Bystander effects, pharmacokinetics, and linker-payload stability of EGFR-targeting antibody-drug conjugates Losatuxizumab vedotin and Depatux-M in glioblastoma models.

PURPOSE: Antibody-drug conjugates (ADCs) are targeted therapies with robust efficacy in solid cancers, and there is intense interest in using EGFR-specific ADCs to target EGFR-amplified glioblastoma (GBM). Given the molecular heterogeneity of GBM, bystander activity of ADCs may be important for determining treatment efficacy. In this study, the activity and toxicity of two EGFR-targeted ADCs, Losatuxizumab vedotin (ABBV-221) and Depatuxizumab mafodotin (Depatux-M), with similar auristatin toxins, were compared in GBM patient-derived xenografts (PDXs) and normal murine brain following direct infusion by convection enhanced delivery (CED). METHODS: EGFRviii-amplified and non-amplified GBM PDXs were used to determine in vitro cytotoxicity, in vivo efficacy, and bystander activities of ABBV-221 and Depatux-M. Non-tumor bearing mice were used to evaluate pharmacokinetics and toxicity of ADCs using LC-MS/MS and immunohistochemistry. RESULTS: CED improved intracranial efficacy of Depatux-M and ABBV-221 in three EGFRviii-amplified GBM PDX models (Median survival: 125 to >300 days vs 20-49 days with isotype-control AB095). Both ADCs had comparable in vitro and in vivo efficacy. However, neuronal toxicity and CD68+ microglia/macrophage infiltration were significantly higher in brains infused with ABBV-221, with the cell-permeable MMAE, as compared to Depatux-M, with the cell-impermeant MMAF. CED infusion of ABBV-221 into brain or incubation of ABBV-221 with normal brain homogenate resulted in significant release of MMAE, which is consistent with linker instability in the brain microenvironment. CONCLUSION: EGFR-targeting ADCs are promising therapeutic options for GBM when delivered intra-tumorally by CED. However, the linker and payload for the ADC must be carefully considered to maximize the therapeutic window.

Convection enhanced delivery of EGFR targeting antibody-drug conjugates Serclutamab talirine and Depatux-M in glioblastoma patient-derived xenografts.

Background: EGFR targeting antibody-drug conjugates (ADCs) are highly effective against EGFR-amplified tumors, but poor distribution across the blood-brain barrier (BBB) limits their efficacy in glioblastoma (GBM) when administered systemically. We studied whether convection-enhanced delivery (CED) can be used to safely infuse ADCs into orthotopic patient-derived xenograft (PDX) models of EGFRvIII mutant GBM. Methods: The efficacy of the EGFR-targeted ADCs depatuxizumab mafodotin (Depatux-M) and Serclutamab talirine (Ser-T) was evaluated in vitro and in vivo. CED was performed in nontumor and tumor-bearing mice. Immunostaining was used to evaluate ADC distribution, pharmacodynamic effects, and normal cell toxicity. Results: Dose-finding studies in orthotopic GBM6 identified single infusion of 2 µg Ser-T and 60 µg Depatux-M as safe and effective associated with extended survival prolongation (>300 days and 95 days, respectively). However, with serial infusions every 21 days, four Ser-T doses controlled tumor growth but was associated with lethal toxicity approximately 7 days after the final infusion. Limiting dosing to two infusions in GBM108 provided profound median survival extension of over 200 days. In contrast, four Depatux-M CED doses were well tolerated and significantly extended survival in both GBM6 (158 days) and GBM108 (310 days). In a toxicity analysis, Ser-T resulted in a profound loss in NeuN+ cells and markedly elevated GFAP staining, while Depatux-M was associated only with modest elevation in GFAP staining. Conclusion: CED of Depatux-M is well tolerated and results in extended survival in orthotopic GBM PDXs. In contrast, CED of Ser-T was associated with a much narrower therapeutic window.

The influence of the blood-brain barrier in the treatment of brain tumours.

