Effect of Trans Sodium Crocetinate on Brain Tumor Oxgenation

J Neurosurg 111:226–229, 2009

Effect of trans sodium crocetinate on brain tumor oxgenation

Laboratory investigation

Ja s o n Sh e e h a n , M.D., Ph.D., Jo n a t h a n Sh e r m a n , M.D., Ch r i s t o p h e r Ci f a r e l l i , M.D., Ph.D., Ja y Ja g a n n a t h a n , M.D., Ka s a n d r a Da s s o u l a s , B.S., Cl a i r e Ol s o n , B.A., Je s s i c a Ra i n e y , a n d Sh a o j i e Ha n , M.S. Department of Neurological Surgery, University of Virginia Health System, Charlottesville, Virginia

Object. Glioblastoma multiforme tumors typically exhibit regions of hypoxia. Hypoxic regions within the tumor make cells less sensitive to radiosurgery and radiation therapy. Trans sodium crocetinate (TSC) has been shown to be a radiosensitizer. The goal of this research was to elucidate the underlying mechanism of TSC’s radiosensitizing effect. Methods. A rat C6 glioma model was used. The C6 glioma cells were stereotactically injected into the rat brain to create a tumor. Two weeks later, MR imaging was used to confirm the presence of a glioma. Following demonstration on MR imaging of a brain tumor, animals were randomized into 1 of 2 groups: 1) TSC alone (100 µg/kg), or 2) saline control. Licox probes were inserted into the brain tumor and contralateral cerebral hemisphere. Tissue oxygenation measurements were recorded before and after intravenous infusion of either TSC or saline. Results. Not surprisingly, tissue oxygenation measurements revealed that the brain tumor was hypoxic relative to the contralateral cerebral hemisphere brain tissue. Two to 8 minutes after TSC was infused, tissue oxygenation measurements in the brain tumor increased above baseline by as much as 60%. After this temporary elevation following TSC infusion, tumor oxygenation measurements returned to baseline. No significant elevations in tissue oxygenation were seen on the contralateral side. Similarly, the saline vehicle was not observed to increase tissue oxygenation in either the brain tumor or the contralateral brain tissue. Conclusions. Administration of TSC transiently improves tissue oxygenation in hypoxic gliomas. Such an effect is one potential mechanism for the radiosensitization previously observed after addition of TSC. (DOI: 10.3171/2009.3.JNS081339)

Ke y Wo r d s • trans sodium crocetinate • glioblastoma multiforme • radiosensitization • oxygen diffusion • rat

lioblastoma multiforme is a very aggressive type plasma.11,17 Because TSC also increases the diffusivity of intracranial tumor. The mainstays of treatment of O2 through other aqueous-based systems, it may also are cytoreductive surgery followed by radiation have an effect on transport through extravascular fluid, Gtherapy and chemotherapy.1,2 Areas within these tumors although TSC appears to remain primarily in the intra- have been found to be hypoxic, and hypoxia may de- vascular space. crease the effectiveness of adjuvant therapy.13 Previously, TSC was found to radiosensitize C6 glio- Oxygen is known to be a powerful radiosensitizer of ma cells in vivo.14 In this study we examined the direct mammalian cells.4,15 It can increase the dose efficiency of effects of TSC on the oxygenation of C6 gliomas in an ionizing radiation by a factor of 3. Considering the many in vivo model. intracranial tumors that have regions of relative hypoxia, the importance of raising partial brain O2 concentrations to physiological levels during initiation of treatment can- Methods not be overstated. Study Design and Animals Trans sodium crocetinate, a carotenoid compound, has been shown to increase the diffusion of O in plasma A total of 30 Sprague-Dawley rats (Charles River 2 Laboratories, Inc.) were included in the study. Each ani- and, hence, to lead to improvements in O2 availability to other tissues. The mechanism for this is believed to be due mal weighed between 200 and 250 g. They were allowed to interactions between the hydrophobic TSC molecules free access to food, and a 12:12 hour light/dark cycle was and water, and it should occur in any aqueous solution.17 maintained. All aspects of this research were performed In vitro measurements confirm that there is approximate- according to the National Institutes of Health animal ex- ly a 30% increase in the diffusivity of O through blood perimentation guidelines and approved by the University 2 of Virginia’s animal care and utilization committee. This article contains some figures that are displayed in color Abbreviations used in this paper: PBS = phosphate-buffered online but in black and white in the print edition. saline; TSC = trans sodium crocetinate.

