Brain cancer is a devastating disease affecting many people worldwide. Effective treatment with chemotherapeutics is limited due to the presence of the blood-brain barrier (BBB) that tightly regulates the diffusion of endogenous molecules but also xenobiotics. Glutathione pegylated liposomal doxorubicin (2B3-101) is being developed as a new treatment option for patients with brain cancer. It is based on already marketed pegylated liposomal doxorubicin (Doxil®/Caelyx®), with an additional glutathione coating that safely enhances drug delivery across the BBB. Uptake of 2B3-101 by human brain capillary endothelial cells in vitro was time-, concentration- and temperature-dependent, while pegylated liposomal doxorubicin mainly remained bound to the cells. In vivo, 2B3-101 and pegylated liposomal doxorubicin had a comparable plasma exposure in mice, yet brain retention 4 days after administration was higher for 2B3-101. 2B3-101 was overall well tolerated by athymic FVB mice with experimental human glioblastoma (luciferase transfected U87MG). In 2 independent experiments a strong inhibition of brain tumor growth was observed for 2B3-101 as measured by bioluminescence intensity. The effect of weekly administration of 5 mg/kg 2B3-101 was more pronounced compared to pegylated liposomal doxorubicin (p<0.05) and saline (p<0.01). Two out of 9 animals receiving 2B3-101 showed a complete tumor regression. Twice-weekly injections of 5 mg/kg 2B3-101 again had a significant effect in inhibiting brain tumor growth (p<0.001) compared to pegylated liposomal doxorubicin and saline, and a complete regression was observed in 1 animal treated with 2B3-101. In addition, twice-weekly dosing of 2B3-101 significantly increased the median survival time by 38.5% (p<0.001) and 16.1% (p<0.05) compared to saline and pegylated liposomal doxorubicin, respectively. Overall, these data demonstrate that glutathione pegylated liposomal doxorubicin enhances the effective delivery of doxorubicin to brain tumors and could become a promising new therapeutic option for the treatment of brain malignancies.

It has been reported that glucocorticoids (GCs) can effectively control seizures in pediatric epilepsy syndromes, possibly by inhibition of inflammation. Since inflammation is supposed to be involved in epileptogenesis, we hypothesized that treatment with GCs would reduce brain inflammation and thereby modify epileptogenesis in a rat model for temporal lobe epilepsy, in which epilepsy gradually develops after electrically induced status epilepticus (SE). To prevent the severe adverse effects that are inevitable with long-term GC treatment, we used liposome nanotechnology (G-Technology(®)) to enhance the sustained delivery to the brain. Starting 4h after onset of SE, rats were treated with glutathione pegylated liposomal methylprednisolone (GSH-PEG liposomal MP) according to a treatment protocol (1× per week; 10mg/kg) that is effective in other models of neuroinflammation. Continuous electro-encephalogram (EEG) recordings revealed that SE duration and onset of spontaneous seizures were not affected by GSH-PEG liposomal MP treatment. The number and duration of spontaneous seizures were also not different between vehicle and GSH-PEG liposomal MP-treated animals. Six weeks after SE, brain inflammation, as assessed by quantification of microglia activation, was not reduced by GSH-PEG liposomal MP-treatment. Also, neuronal cell loss and mossy fiber sprouting were not affected. Our study shows that the selected GSH-PEG liposomal MP treatment regimen that was administered beyond the acute SE phase does not reduce brain inflammation and development of temporal lobe epilepsy.

Hallmarks of CNS inflammation, including microglial and astrocyte activation, are prominent features in post-mortem tissue from amyotrophic lateral sclerosis (ALS) patients and in mice overexpressing mutant superoxide dismutase-1 (SOD1 G93A ). Administration of non-targeted glucocorticoids does not significantly alter disease progression, but this may reflect poor CNS delivery. Here, we sought to discover whether CNS-targeted, liposomal encapsulated glucocorticoid would inhibit the CNS inflammatory response and reduce motor neuron loss. SOD1 G93A mice were treated with saline, free methylprednisolone (MP, 10 mg/kg/week) or glutathione PEGylated liposomal MP (2B3-201, 10 mg/kg/week) and compared to saline treated wild-type animals. Animals were treated weekly with intravenous injections for 9 weeks from 60 days of age. Weights and motor performance were monitored during this period. At the end of the experimental period (116 days) mice were imaged using T2-weighted MRI for brainstem pathology; brain and spinal cord tissue were then collected for histological analysis.

