Pharmacokinetics of Enarodustat in Non-Japanese and Japanese Healthy Subjects and in Patients With End-Stage Renal Disease on Hemodialysis.

The pharmacokinetics of enarodustat were elucidated in healthy subjects and in patients with end-stage renal disease (ESRD) on hemodialysis in phase 1 studies conducted in the United States and Japan. In healthy non-Japanese and Japanese subjects, following single oral administration up to 400 mg, enarodustat was rapidly absorbed. Maximum plasma concentration and area under the plasma concentration-time curve from the time of dosing to infinity were dose-dependent, renal excretion of unchanged enarodustat was substantial (on average ≈45% of dose), and mean t1/2 of <10 hours indicated negligible accumulation with once-daily dosing. In general, with daily dosing (25, 50 mg), accumulation at steady-state was ≈1.5-fold (t1/2(eff) ≈15 hours), presumably due to a decrease in renal drug excretion which is not clinically relevant in patients with ESRD. In the single- and multiple-dose studies, plasma clearance (CL/F) was lower in healthy Japanese subjects. In non-Japanese patients with ESRD on hemodialysis, following once-daily dosing (2-15 mg), enarodustat was rapidly absorbed, steady-state maximum plasma concentration and area under the plasma concentration-time curve during the dosing interval were dose-dependent, and interindividual variability in the exposure parameters was low-to-moderate (coefficient of variation, 27%-39%). Steady-state CL/F was similar across doses, renal drug excretion was not significant (<10% of dose), mean t1/2 and t1/2(eff) were similar (overall, 8.97-11.6 hours), and accumulation was minimal (≈20%), demonstrating predictable pharmacokinetics. Japanese patients with ESRD on hemodialysis (15 mg, single dose) exhibited similar pharmacokinetics with mean t1/2 of 11.3 hours and low interindividual variability in the exposure parameters, albeit with lower CL/F versus non-Japanese patients. Body weight-adjusted clearance values were generally similar in non-Japanese and Japanese healthy subjects and also in patients with ESRD on hemodialysis.

Nanoparticle-based targeting of microglia improves the neural regeneration enhancing effects of immunosuppression in the zebrafish retina.

Retinal Müller glia function as injury-induced stem-like cells in zebrafish but not mammals. However, insights gleaned from zebrafish have been applied to stimulate nascent regenerative responses in the mammalian retina. For instance, microglia/macrophages regulate Müller glia stem cell activity in the chick, zebrafish, and mouse. We previously showed that post-injury immunosuppression by the glucocorticoid dexamethasone accelerated retinal regeneration kinetics in zebrafish. Similarly, microglia ablation enhances regenerative outcomes in the mouse retina. Targeted immunomodulation of microglia reactivity may therefore enhance the regenerative potential of Müller glia for therapeutic purposes. Here, we investigated potential mechanisms by which post-injury dexamethasone accelerates retinal regeneration kinetics, and the effects of dendrimer-based targeting of dexamethasone to reactive microglia. Intravital time-lapse imaging revealed that post-injury dexamethasone inhibited microglia reactivity. The dendrimer-conjugated formulation: (1) decreased dexamethasone-associated systemic toxicity, (2) targeted dexamethasone to reactive microglia, and (3) improved the regeneration enhancing effects of immunosuppression by increasing stem/progenitor proliferation rates. Lastly, we show that the gene rnf2 is required for the enhanced regeneration effect of D-Dex. These data support the use of dendrimer-based targeting of reactive immune cells to reduce toxicity and enhance the regeneration promoting effects of immunosuppressants in the retina.

Dendrimer-enabled targeted delivery attenuates glutamate excitotoxicity and improves motor function in a rabbit model of cerebral palsy.