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After the tumor implantation, the animals were ran- the lesion’s depth and cross-sectional diameters were domly assigned to 1 of 2 groups: 1) TSC alone; or 2) con- measured to determine the appropriate insertion depth trol (that is, saline infusion) alone. for the Licox monitor probe’s tip. Probes were inserted into the tumor side through the previously placed bur Cell Line hole, which had been used for stereotactic insertion of the The tumor cell line used was the C6 glioma line glioma cells. from American Type Culture Collection. Cells were cul- tured for 4–5 passages in F-12 Kaighn nutrient mixture Administration of TSC medium (GIBCO) supplemented with 15% horse serum, The TSC was provided as a gift by Diffusion Phar- 2.5% fetal bovine serum, and 1% antibiotic-antimycotic maceuticals. The TSC was administered to the animals solution. The medium was changed twice per week, and by tail vein injection starting on the 14th day after tumor the cells were grown in 75-cm2 culture flasks. Cells were implantation. Following 20 minutes of baseline Licox tis- incubated at 37°C in 5% CO2, passaged weekly by wash- sue measurements in each animal, the TSC was delivered ing with PBS, detached in 10 ml of 0.5 mM ethylenedi- intravenously in bolus form. This 20-minute interval per- aminetetraacetic acid, and subcultured at a ratio of 1:10. mitted equilibration of the probe in the brain or tumor Cells for implantation were obtained by trypsiniza- tissues. Rats in the TSC group were treated with 100 µg/ tion from subconfluent cultures, washed twice with PBS, kg of the drug. Control animals received a comparable and resuspended in 10 mM PBS–glucose to a final con- volume of saline intravenously. centration of 5 × 105/5 μl. The cell density was deter- mined using the microscope stage micrometer. The time Licox Measurements between removal of the cells from culture to implantation A total of 9 animals were excluded from the tissue ox- in the rat host was < 90 minutes. ygenation measurements due to either poor tumor grafting as noted on MR imaging or a substantial clinical deterio- Stereotactic Tumor Implantation Technique ration within the first 2 weeks following surgery. During Prior to the operative procedure, all animals were tissue oxygenation measurements, animals were anesthe- anesthetized by intraperitoneal injection of a mixture tized with ketamine/xylazine for ~ 60 minutes. The previ- of ketamine/xylazine (60–80 mg/5–10 mg/kg). The rat’s ous cranial incision was opened and the bur hole overly- head was fixed in a stereotactic frame (David Kopf In- ing the tumor was localized. A contralateral bur hole was struments). Using sterile technique, a small craniectomy placed overlying the opposite cerebral hemisphere. was drilled (2 × 1 mm) at 3 mm from the midline and 1 Small-animal Licox probes (model #CC1, Integra) mm anterior to the coronal suture. The dura mater was were used. The probe is 0.5 