Partly due to poor blood-brain barrier drug penetration the treatment options for many brain diseases are limited. To safely enhance drug delivery to the brain, glutathione PEGylated liposomes (G-Technology (R)) were developed. In this study, in rats, we compared the pharmacokinetics and organ distribution of GSH-PEG liposomes using an autoquenched fluorescent tracer after intraperitoneal administration and intravenous administration. Although the appearance of liposomes in the circulation was much slower after intraperitoneal administration, comparable maximum levels of long circulating liposomes were found between 4 and 24 h after injection. Furthermore, 24 h after injection a similar tissue distribution was found. To investigate the effect of GSH coating on brain delivery in vitro uptake studies in rat brain endothelial cells (RBE4) and an in vivo brain microdialysis study in rats were used. Significantly more fluorescent tracer was found in RBE4 cell homogenates incubated with GSH-PEG liposomes compared to non-targeted PEG liposomes (1.8-fold, p<0.001). In the microdialysis study 4-fold higher (p<0.001) brain levels of fluorescent tracer were found after intravenous injection of GSH-PEG liposomes compared with PEG control liposomes. The results support further investigation into the versatility of GSH-PEG liposomes for enhanced drug delivery to the brain within a tolerable therapeutic window.

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.

Methylprednisolone (MP) pulses are the mainstay for relapse therapy in multiple sclerosis (MS). To improve the efficacy of treatment and reduce the side effects of MP, a long circulating brain-targeted formulation was developed; glutathione polyethylene glycol (PEG)ylated liposomal MP (2B3-201). Here we investigate the efficacy of 2B3-201 in murine myelin oligodendrocyte induced experimental autoimmune encephalomyelitis (MOG-EAE), an animal model mimicking inflammatory features and neurodegenerative aspects of MS. After disease onset, mice were randomized to receive either saline, three injections of free MP (high dose MP, 100mg/kg i.v.), two injections of free MP (low dose MP, 10mg/kg; i.v.), or two injections of 2B3-201 (10mg/kg i.v.). Treatment with a low dose of 2B3-201 significantly reduced the severity of EAE as compared to saline control, similar to treatment with high dose free MP, while a low dose of free MP was not effective. In a histological analysis of the spinal cord, treatment with 2B3-201 significantly decreased T cell as well as macrophage/microglia infiltration in the CNS by about 50%. Moreover, application of a low dose of 2B3-201 or a high dose of free MP reduced the amount of astrocyte activation as well as the extent of axonal loss and also demyelination in spinal cord lesions as compared to low dose MP or sham treatment. In summary, in the murine MOG-EAE model of MS, a glutathione PEGylated liposomal formulation of MP (2B3-201) is clinically and histologically as effective as free MP at one tenth of the dosage as well as at a lower application frequency and clearly more effective than the same dosage of free MP. These positive proof-of-concept efficacy studies warrant further development of 2B3-201 for the treatment of neuroinflammatory conditions such as MS.

PURPOSE:Ocular inflammation is associated with the loss of visual acuity and subsequent blindness. Since their development, glucocorticoids have been the mainstay of therapy for ocular inflammatory diseases. However, the clinical benefit is limited by side effects due to the chronic use and generally high dosage that is required for effective treatment. We have developed the G-Technology to provide a means for sustained drug delivery, increased drug half-life, and reduced bodily drug exposure. Glutathione PEGylated liposomal methylprednisolone (2B3-201) has been developed as treatment for neuroinflammatory conditions and was evaluated in ocular inflammation.METHODS:The efficacy of 2B3-201 was investigated in rats with experimental autoimmune uveitis (EAU). Rats received 10 mg/kg of 2B3-201 intravenously at disease onset and at peak of the disease. The same dose of free methylprednisolone served as control treatment. Clinical signs of ocular inflammation were assessed by slit-lamp and immunohistochemistry.RESULTS:Whereas free methylprednisolone was ineffective, two doses of 2B3-201 almost completely abolished clinical signs of EAU. This was corroborated further by immunohistochemical analyses of isolated eyes. Treatment with 2B3-201 significantly reduced the infiltration of inflammatory cells and subsequent destruction of the retina cell layers.CONCLUSIONS:In this study, we show that systemic treatment with 2B3-201, a glutathione PEGylated liposomal methylprednisolone formulation, resulted in a superior efficacy in rats with EAU. Altogether, our findings hold promise for the development of a safe and more convenient systemic treatment for uveitis.