Glutamate carboxypeptidase II (GCPII), localized on the surface of astrocytes and activated microglia, regulates extracellular glutamate concentration in the central nervous system (CNS). We have previously shown that GCPII is upregulated in activated microglia in the presence of inflammation. Inhibition of GCPII activity could reduce glutamate excitotoxicity, which may decrease inflammation and promote a 'normal' microglial phenotype. 2-(3-Mercaptopropyl) pentanedioic acid (2-MPPA) is the first GCPII inhibitor that underwent clinical trials. Unfortunately, immunological toxicities have hindered 2-MPPA clinical translation. Targeted delivery of 2-MPPA specifically to activated microglia and astrocytes that over-express GCPII has the potential to mitigate glutamate excitotoxicity and attenuate neuroinflammation. In this study, we demonstrate that 2-MPPA when conjugated to generation-4, hydroxyl-terminated polyamidoamine (PAMAM) dendrimers (D-2MPPA) localize specifically in activated microglia and astrocytes only in newborn rabbits with cerebral palsy (CP), not in controls. D-2MPPA treatment led to higher 2-MPPA levels in the injured brain regions compared to 2-MPPA treatment, and the extent of D-2MPPA uptake correlated with the injury severity. D-2MPPA was more efficacious than 2-MPPA in decreasing extracellular glutamate level in ex vivo brain slices of CP kits, and in increasing transforming growth factor beta 1 (TGF-ß1) level in primary mixed glial cell cultures. A single systemic intravenous dose of D-2MPPA on postnatal day 1 (PND1) decreased microglial activation and resulted in a change in microglial morphology to a more ramified form along with amelioration of motor deficits by PND5. These results indicate that targeted dendrimer-based delivery specifically to activated microglia and astrocytes can improve the efficacy of 2-MPPA by attenuating glutamate excitotoxicity and microglial activation.

Targeted Microglial Attenuation through Dendrimer-Drug Conjugates Improves Glaucoma Neuroprotection.

Retinal microglial/macrophage activation and optic nerve (ON) microglial/macrophage activation are glaucoma biomarkers and potential therapeutic targets for this blinding disease. We report targeting of activated microglia by PAMAM dendrimers in a rat glaucoma model and neuroprotection by N-acetylcysteine-conjugated dendrimer (D-NAC) conjugates in a post-injury rescue experiment. Intravitreally delivered fluorescently labeled dendrimer (D-Cy5) conjugates targeted and were retained in Iba-1-positive cells (90% at 7 days and 55% after 28 days) in the retina following intraocular pressure (IOP) elevation, while systemically delivered D-Cy5 targeted ON cells. A single intravitreal D-NAC dose given 1 week after IOP elevation significantly reduced transcription of pro-inflammatory (IL-6, MCP-1, IL-1ß) and A1 astrocyte (Serping1, Fkbp5, Amigo2) markers and increased survival of retinal ganglion cells (39 ± 12%) versus BSS- (20 ± 15%, p = 0.02) and free NAC-treated (26 ± 14%, p = 0.15) eyes. These results highlight the potential of dendrimer-targeted microglia and macrophages for early glaucoma detection and as a neuroprotective therapeutic target.

Dendrimer-2PMPA Delays Muscle Function Loss and Denervation in a Murine Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease where muscle weakness and neuromuscular junction (NMJ) denervation precede motor neuron cell death. Although acetylcholine is the canonical neurotransmitter at the mammalian NMJ synapse, glutamate has recently been identified as a critical neurotransmitter for NMJ development and maintenance. One source of glutamate is through the catabolism of N-acetyl-aspartyl-glutamate (NAAG), which is found in mM concentrations in mammalian motoneurons, where it is released upon stimulation and hydrolyzed to glutamate by the glial enzyme glutamate carboxypeptidase II (GCPII). Using the SOD1G93A model of ALS, we found an almost fourfold elevation of GCPII enzymatic activity in SOD1G93A versus WT muscle and a robust increase in GCPII expression which was specifically associated with activated macrophages infiltrating the muscle. 2-(Phosphonomethyl)pentanedioic acid (2PMPA) is a potent GCPII inhibitor which robustly blocks glutamate release from NAAG but is highly polar with limited tissue penetration. To improve this, we covalently attached 2PMPA to a hydroxyl polyamidoamine (PAMAM-G4-OH) dendrimer delivery system (D-2PMPA) which is known to target activated macrophages in affected tissues. Systemic D-2PMPA therapy (20 mg/kg 2PMPA equivalent; IP 2 × /week) was found to localize in muscle macrophages in SOD1G93A mice and completely normalize the enhanced GCPII activity. Although no changes in body weight or survival were observed, D-2PMPA significantly improved grip strength and inhibited the loss of NMJ innervation in the gastrocnemius muscles. Our finding that inhibiting elevated GCPII activity in SOD1G93A muscle can prolong muscle function and delay NMJ denervation may have early therapeutic implications for ALS patients.

Dendrimer-2PMPA selectively blocks upregulated microglial GCPII activity and improves cognition in a mouse model of multiple sclerosis.