mm in diameter and has a 2 then pierced with a small-gauge needle. A 5-μl volume PO2 sensitive area of 7 mm . Probe error is typically 3.8 ± of 5 × 105 cells were implanted at a rate of 1 μl/minute 3.5%.19 There is a tendency for the probe to underestimate to a depth of 4 mm below the craniectomy into the left O2 tension; there is typically little in the way of drift in frontotemporal region. The needle was left in place for 2 the probe readings during the time span of these experi- minutes before being removed. The craniectomy was then ments (that is, 1–2 hours).19 Twenty minutes of baseline sealed with bone wax, and the scalp was closed with an measurements were taken with each probe, and stabili- interrupted nylon suture. zation of the PO2 measurements was confirmed prior to injection of the agent or vehicle. Two Licox probes were Magnetic Resonance Imaging inserted, one through each bur hole, thereby allowing for Each rat’s brain was imaged 13 days after tumor simultaneous measurements of O2 on the ipsilateral (that implantation by using a Varian MR imaging system (4.7- is, with tumor) and contralateral (without tumor) cerebral T, 40-cm-bore magnet; accessible diameter of 32.4 cm) hemispheres. On the tumor side, the Licox probe was (Varian Medical System) in the University of Virginia’s inserted at a depth derived from the MR imaging that Small Animal Multimodality Imaging Core. would put the probe tip within the tumor itself (Fig. 1). A Gd-enhanced, T1-weighted pulse sequence was Animals spontaneously breathed room air during tissue used for imaging (TR/TE 400/20, number of excitations oxygenation measurements. 2, field of view 8 cm, matrix size 256 × 192, slice thick- Ultimately, tissue oxygenation measurements were ness 3 mm). The pulse sequences allowed 30 slices per made in 12 TSC-treated animals and in 9 saline-treated scan, which covered the entire brain. The average scan controls. Tissue oxygenation changes were expressed as a time was 3 minutes. All imaging was performed 1 minute ratio of the tissue oxygenation at a single time point fol- after an intraperitoneal injection of 1 ml (70 mg) of gado- lowing TSC or saline infusion, divided by the mean tissue teridol (Prohance; Bracco Diagnostics, Inc.). The images oxygenation over a 20-minute time period prior to the in- obtained in the sessions were analyzed to verify tumor fusion. Measurements were taken at 2-minute intervals. implantation in each animal. Animals were excluded from the tissue oxygenation Statistical Analysis measurements if there was no discernible evidence of To evaluate for differences in tissue oxygenation be- tumor, as indicated by the presence of a circumscribed tween treatment groups, a commercial statistical package lesion with Gd enhancement on the postoperative MR im- (SPSS version 15.0; SPSS, Inc.) was used. A probability val- ages. In those animals with tumor visible on MR images, ue of ≤ 0.05 was considered to be statistically significant.