Chemotherapy-induced peripheral neuropathy is a dose-limiting debilitating symptom that can affect many patients treated with anticancer treatments. The resulting neuropathic pain is thought to be a peripheral symptom, however, central sensitization has recently been suggested as a possible causal mechanism for chronic pain conditions; nociceptive neurons in the dorsal horns of the spinal cord become sensitized by a neuroinflammatory response through chronic peripheral tissue damage or inflammation [Zhuo, 2012]. Centrally active anti-inflammatory therapies, such as methylprednisolone (MP), may therefore have beneficial therapeutic properties, but its effective use is limited by several (severe) acute and chronic side effects or highly invasive local delivery routes. Systemic administrations of encapsulated MP in glutathione PEGylated liposomes (2B3-201) have recently resulted in superior efficacy and reduced side effects compared to the free MP in rodent models with neuroinflammation [Gaillard, 2012]. Therefore, 2B3-201 was hypothesized to have therapeutic value in preventing chemo-induced neuropathic pain as well. 2B3-201 was initially investigated in a pharmacokinetic and biodistribution study and compared to free MP, showing an enhanced plasma circulation (half life of ∼7 hours vs. several minutes for free MP), and higher sustained levels of 2B3-201 in brain and spinal cord. Furthermore, 2B3-201 did not result in psychotic-like behavioral effects in rats, as were caused by free MP. Also, repeated weekly administrations of 2B3-201 were well tolerated in rats, while the same weekly doses of free MP were causing side effects. The efficacy of 2B3-201 was investigated at WuXi AppTec (Shanghai, China) in two chemo-induced neuropathic pain models with demonstrated spinal cord pathology as a consequence of repeated bortezomib or paclitaxel administrations. Rats received paclitaxel (1 mg/kg/day, 8x) or bortezomib (0.2 mg/kg/day, 8x), and the effect of 2B3-201 on mechanical allodynia was evaluated at three dose levels (10, 20, or 30 mg/kg). Rats were monitored daily and allodynic measurements were taken at baseline and from days 4 to 8 after treatment start. The body weight gain of the rats was slightly inhibited by paclitaxel and bortezomib, and co-therapy with 2B3-201 showed a dose-dependent larger inhibition of weight gain. Despite the increased signs of chemotherapeutic side effects on body weight, 2B3-201 dose-dependently relieved neuropathic pain in both models. At the highest dose, the significant effect of 2B3-201 on neuropathic pain was no longer observed, which was probably due to the more severe chemotherapeutic side effects. In conclusion, the positive treatment effect of 2B3-201 against chemotherapy-induced neuropathic pain, in combination with a good PK, biodistribution and safety profile, warrant the further development of 2B3-201 for this indication. Citation Format: Pieter J. Gaillard, Rick Dorland, Chantal C. Appeldoorn. 2B3-201, glutathione pegylated liposomal methylprednisolone, prevents chemotherapy-induced neuropathic pain. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4342. doi:10.1158/1538-7445.AM2013-4342

Neuroinflammation contributes to a wide range of disorders of the central nervous system (CNS). Of the available anti-inflammatory drugs, only glucocorticoids have shown central efficacy in CNS-related disorders, such as multiple sclerosis (MS). However, their side effects are dose limiting. To optimally improve the therapeutic window of methylprednisolone, we enhanced its CNS delivery by using pegylated liposomes conjugated to the brain-targeting ligand glutathione. In healthy rats, plasma circulation and brain uptake were significantly increased after encapsulating methylprednisolone in glutathione pegylated (GSH-PEG) liposomes. Furthermore, the efficacy of GSH-PEG liposomal methylprednisolone was investigated in rats with acute experimental autoimmune encephalomyelitis (EAE), an animal model of MS; rats received treatment (10 mg/kg; i.v. injection), before disease onset, at disease onset, or at the peak of disease. Free methylprednisolone and non-targeted pegylated (PEG) liposomal methylprednisolone served as control treatments. When treatment was initiated at disease onset, free methylprednisolone showed no effect, while GSH-PEG liposomal methylprednisolone significantly reduced the clinical signs to 42 ± 6.4% of saline control. Moreover, treatment using GSH-PEG liposomes was significantly more effective compared to PEG liposomes. Our findings hold promise for MS treatment and warrant further investigations into this brain delivery system for the treatment of neuroinflammation.

High-grade glioma is a uniformly fatal disease with an unmet need for better therapy. A major reason for this poor outcome is the invasive nature of gliomas. Novel therapies that target invasive brain tumor cells should be invented, but a major impediment to the delivery of adequate amounts of therapeutics is the blood brain barrier (BBB), which is largely intact in regions where invasive cells reside. Glutathione (GSH)-conjugated PEGylated liposomes (G-TechnologyTM) may be suitable vehicles for targeted delivery of small molecule cytotoxic drugs across the BBB. GSH is a natural anti-oxidant that is found at high levels in the brain and its active transporter is abundantly expressed at the BBB. Previous studies using microdialysis with an increasing % of GSH conjugated to liposomes carrying ribavirin have shown a %GSH-dependent increase of drug levels in brain interstitial fluid (up to 5-fold higher), and GSH-liposomes carrying endomorphin-1 were more effective in hot-plate tests when compared to unconjugated liposomes. We have now tested GSH-conjugated PEGylated liposomes containing doxorubicin (GSH-Doxil) for treatment of mice carrying intracranial U87 xenografts. In a first series, we compared 5%GSH-Doxil to conventional Doxil, free doxorubicin (Dx) and untreated controls. Mice were injected with 10^5 U87-luc cells and bioluminescence (BL) imaging was used for follow up. After 11 days, mice were stratified into control or test groups (n=9 / group). Mice received 3 consecutive weekly dosings of 5 mg/kg Dx-equivalents. The cohorts receiving Doxil and Dx showed a marginal growth delay relative to controls. The response with 5%GSH-Doxil was more promising but variable: two animals receiving 5%GSH-Doxil showed complete regression, which was not observed in any of the other cohorts, whereas other tumors in this cohort responded more similar to the other treatment groups. Since the treatment was well tolerated, we performed another more dose-intense series, administering biweekly 5 mg/kg Dx equivalents. Moreover, 5%GSH-Doxil and 3%GSH-Doxil were tested relative to Doxil and untreated controls. Treatment started at day 14 after tumor cell injection, stratifying only animals whose tumor BL signals increased relative to day 11. After day 25 the animals experienced weight loss that precluded further dosing. In this series, the variation in tumor response was small. There was again one complete regression in the cohort of 5%GSH and not in any of the other cohorts. Moreover, the growth delay in the other tumors in this 5%GSH-Doxil cohort was significantly longer than in any of the other groups. This growth delay translated into a significantly increased median survival of 32.5 days relative to 27 days for untreated controls. The response in the 3%GSH and Doxil cohorts was marginally better relative to controls. Overall these results warrant further preclinical and clinical investigation using 5%GSH-Doxil liposomes. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5537.