Cognitive impairment is a common aspect of multiple sclerosis (MS) for which there are no treatments. Reduced brain N-acetylaspartylglutamate (NAAG) levels are linked to impaired cognition in various neurological diseases, including MS. NAAG levels are regulated by glutamate carboxypeptidase II (GCPII), which hydrolyzes the neuropeptide to N-acetyl-aspartate and glutamate. GCPII activity is upregulated multifold in microglia following neuroinflammation. Although several GCPII inhibitors, such as 2-PMPA, elevate brain NAAG levels and restore cognitive function in preclinical studies when given at high systemic doses or via direct brain injection, none are clinically available due to poor bioavailability and limited brain penetration. Hydroxyl-dendrimers have been successfully used to selectively deliver drugs to activated glia. Methods: We attached 2-PMPA to hydroxyl polyamidoamine (PAMAM) dendrimers (D-2PMPA) using a click chemistry approach. Cy5-labelled-D-2PMPA was used to visualize selective glial uptake in vitro and in vivo. D-2PMPA was evaluated for anti-inflammatory effects in LPS-treated glial cultures. In experimental autoimmune encephalomyelitis (EAE)-immunized mice, D-2PMPA was dosed biweekly starting at disease onset and cognition was assessed using the Barnes maze, and GCPII activity was measured in CD11b+ hippocampal cells. Results: D-2PMPA showed preferential uptake into microglia and robust anti-inflammatory activity, including elevations in NAAG, TGFß, and mGluR3 in glial cultures. D-2PMPA significantly improved cognition in EAE mice, even though physical severity was unaffected. GCPII activity increased >20-fold in CD11b+ cells from EAE mice, which was significantly mitigated by D-2PMPA treatment. Conclusions: Hydroxyl dendrimers facilitate targeted drug delivery to activated microglia. These data support further development of D-2PMPA to attenuate elevated microglial GCPII activity and treat cognitive impairment in MS.

Dendrimer-Triamcinolone Acetonide Reduces Neuroinflammation, Pathological Angiogenesis, and Neuroretinal Dysfunction in Ischemic Retinopathy.

Diabetic retinopathy (DR) is the leading cause of blindness in working-age adults. Severe visual loss in DR is primarily due to proliferative diabetic retinopathy, characterized by pathologic preretinal angiogenesis driven by retinal ischemia. Microglia, the resident immune cells of the retina, have emerged as a potentially important regulator of pathologic retinal angiogenesis. Corticosteroids including triamcinolone acetonide (TA), known for their antiangiogenic effects, are used in treating retinal diseases, but their use is significantly limited by side effects including cataracts and glaucoma. Generation-4 hydroxyl polyamidoamine dendrimer nanoparticles are utilized to deliver TA to activated microglia in the ischemic retina in a mouse model of oxygen-induced retinopathy (OIR). Following intravitreal injection, dendrimer-conjugated TA (D-TA) exhibits selective localization and sustained retention in activated microglia in disease-associated areas of the retina. D-TA, but not free TA, suppresses inflammatory cytokine production, microglial activation, and preretinal neovascularization in OIR. In addition, D-TA, but not free TA, ameliorates OIR-induced neuroretinal and visual dysfunction. These results indicate that activated microglia are a promising therapeutic target for retinal angiogenesis and neuroprotection in ischemic retinal diseases. Furthermore, dendrimer-based targeted therapy and specifically D-TA constitute a promising treatment approach for DR, offering increased and sustained drug efficacy with reduced side effects.

Systemic dendrimer nanotherapies for targeted suppression of choroidal inflammation and neovascularization in age-related macular degeneration.

Inflammation and neovascularization are key pathological events in human age-related macular degeneration (AMD). Activated microglia/macrophages (mi/ma) and retinal pigmented epithelium (RPE) play an active role in every stage of disease progression. Systemic therapies that can target these cells and address both inflammation and neovascularization will broaden the impact of existing therapies and potentially open new avenues for early AMD where there are no viable therapies. Utilizing a clinically relevant rat model of AMD that mirrors many aspects that of human AMD pathological events, we show that systemic hydroxyl-terminated polyamidoamine dendrimer-triamcinolone acetonide conjugate (D-TA) is selectively taken up by the injured mi/ma and RPE (without the need for targeting ligands). D-TA suppresses choroidal neovascularization significantly (by >80%, >50-fold better than free drug), attenuates inflammation in the choroid and retina, by limiting macrophage infiltration in the pathological area, significantly suppressing pro-inflammatory cytokines and pro-angiogenic factors, with minimal side effects to healthy ocular tissue and other organs. In ex vivo studies on human postmortem diabetic eyes, the dendrimer is also taken up into choroidal macrophages. These results suggest that the systemic hydroxyl dendrimer-drugs can offer new avenues for therapies in treating early/dry AMD and late/neovascular AMD alone, or in combination with current anti-VEGF therapies. This hydroxyl dendrimer platform but conjugated to a different drug is undergoing clinical trials for severe COVID-19, potentially paving the way for faster clinical translation of similar compounds for ocular and retinal disorders.