Brain tumours have a poor prognosis and lack effective treatments. The blood-brain barrier (BBB) represents a major hurdle to drug delivery to brain tumours. In some locations in the tumour, the BBB may be disrupted to form the blood-brain tumour barrier (BBTB). This leaky BBTB enables diagnosis of brain tumours by contrast enhanced magnetic resonance imaging; however, this disruption is heterogeneous throughout the tumour. Thus, relying on the disrupted BBTB for achieving effective drug concentrations in brain tumours has met with little clinical success. Because of this, it would be beneficial to design drugs and drug delivery strategies to overcome the 'normal' BBB to effectively treat the brain tumours. In this review, we discuss the role of BBB/BBTB in brain tumour diagnosis and treatment highlighting the heterogeneity of the BBTB. We also discuss various strategies to improve drug delivery across the BBB/BBTB to treat both primary and metastatic brain tumours. Recognizing that the BBB represents a critical determinant of drug efficacy in central nervous system tumours will allow a more rapid translation from basic science to clinical application. A more complete understanding of the factors, such as BBB-limited drug delivery, that have hindered progress in treating both primary and metastatic brain tumours, is necessary to develop more effective therapies.

Central Nervous System Distribution of an Opioid Agonist Combination with Synergistic Activity.

Novel combinations of specific opioid agonists like loperamide and oxymorphindole targeting the µ- and δ-opioid receptors, respectively, have shown increased potency with minimized opioid-associated risks. However, whether their interaction is pharmacokinetic or pharmacodynamic in nature has not been determined. This study quantitatively determined whether these drugs have a pharmacokinetic interaction that alters systemic disposition or central nervous system (CNS) distribution. We performed intravenous and oral in vivo pharmacokinetic assessments of both drugs after discrete dosing and administration in combination to determine whether the combination had any effect on systemic pharmacokinetic parameters or CNS exposure. Drugs were administered at 5 or 10 mg/kg i.v. or 30 mg/kg orally to institute for cancer research (ICR) mice and 5 mg/kg i.v. to Friend leukemia virus strain B mice of the following genotypes: wild-type, breast cancer resistance protein (Bcrp-/- ) (Bcrp knockout), Mdr1a/b-/- [P-glycoprotein (P-gp) knockout], and Bcrp-/- Mdr1a/b-/- (triple knockout). In the combination, clearance of oxymorphindole (OMI) was reduced by approximately half, and the plasma area under the concentration-time curve (AUC) increased. Consequently, brain and spinal cord AUCs for OMI in the combination also increased proportionately. Both loperamide and OMI are P-gp substrates, but administration of the two drugs in combination does not alter efflux transport at the CNS barriers. Because OMI alone shows appreciable brain penetration but little therapeutic efficacy on its own, and because loperamide's CNS distribution is unchanged in the combination, the mechanism of action for the increased potency of the combination is most likely pharmacodynamic and most likely occurs at receptors in the peripheral nervous system. This combination has favorable characteristics for future development. SIGNIFICANCE STATEMENT: Opioids have yet to be replaced as the most effective treatments for moderate-to-severe pain and chronic pain, but their side effects are dangerous. Combinations of opioids with peripheral activity, such as loperamide and oxymorphindole, would be valuable in that they are effective at much lower doses and have reduced risks for dangerous side effects because the µ-opioid receptor agonist is largely excluded from the CNS.

Efflux Limits Tumor Drug Delivery Despite Disrupted BBB.

Apparent blood-brain barrier (BBB) disruption is common in glioblastoma (GBM), but has not translated to improved drug delivery efficacy. Recently, de Gooijer et al. demonstrated that efflux transporters can have a prominent role in limiting drug delivery. These transport systems contribute to ineffective drug delivery to tumor cells in the brain.

Addressing BBB Heterogeneity: A New Paradigm for Drug Delivery to Brain Tumors.

Effective treatments for brain tumors remain one of the most urgent and unmet needs in modern oncology. This is due not only to the presence of the neurovascular unit/blood-brain barrier (NVU/BBB) but also to the heterogeneity of barrier alteration in the case of brain tumors, which results in what is referred to as the blood-tumor barrier (BTB). Herein, we discuss this heterogeneity, how it contributes to the failure of novel pharmaceutical treatment strategies, and why a "whole brain" approach to the treatment of brain tumors might be beneficial. We discuss various methods by which these obstacles might be overcome and assess how these strategies are progressing in the clinic. We believe that by approaching brain tumor treatment from this perspective, a new paradigm for drug delivery to brain tumors might be established.

Permeability changes and effect of chemotherapy in brain adjacent to tumor in an experimental model of metastatic brain tumor from breast cancer.