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ably require a better understanding of tumor hypoxia and hypoxia-induced gene expression.10 The ability to alter hypoxia in tissue has many potential roles for CNS lesions. In the setting of a brain tumor, shifting hypoxic tissue to normoxia may permit more beneficial effects from radiation or chemotherapy. Simi- larly, increased oxygenation may facilitate improvements in the rate and extent of recovery from hypoxic injury associated with a stroke or neurotrauma. Normoxia rather than hyperoxia appears to be more appropriate in the clinical arena for pathological entities in the CNS.5 Normal brain tissue is very well perfused and therefore well oxygenated. Quantitative MR imaging of human brain perfusion reveals that gray matter is perfused at a rate of 93 ± 16 ml/100 g/min, white matter at a rate of 38 ± 10 ml/100 g/min, and whole brain at a rate of 52 ± 8 ml/100 g/min.12 This translates to a typical tissue oxygenation in the brain of > 25 mm Hg. In the setting of normoxic brain tissue, TSC did not appreciably Fig. 1. Coronal T1-weighted contrast-enhanced MR image illustrat- alter partial tissue oxygenation. However, in the context ing the location of tissue oxygenation measurements of the tumor (T) and brain (B). of hypoxia associated with a high-grade glioma, a single intravenous bolus of TSC temporarily elevated tissue oxygenation above its baseline hypoxic state. Results The selection of the 100 µg/kg dose of TSC is based on a dose-response curve generated in an ischemic rat Licox measurements were taken in the cerebral hemi- model and the effective dose for radiosensitization seen spheres ipsilateral and contralateral to the C6 glioma. by this group (unpublished data).14 It is also within the Absolute tissue oxygenation measurements of C6 glioma effective TSC range of 60–180 µg/kg reported by other tissue were consistent with hypoxic tissue (< 25 mm Hg), groups.7–9 The short duration of the elevation in tumor ox- whereas the contralateral cerebral hemisphere typically ygenation following TSC administration may have been a showed normal partial tissue oxygenation. During the ~ function of the method of administration (bolus) and the 1 hour of tissue oxygenation monitoring, there were no pharmacokinetics of the drug. Refinements in delivery, statistically significant differences noted in the normal- dose, and half-life of the agent may improve the therapeu- ized oxygenation values among the following groups: 1) tic window of improved tissue oxygenation. brain parenchyma in saline-treated rats; 2) tumor in sa- The increased order within plasma as facilitated by line-treated rats; and 3) brain parenchyma in TSC-treated TSC would not seem to increase the oxygenation of al- animals. ready well-perfused brain tissue. The TSC appears to al- Nevertheless, a marked increase in tissue oxygenation ter the hydrogen bonding patterns in serum and thereby measurements was observed within the brain tumors of cause an increase in the rate of O2 diffusion across cap- those animals in which TSC was administered. A statisti- illary membranes.7 In the setting of hypoxia, the effect cally significant elevation in tissue oxygenation was seen of increased O2 diffusion seems to be accentuated. In from 22 to 28 minutes into the experiments (p < 0.05; a previous study,17 TSC has been shown to increase the t-test). Because the agent (or saline) was administered diffusion of glucose and O2 through water by 25–30%. after 20 minutes of baseline oxygenation monitoring, This effect is probably achieved through induction of or- this amounted to elevations in tissue oxygenation ~ 2–8 der in the surrounding water structure through increased minutes after administration of TSC. The TSC increased hydrogen bonding of water molecules.17,18 In the setting brain tumor oxygenation measurements by factors of 1.66, of hemorrhagic shock, TSC has been shown to reduce 1.48, 1.38, and 1.33 at 2, 4, 6, and 8 minutes, respectively, lactate levels, presumably through increased O2 diffusiv- 7,15 following TSC bolus (p < 0.05). Tissue oxygenation in the ity. Also, in animals receiving supplemental O2, TSC brain tumors of the TSC-treated animals was increased was found to increase O2 delivery to baseline normoxic by as much as 66% before returning to baseline (Fig. 2). brain tissue.11 In previous work by our group, TSC was seen to in- 14 Discussion crease the sensitivity of the C6 glioma to radiation. The current results would serve as a reasonable explanation High-grade gliomas are well known to have regions for this effect. During irradiation, tumor cell death results of tissue hypoxia, which is likely to contribute to the re- from direct ionizations within the pathological tissue. In- sistance of gliomas to radiosurgery, radiation therapy, creased oxygenation can enhance free-radical generation and chemotherapy.16 It may also stimulate angiogenesis within the treated volume.3 Thus, temporary improve- through vascular endothelial growth factor upregulation ment in oxygenation of a hypoxic tumor appears to afford and lead to glioma cell invasion.6,20 The development of a therapeutic advantage during delivery of certain forms antiangiogenic therapies for CNS malignancies will prob- of adjuvant treatment. Further study of this compound in

