poor prognosis in humans despite current treatment options. Here, we present magnetic resonance imaging (MRI) data regarding the regression of aggressive rat F98 gliomas and human U87 glioma xenografts after treatment with the nitrone compound OKN-007, a disulfonyl derivative of α-phenyl-tert-butyl nitrone.

Methods: MRI was used to assess tumor volumes in F98 and U87 gliomas, and bioluminescence imaging was used to measure tumor volumes in F98 gliomas encoded with the luciferase gene (F98(luc)). Immunohistochemistry was used to assess angiogenesis (vascular endothelial growth factor [VEGF] and microvessel density [MVD]), cell differentiation (carbonic anhydrase IX [CA-IX]), hypoxia (hypoxia-inducible factor-1α [HIF-1α]), cell proliferation (glucose transporter 1 [Glut-1] and MIB-1), proliferation index, and apoptosis (cleaved caspase 3) markers in F98 gliomas. VEGF, CA-IX, Glut-1, HIF-1α, and cleaved caspase 3 were assessed in U87 gliomas.

Results: Animal survival was found to be significantly increased (P < .001 for F98, P < .01 for U87) in the group that received OKN-007 treatment compared with the untreated groups. After MRI detection of F98 gliomas, OKN-007, administered orally, was found to decrease tumor growth (P < .05). U87 glioma volumes were found to significantly decrease (P < .05) after OKN-007 treatment, compared with untreated animals. OKN-007 administration resulted in significant decreases in tumor hypoxia (HIF-1α [P < .05] in both F98 and U87), angiogenesis (MVD [P < .05], but not VEGF, in F98 or U87), and cell proliferation (Glut-1 [P < .05 in F98, P < .01 in U87] and MIB-1 [P < .01] in F98) and caused a significant increase in apoptosis (cleaved caspase 3 [P < .001 in F98, P < .05 in U87]), compared with untreated animals.

Conclusions: OKN-007 may be considered as a promising therapeutic addition or alternative for the treatment of aggressive human gliomas.

Fig. 1.

(A) Chemical structure of OKN-007 (disodium 4-[(tert-butylimino)methyl]benzene-1.3-disulfonate N-oxide). (B–D) Pharmacokinetic analysis of plasma levels of OKN-007 in Fisher 344 rats collected from blood samples at 0, 5, 15, and 30 min and 1, 2, 4, 8, 24, and 48 h. (C) Pharmacokinetic curve of OKN-007 plasma concentration over 4 h. (D) One-compartment model with a lag time with equal weighting of data. AUC = area under the plasma concentration versus time curve, AUMC = area under the concentration times time versus time curve (using the trapezoidal rule), MRT = mean residence time ( = AUMC/AUC), CL/F = oral clearance ( = Dose/AUC), first-order rate constants, k_a = absorption and k_el = elimination, V/F = an apparent volume of distribution divided by bioavailabilty and a lag time (t_lag), and t_lag = delay before absorption starts.

Fig. 2.

Survival of F98 (A) and U87 (B) glioma-bearing rats that were either untreated (UT) (F98: n = 4; U87: n = 5) or treated with OKN-007 (OKN; oral administration via the drinking water; 18 mg/kg/day; F98: n = 8; U87: n = 5). A significant difference (F98: ***P < .001; U87: **P < .01) was established when comparing the 2 treatment groups over the course of the study, where OKN-treated rats were found to survive longer. Treatment was started when tumors reached 20–50 mm³ in volume, which was at 11–15 days after intracerebral cell implantation of F98 or U87 cells.

Fig. 3.