BACKGROUND: Brain tumor vasculature can be significantly compromised and leakier than that of normal brain blood vessels. Little is known if there are vascular permeability alterations in the brain adjacent to tumor (BAT). Changes in BAT permeability may also lead to increased drug permeation in the BAT, which may exert toxicity on cells of the central nervous system. Herein, we studied permeation changes in BAT using quantitative fluorescent microscopy and autoradiography, while the effect of chemotherapy within the BAT region was determined by staining for activated astrocytes. METHODS: Human metastatic breast cancer cells (MDA-MB-231Br) were injected into left ventricle of female NuNu mice. Metastases were allowed to grow for 28 days, after which animals were injected fluorescent tracers Texas Red (625 Da) or Texas Red dextran (3 kDa) or a chemotherapeutic agent 14C-paclitaxel. The accumulation of tracers and 14C-paclitaxel in BAT were determined by using quantitative fluorescent microscopy and autoradiography respectively. The effect of chemotherapy in BAT was determined by staining for activated astrocytes. RESULTS: The mean permeability of texas Red (625 Da) within BAT region increased 1.0 to 2.5-fold when compared to normal brain, whereas, Texas Red dextran (3 kDa) demonstrated mean permeability increase ranging from 1.0 to 1.8-fold compared to normal brain. The Kin values in the BAT for both Texas Red (625 Da) and Texas Red dextran (3 kDa) were found to be 4.32 ± 0.2 × 105 mL/s/g and 1.6 ± 1.4 × 105 mL/s/g respectively and found to be significantly higher than the normal brain. We also found that there is significant increase in accumulation of 14C-Paclitaxel in BAT compared to the normal brain. We also observed animals treated with chemotherapy (paclitaxel (10 mg/kg), erubilin (1.5 mg/kg) and docetaxel (10 mg/kg)) showed activated astrocytes in BAT. CONCLUSIONS: Our data showed increased permeation of fluorescent tracers and 14C-paclitaxel in the BAT. This increased permeation lead to elevated levels of activated astrocytes in BAT region in the animals treated with chemotherapy.

Liposomal Irinotecan Accumulates in Metastatic Lesions, Crosses the Blood-Tumor Barrier (BTB), and Prolongs Survival in an Experimental Model of Brain Metastases of Triple Negative Breast Cancer.

PURPOSE: The blood-tumor barrier (BTB) limits irinotecan distribution in tumors of the central nervous system. However, given that the BTB has increased passive permeability we hypothesize that liposomal irinotecan would improve local exposure of irinotecan and its active metabolite SN-38 in brain metastases relative to conventional irinotecan due to enhanced-permeation and retention (EPR) effect. METHODS: Female nude mice were intracardially or intracranially implanted with human brain seeking breast cancer cells (brain metastases of breast cancer model). Mice were administered vehicle, non-liposomal irinotecan (50 mg/kg), liposomal irinotecan (10 mg/kg and 50 mg/kg) intravenously starting on day 21. Drug accumulation, tumor burden, and survival were evaluated. RESULTS: Liposomal irinotecan showed prolonged plasma drug exposure with mean residence time (MRT) of 17.7 ± 3.8 h for SN-38, whereas MRT was 3.67 ± 1.2 for non-liposomal irinotecan. Further, liposomal irinotecan accumulated in metastatic lesions and demonstrated prolonged exposure of SN-38 compared to non-liposomal irinotecan. Liposomal irinotecan achieved AUC values of 6883 ± 4149 ng-h/g for SN-38, whereas non-liposomal irinotecan showed significantly lower AUC values of 982 ± 256 ng-h/g for SN-38. Median survival for liposomal irinotecan was 50 days, increased from 37 days (p<0.05) for vehicle. CONCLUSIONS: Liposomal irinotecan accumulates in brain metastases, acts as depot for sustained release of irinotecan and SN-38, which results in prolonged survival in preclinical model of breast cancer brain metastasis.

Trastuzumab distribution in an in-vivo and in-vitro model of brain metastases of breast cancer.