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

The blood–brain barrier presents a significant hurdle in CNS drug development. Blood-to-brain delivery by effectively crossing this barrier allows therapeutics to reach a large area of the brain. Over the past decades several drug delivery technologies have been developed, some more successful than others, which we hold against 10 key development criteria. Adhering to these criteria will allow a more successful development of therapeutics for patients with devastating brain diseases.

Drug delivery to the brain remains challenging due to the presence of the blood-brain barrier. In this review, 10 key development criteria are presented that are important for successful drug development to treat CNS diseases by targeted drug delivery systems. Although several routes of delivery are being investigated, such as intranasal delivery, direct injections into the brain or CSF, and transient opening of the blood-brain barrier, the focus of this review is on physiological strategies aiming to target endogenous transport mechanisms. Examples from literature, focusing on targeted drug delivery systems that are being commercially developed, will be discussed to illustrate the 10 key development criteria. The first four criteria apply to the targeting of the blood-brain barrier: (1) a proven inherently safe receptor biology, (2) a safe and human applicable ligand, (3) receptor specific binding, and (4) applicable for acute and chronic indications. Next to an efficient and safe targeting strategy, as captured in key criteria 1 to 4, a favorable pharmacokinetic profile is also important (key criterion 5). With regard to the drug carriers, two criteria are important: (6) no modification of active ingredient and (7) able to carry various classes of molecules. The final three criteria apply to the development of a drug from lab to clinic: (8) low costs and straightforward manufacturing, (9) activity in all animal models, and (10) strong intellectual property (IP) protection. Adhering to these 10 key development criteria will allow for a successful brain drug development.

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.

Abstract Treatment options for brain cancer are limited and overall prognoses are poor. Glutathione pegylated liposomal doxorubicin (2B3-101) is being developed as a new treatment option for patients with brain cancer. It is essentially based on the already marketed pegylated liposomal doxorubicin (Doxil), with an additional glutathione (GSH) coating to safely enhance delivery across the blood-brain barrier. In a GLP safety study 2B3-101 was administered by intravenous bolus injection on days 1, 15 and 29 to Wistar rats at 1.75, 3.5 and 7 mg/kg. Doxil (7 mg/kg), empty GSH-PEG-liposomes, and vehicle were included, resulting in 6 groups (10 animals/sex/group). Additional animals were allowed 4 weeks of recovery or were used for toxicokinetic analysis. From observations during the in-life part of the study, as well as after histopathological investigations, no major differences were noted between 2B3-101 and Doxil at 7 mg/kg q14dx3. No treatment-related changes in the brain were observed neither after repeated 2B3-101 exposure, nor after treatment with empty GSH-PEG liposomes. In addition, 2B3-101 did not result in doxorubicin-related toxicity in heart tissue. After 1st and 3rd administration of 7 mg/kg, plasma AUC values were 10-44% higher for Doxil when compared to 2B3-101, while C0 values were considered similar for both drug substances. Apparent half-lives for Doxil were 36.1 and 32.6 hours in males and females after the 3rd administration, respectively. For 2B3-101 these values were slightly shorter at 30.0 and 28.8 hours in males and females, respectively. The doxorubicin brain/plasma ratio showed retention of both 2B3-101 and Doxil after repeated doses, where 2B3-101 resulted in significantly higher doxorubicin retention in the brain 2 weeks after the 3rd administration (p<0.02). A modified Irwin test in male Wistar rats (n=8 per group) showed no neurobehavioral effects and no effects of 2B3-101 on body temperature after administration of 1.75 mg/kg, 3.5 mg/kg and 7 mg/kg. Also, empty GSH-PEG liposomes and Doxil did not result in neurobehavioral effects and effects on body temperature. Efficacy of GMP grade 2B3-101 at the maximum tolerated dose was confirmed in mice with intracranial U87 xenografts; compared to saline 10 mg/kg q4dx4 and 18 mg/kg q8dx2 showed a survival benefit of up to 60%, where 18 mg/kg q8dx2 seemed superior. In addition, in mice carrying subcutaneous MDA-MB-231 xenografts it was shown that 10 mg/kg q4dx3 2B3-101 and Doxil both produced significant anti-tumor activity, were well tolerated, and gave similar weight loss profiles. In conclusion, the therapeutic benefit and predictable safety profile of clinical-scale 2B3-101 was successfully demonstrated in preclinical studies. A clinical trial to determine the safety, tolerability and pharmacokinetics of 2B3-101 in patients with solid tumors and brain metastases or recurrent malignant glioma was initiated in June 2011. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5687. doi:1538-7445.AM2012-5687