Bioluminescence imaging (mean radiance [p/s/cm²/sr]) of F98 glioma-bearing rats that were either (A) untreated (UT; n = 4; representative animals 19 days after intracerebral implantation of F98 cells) or (B) treated with OKN-007 (OKN; oral administration via the drinking water; n = 8). F98 cells were transfected with the luciferase gene. Totally regressed tumors are shown at 19 days after cell implantation/4 days after the initiation of OKN-007 treatment (Bi); at day 29 after implantation/14 days after initiation of treatment) (Bii); and at day 30 after implantation/15 days after initiation of treatment (Biv). (Biii) Slow response to OKN-007 (26 days after cell implantation/11 days after initiation of treatment). (C) The mean radiance of OKN-treated rats was evaluated including the total cohort of animals (n = 8) and again with only those animals that were responsive (n = 6). A significant difference (*P < .05) in the mean radiance was obtained when comparing untreated rats with the responsive OKN-treated rats.

Fig. 4.

T2-weighted MR images of (A) untreated (UT; representative rats at 23–24 days after intracerebral implantation of F98 cells; n = 4) and (B) OKN-007 (OKN)–treated F98 glioma-bearing rats (representative responsive [R] and slow- or nonresponsive [NR] rats at [i] 29–30 days and [ii] 23–24 days after cell implantation; n = 8). Glioma boundaries are outlined in light gray dotted lines. (C) Mean F98 tumor volumes calculated from MR images indicate a significant decrease in OKN-treated rats (*P < .05 for the total treated cohort of animals and ***P < .001 for the responsive rats; n = 6; 20–34 days after cell implantation) when compared with UT animals (15–24 days after cell implantations). (D) Mean U87 tumor volumes calculated from MR images indicate a significant decrease in OKN-treated rats (*P < .05 for all rats; n = 5; 35–61 days after cell implantation) when compared with UT animals (n = 5; 18–34 days after cell implantation).

Fig. 5.

Representative IHC-stained histological brain tissue slices from untreated (1) or OKN-007–treated (2) F98 (A) or U87 (B) glioma-bearing rats. (i) Hematoxylin and eosin–stained tissues (H&E) indicated a decrease in cell population after OKN treatment in F98 (A) and U87 (B) gliomas (both at 20× magnification). Decreases in the staining of markers for (ii) cell proliferation (Glut-1) and (iii) hypoxia (HIF-1α) were observed after OKN treatment. An increase in (iv) apoptosis (cleaved caspase 3) after OKN treatment was also observed. Decreases in staining for (v) MVD a marker for angiogenesis were also observed. (vi) VEGF, a marker for angiogenesis, and (vii) CA-IX, a marker for cell differentiation, were also examined. All of the IHC images are at 40× magnification.

Fig. 6.

(A) Mean IHC scores for VEGF, CA-IX, Glut-1, and HIF-1α (±SD) in untreated (dark gray bars; n = 5) and OKN-treated (light gray bars; n = 5) F98 glioma-bearing rats. Significant decreases (*P < .05) were observed for Glut-1 and HIF-1α in OKN-treated rats, compared with untreated animals. (B) Mean IHC index scores (±SD) for MVD (angiogenesis index), MIB-1 (cell proliferation index), and apoptosis (cleaved caspase 3) in untreated (dark gray bars; n = 5) and OKN-treated (light gray bars; n = 5) F98 glioma-bearing rats. (C) Mean IHC scores for VEGF, CA-IX, Glut-1, and HIF-1α (±SD) in untreated (dark gray bars; n = 5) and OKN-treated (light gray bars; n = 5) U87 glioma-bearing rats. (D) Mean IHC index scores (±SD) for apoptosis (cleaved caspase 3) in untreated (dark gray bars; n = 5) and OKN-treated (light gray bars; n = 5) U87 glioma-bearing rats. Significant decreases were observed for Glut-1 (*P < .05 in F98 gliomas, and **P < .01 in U87 gliomas) and HIF-1α (*P < .05 for both F98 and U87 gliomas) in OKN-treated rats, compared with untreated animals. Significant decreases were observed for MVD (*P < .05) and MIB-1 (**P < .01) in F98 OKN-treated rats when compared with untreated animals. A significant increase in apoptosis (***P < .001 for F98 gliomas and *P < .05 for U87 gliomas) was also observed in OKN-treated rats, compared with untreated animals.