BACKGROUND: Drug and antibody delivery to brain metastases has been highly debated in the literature. The blood-tumor barrier (BTB) is more permeable than the blood-brain barrier (BBB), and has shown to have highly functioning efflux transporters and barrier properties, which limits delivery of targeted therapies. METHODS: We characterized the permeability of 125I-trastuzumab in an in-vivo, and fluorescent trastuzumab-Rhodamine123 (t-Rho123) in a novel microfluidic in-vitro, BBB and BTB brain metastases of breast cancer model. In-vivo: Human MDA-MB-231-HER2+ metastatic breast cancer cells were grown and maintained under static conditions. Cells were harvested at 80% confluency and prepped for intra-cardiac injection into 20 homozygous female Nu/Nu mice. In-vitro: In a microfluidic device (SynVivo), human umbilical vein endothelial cells were grown and maintained under shear stress conditions in the outer compartment and co-cultured with CTX-TNA2 rat brain astrocytes (BBB) or Met-1 metastatic HER2+ murine breast cancer cells (BTB), which were maintained in the central compartment under static conditions. RESULTS: Tissue distribution of 125I-trastuzumab revealed only ~3% of injected dose reached normal brain, with ~5% of injected dose reaching brain tumors. No clear correlation was observed between size of metastases and the amount of 125I-trastuzumab localized in-vivo. This heterogeneity was paralleled in-vitro, where the distribution of t-Rho123 from the outer chamber to the central chamber of the microfluidic device was qualitatively and quantitatively analyzed over time. The rate of t-Rho123 linear uptake in the BBB (0.27 ± 0.33 × 104) and BTB (1.29 ± 0.93 × 104) showed to be significantly greater than 0 (p < 0.05). The BTB devices showed significant heterogenetic tendencies, as seen in in-vivo. CONCLUSIONS: This study is one of the first studies to measure antibody movement across the blood-brain and blood-tumor barriers, and demonstrates that, though in small and most likely not efficacious quantities, trastuzumab does cross the blood-brain and blood-tumor barriers.

Permeability across a novel microfluidic blood-tumor barrier model.

BACKGROUND: The lack of translatable in vitro blood-tumor barrier (BTB) models creates challenges in the development of drugs to treat tumors of the CNS and our understanding of how the vascular changes at the BBB in the presence of a tumor. METHODS: In this study, we characterize a novel microfluidic model of the BTB (and BBB model as a reference) that incorporates flow and induces shear stress on endothelial cells. Cell lines utilized include human umbilical vein endothelial cells co-cultured with CTX-TNA2 rat astrocytes (BBB) or Met-1 metastatic murine breast cancer cells (BTB). Cells were capable of communicating across microfluidic compartments via a porous interface. We characterized the device by comparing permeability of three passive permeability markers and one marker subject to efflux. RESULTS: The permeability of Sulforhodamine 101 was significantly (p < 0.05) higher in the BTB model (13.1 ± 1.3 × 10-3, n = 4) than the BBB model (2.5 ± 0.3 × 10-3, n = 6). Similar permeability increases were observed in the BTB model for molecules ranging from 600 Da to 60 kDa. The function of P-gp was intact in both models and consistent with recent published in vivo data. Specifically, the rate of permeability of Rhodamine 123 across the BBB model (0.6 ± 0.1 × 10-3, n = 4), increased 14-fold in the presence of the P-gp inhibitor verapamil (14.7 ± 7.5 × 10-3, n = 3) and eightfold with the addition of Cyclosporine A (8.8 ± 1.8 × 10-3, n = 3). Similar values were noted in the BTB model. CONCLUSIONS: The dynamic microfluidic in vitro BTB model is a novel commercially available model that incorporates shear stress, and has permeability and efflux properties that are similar to in vivo data.

Identification of Novel Agents for the Treatment of Brain Metastases of Breast Cancer.

BACKGROUND: Brain cancer from metastasized breast cancer has a high mortality rate in women. The treatment of lesions is hampered in large part by the blood-brain barrier (BBB), which prevents adequate distribution of anti-cancer compounds to brain metastases. METHOD: In this study we used a novel screening method to identify candidate molecules that are well-suited to utilizing the BBB choline transporter for distribution into the brain parenchyma. RESULTS: From our screen we identified two compounds, Ch-1 and Ch-2 that were able to reduce the brain tumor burden in a murine mouse model of brain metastasis of breast cancer. These compounds also significantly increased the survival of mice by more than 10 days. Mechanistic studies indicated that Ch-1 is able to prevent the activation of the pro-survival mitogen-activated kinases (MAPKs) by osteoactivin (OA; Glycoprotein nonmetastatic melanoma protein B GPNMB). CONCLUSION: The results from this study show that nutrient transporter virtual screening is a viable novel alternative to traditional drug screening programs to identify anti-cancer compounds for the treatment of brain cancers.