Burst Pressures in Eyes with Clear Corneal Incisions Treated with ReSure Glue.

PURPOSE: This study aims to measure burst pressures in 3 mm clear corneal incisions sealed with ReSure, a biodegradable hydrogel sealant, and to compare it to traditional 10-0 nylon sutures and unsealed controls. DESIGN: An ex vivo animal study. METHODS: 3 mm clear corneal incisions were performed in rabbit eyes (ex vivo). The burst pressure was determined, and then, the incisions were sealed with either ReSure glue or a single 10-0 nylon suture. Burst pressure measurements were repeated. RESULTS: Fourteen eyes were included. The median burst pressure in the suture-control group (7 eyes) prior to suture application was 7 mmHg (range: 0-45); the median burst pressure in the 7 glue-controls was 36 mmHg (range: 5-61, p = 0.08 for the comparison of the two control groups). The median burst pressure in the glue group was 93 mmHg (range: 39-129, p = 0.043 when compared to glue-control). The median burst pressure in the suture group was 158 mmHg (range: 70-180, p = 0.018 when compared to suture-control). There was no statistically significant difference in burst pressure values between the glue and suture groups (p = 0.08). CONCLUSION: In this study, ReSure glue applied to 3 mm clear corneal incisions provided sufficient resistance to elevated intraocular pressure when compared to controls. The results of this study suggest that ReSure glue may be comparable to a single 10-0 nylon suture in resisting fluid egress during the early postoperative period.

Thermosensitive and biodegradable hydrogel encapsulating targeted nanoparticles for the sustained co-delivery of gemcitabine and paclitaxel to pancreatic cancer cells.

Pancreatic cancer represents a life threatening disease with rising mortality. Although the synergistic combination of gemcitabine and albumin-bound paclitaxel has proven to enhance the median survival rates as compared to gemcitabine alone, their systemic and repeated co-administration has been associated with serious toxic side effects and poor patient compliance. For this purpose, we designed a thermosensitive and biodegradable hydrogel encapsulating targeted nanoparticles for the local and sustained delivery of gemcitabine (GEM) and paclitaxel (PTX) to pancreatic cancer. GEM and PTX were loaded into PR_b-functionalized liposomes targeting integrin α5ß1, which was shown to be overexpressed in pancreatic cancer. PR_b is a fibronectin-mimetic peptide that binds to α5ß1 with high affinity and specificity. The PR_b liposomes were encapsulated into a poly(δ-valerolactone-co-D,L-lactide)-b-poly(ethylene glycol)-b-poly(δ-valerolactone-co-D,L-lactide) (PVLA-PEG-PVLA) hydrogel and demonstrated sustained release of both drugs compared to PR_b-functionalized liposomes free in solution or free drugs in the hydrogel. Moreover, the hydrogel-nanoparticle system was proven to be very efficient towards killing monolayers of human pancreatic cancer cells (PANC-1), and showed a significant reduction in the growth pattern of PANC-1 tumor spheroids as compared to hydrogels encapsulating non-targeted liposomes with GEM/PTX or free drugs, after a one week treatment period. Our hybrid hydrogel-nanoparticle system is a promising platform for the local and sustained delivery of GEM/PTX to pancreatic cancer, with the goal of maximizing the therapeutic efficacy of this synergistic drug cocktail while potentially minimizing toxic side effects and eliminating the need for repeated co-administration.

Evolution of oxidative stress, inflammation and neovascularization in the choroid and retina in a subretinal lipid induced age-related macular degeneration model.