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Okonkwo DO, Wagner J, Melon DE, Alden T, Stone JR, Helm GA, et al: Trans-sodium crocetinate increases oxygen delivery Fig. 2. Graph showing normalized partial tissue oxygenation follow- to brain parenchyma in rats on oxygen supplementation. Neu ing administration of TSC in a rat model of C6 glioma. The TSC was rosci Lett 352:97–100, 2003 administered 20 minutes after the start of Licox monitoring. Statistically 12. Roberts DA, Detre JA, Bolinger L, Insko EK, Leigh JS Jr: significant differences (denoted by asterisks) were noted at 22-, 24-, Quantitative magnetic resonance imaging of human brain 26-, and 28-minute time points in the tumor tissue of the animals receiv- perfusion at 1.5T using steady state inversion of arterial water. ing TSC. Proc Natl Acad Sci U S A 91:33–37, 1994 13. Rong Y, Durden DL, Van Meir EG, Brat DJ: 'Pseudopalisad- the setting of hypoxic conditions within the CNS seems ing' necrosis in glioblastoma: a familiar morphologic feature that links vascular pathology, hypoxia, and angiogenesis. J warranted. Neuropathol Exp Neurol 65:529–539, 2006 14. Sheehan J, Ionescu A, Pouratian N, Hamilton DK, Schlesinger D, Oskouian RJ Jr, et al: Use of trans sodium crocetinate for Conclusions sensitizing glioblastoma multiforme to radiation. Laboratory Intratumoral hypoxia is a common finding in the set- investigation. J Neurosurg 108:972–978, 2008 ting of high-grade gliomas, and has been shown to dimin- 15. Shrieve DC, Klish M, Wendland MW, Watson GA: Basic prin- ciples of radiobiology, radiotherapy, and radiosurgery. Neuro ish the effectiveness of radiosurgery. Treatment with TSC surg Clin N Am 15:467–479, 2004 appears to increase the tissue oxygenation temporarily in 16. Spence AM, Muzi M, Swanson KR, O'Sullivan F, Rockhill hypoxic C6 gliomas. Under a normal fraction of inspired JK, Rajendran JG, et al: Regional hypoxia in glioblastoma oxygen, TSC did not elevate the oxygenation concentra- multiforme quantified with [18F]fluoromisonidazole positron tion of normal brain tissue. Additional testing of TSC ap- emission tomography before radiotherapy: correlation with pears warranted for evaluation of therapeutic advantages time to progression and survival. Clin Cancer Res 14:2623– afforded by a compound that reduces hypoxia in patho- 2630, 2008 logical tissue. 17. Stennett AK, Dempsey GL, Gainer JL: trans-Sodium crocetinate and diffusion enhancement. J Phys Chem B 110:18078– 18080, 2006 Disclosure 18. Stennett AK, Gainer JL: TSC for hemorrhagic shock: effects on cytokines and blood pressure. Shock 22:569–574, 2004 The TSC was provided by Diffusion Pharmaceuticals (Charlot 19. Stewart C, Haitsma I, Zador Z, Morabito D, Manley G, Rosen- tesville, VA), and Licox probes were provided by Integra Lifesciences thal G, et al: The new licos brain tissue oxygenation and brain Corp. (Plainsboro, NJ). Also, Diffusion provided a grant to fund a temperature monitor: assessment of in vitro accuracy and clin portion of this research. Diffusion Pharmaceuticals did not devise ical experience in severe traumatic brain injury. Neurosur the scientific methodology and had no role in the data analysis or gery 63:1159–1165, 2008 preparation of the manuscript. The investigators have not received 20. Zagzag D, Lukyanov Y, Lan L, Ali MA, Esencay M, Men- any financial payment from these companies. dez O, et al: Hypoxia-inducible factor 1 and VEGF upregulate CXCR4 in glioblastoma: implications for angiogenesis and References glioma cell invasion. Lab Invest 86:1221–1232, 2006 1. Burton EC, Prados MD: Malignant gliomas. Curr Treat Op tions Oncol 1:459–468, 2000 2. Butowski NA, Sneed PK, Chang SM: Diagnosis and treatment Manuscript submitted October 18, 2008. of recurrent high-grade astrocytoma. J Clin Oncol 24:1273– Accepted March 6, 2009. 1280, 2006 Please include this information when citing this paper: published 3. Chapman JD, Reuvers AP, Borsa J, Greenstock JL: Chemi- online March 27, 2009; DOI: 10.3171/2009.3.JNS081339 . cal radioprotection and radiosensitization of mammalian cells Address correspondence to: Jason Sheehan, M.D., Box 800-212, growing in vitro. Radiat Res 56:291–306, 1973 Health Sciences Center, Charlottesville, Virginia 22908. email: 4. Coleman CN, Turrisi AT: Radiation and chemotherapy sensi- [email protected].

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