Drug delivery to the brain remains challenging due to the presence of the blood-brain barrier. In this review, 10 key development criteria are presented that are important for successful drug development to treat CNS diseases by targeted drug delivery systems. Although several routes of delivery are being investigated, such as intranasal delivery, direct injections into the brain or CSF, and transient opening of the blood-brain barrier, the focus of this review is on physiological strategies aiming to target endogenous transport mechanisms. Examples from literature, focusing on targeted drug delivery systems that are being commercially developed, will be discussed to illustrate the 10 key development criteria. The first four criteria apply to the targeting of the blood-brain barrier: (1) a proven inherently safe receptor biology, (2) a safe and human applicable ligand, (3) receptor specific binding, and (4) applicable for acute and chronic indications. Next to an efficient and safe targeting strategy, as captured in key criteria 1 to 4, a favorable pharmacokinetic profile is also important (key criterion 5). With regard to the drug carriers, two criteria are important: (6) no modification of active ingredient and (7) able to carry various classes of molecules. The final three criteria apply to the development of a drug from lab to clinic: (8) low costs and straightforward manufacturing, (9) activity in all animal models, and (10) strong intellectual property (IP) protection. Adhering to these 10 key development criteria will allow for a successful brain drug development.

RNA interference (RNAi) allows the specific knockdown of tumor relevant genes. To induce RNAi, the delivery of small interfering RNAs (siRNAs) is of crucial importance. This is particularly challenging for their therapeutic applications in vivo. Low molecular weight branched polyethylenimine (PEI) is safe and efficient for nucleic acid delivery including small RNA molecules, based on its ability to electrostatically complex siRNA molecules, thereby protecting them from nuclease degradation. The nanoscale PEI/siRNA complexes are endocytosed by cells prior to intracellular complex release from the lysosome and cytoplasmic release of the siRNAs from the complexes. Chemical modification and ligand decoration of the complexes aim at introducing target tissue specificity and further increased efficacy of PEI-mediated siRNA delivery. CRM197 is a mutated, non-toxic diphtheria toxin (DT) that binds to the membrane-bound precursor of HB-EGF-like growth factor/diphtheria toxin receptor highly expressed in glioblastoma cells. Likewise, the growth factor pleiotrophin (PTN/HB-GAM/HARP) is overexpressed in glioblastoma and is rate limiting for tumor growth, thus representing an attractive target gene for therapeutic knockdown approaches. PEGylation of PEI was performed to reduce the surface charge, and by CRM197 coupling we prepared a modified PEI for siRNA delivery into glioblastoma cells. The novel PEI conjugates were analyzed for their complexation efficiency and optimal mixing ratios, and complexes were physicochemically characterized regarding stability, size and zeta potential. The biological activity of the complexes was confirmed in cell culture by reporter gene knockdown. For the therapeutic treatment of subcutaneous human gliobastoma xenografts in athymic nude mice, we systemically injected the modified PEI/siRNA complexes targeting PTN. Antitumor effects based on PTN knockdown demonstrated the advantage of tumor-targeted CRM197-PEG-PEI/siRNA over untargeted PEG-PEI polyplexes. Thus, we establish targeted CRM197-PEG-PEI-based complexes for siRNA delivery in vivo, and show therapeutic effects of CRM197-PEG-PEI/siRNA-mediated knockdown of PTN.

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.

In this report, we describe a novel phage display strategy for the identification of dedicated protease inhibiting peptides, based on degradation-aided enrichment of protease resistant phages. Phages were directly incubated with a range of phage-degrading proteases, after which non-degraded phages were used for the next selection round. For proteinase-K we identified after only four selection rounds a peptide (VLIMPVLLGIPLLC) that inhibits proteinase-K activity with an inhibition constant of 4 microM. In analogy, we identified a peptide capable of inhibiting substrate degradation by cathepsin-S (VWNCERITISRLIN), which showed functional inhibition of cathepsin-S induced sprouting of endothelial cells. We envision that the pursued strategy of degradation-aided selection of protease inhibitors (DASPI) represents an effective approach in the design of new protease inhibitors but also of new strategies to render gene and drug vectors protease resistant.