Oxidative stress, inflammation and neovascularization are the key pathological events that are implicated in human age-related macular degeneration (AMD). There are a limited number of animal models available for evaluating and developing new therapies. Most models represent late exudative or neovascular AMD (nAMD) but there is a relative paucity of models that mimic early events in AMD. The purpose of this study is to characterize the evolution of oxidative stress, inflammation, retinal degeneration and neovascularization in a rat model of AMD, created by subretinal injection of human lipid hydroperoxide (HpODE) that found in the sub-macular region in aged and AMD patients. Subretinal HpODE induced retinal pigment epithelium (RPE) and retinal degeneration resulting in loss of RPE cells, photoreceptors and retinal thinning. RPE degeneration and atrophy were detected by day 5, followed by neural tissue degeneration at day 12 with robust TUNEL positive cells. Western blot analysis confirmed an increase in pro-apoptotic Bak protein at day 12 in retinal tissues. Oxidative damage biomarkers (4-hydroxynonenal, malondialdehyde, 8-hydroxy-2'-deoxyguanosine, and nitrotyrosine) increased in retinal tissue from days 5-12. Müller glial activation was observed in the HpODE injected area at day 5 followed by its remodeling and migration in the outer retina by day 20. RT-qPCR analysis further indicated upregulation of pro-inflammatory genes (TNF-α, IL-1ß and IL-6) both in retinal and RPE/choroidal tissue as early as day 2 and persisted until day 12. Upregulation of oxidative stress markers such as NADPH oxidase (NOX and DOUX family) was detected early in retinal tissue by day 2 followed by its upregulation in choroidal tissue at day 5. Neovascularization was demonstrated from day 12 to day 20 post HpODE injection in choroidal tissue. The results from this study indicate that subretinal HpODE induces advanced AMD phenotypes comprising many aspects of both dry/early and late) and neovascular/late AMD as observed in humans. Within 3 weeks via oxidative damage, upregulation of reactive oxygen species and pro-inflammatory genes, pro-apoptotic Bak and pro-angiogenic VEGF upregulation occurs leading to CNV formation. This experimental model of subretinal HpODE is an appropriate model for the study of AMD and provides an important platform for translational and basic research in developing new therapies particularly for early/dry AMD where currently no viable therapies are available.

Development and Application of a Mechanistic Population Modeling Approach to Describe Abemaciclib Pharmacokinetics.

Abemaciclib is an oral anticancer drug that inhibits cyclin dependent kinases 4 and 6 and is metabolized by cytochrome P450 3A in the intestines and liver to active metabolites. The objectives were (1) to develop a mechanistic model to characterize the pharmacokinetics (PK) of the active moieties and investigate the effect of patient factors and (2) apply the model to dat from two phase III breast cancer trials of abemaciclib in combination with endocrine therapy. To develop the model, data from seven phase I studies and two phase II studies including 421 patients with cancer and 65 healthy individuals were pooled for nonlinear mixed effects modeling. The PK was similar between patients and healthy subjects, and the effects of diarrhea, formulation, race, and patient covariates on exposure were negligible. Application of the model confirmed its predictive performance and that abemaciclib PK did not change when coadministered with endocrine therapy.

A role for mast cells in geographic atrophy.

Mast cells (MCs) are the initial responders of innate immunity and their degranulation contribute to various etiologies. While the abundance of MCs in the choroid implies their fundamental importance in the eye, little is known about the significance of MCs and their degranulation in choroid. The cause of geographic atrophy (GA), a progressive dry form of age-related macular degeneration is elusive and there is currently no therapy for this blinding disorder. Here we demonstrate in both human GA and a rat model for GA, that MC degranulation and MC-derived tryptase are central to disease progression. Retinal pigment epithelium degeneration followed by retinal and choroidal thinning, characteristic phenotypes of GA, were driven by continuous choroidal MC stimulation and activation in a slow release fashion in the rat. Genetic manipulation of MCs, pharmacological intervention targeting MC degranulation with ketotifen fumarate or inhibition of MC-derived tryptase with APC 366 prevented all of GA-like phenotypes following MC degranulation in the rat model. Our results demonstrate the fundamental role of choroidal MC involvement in GA disease etiology, and will provide new opportunities for understanding GA pathology and identifying novel therapies targeting MCs.

Dendrimer mediated targeted delivery of sinomenine for the treatment of acute neuroinflammation in traumatic brain injury.

Traumatic brain injury (TBI) is a significant medical problem with limited treatment options and is one of the main causes of life-long disability. Neuroinflammation orchestrated by activated microglia/macrophages at the site of injury plays a critical role in the onset of many pathological events following TBI, leading to blood brain barrier (BBB) dysfunction, neuronal damage and long term neuronal and behavioral deficits. Current treatment involves intravenous administration of anti-inflammatory drugs which have limited clinical outcomes only when dosed within the early time window after injury. Hence there is an urgent need to develop improved drug delivery systems which have potential to cross impaired BBB, target and deliver drugs selectively to activated microglia/macrophages at the sites of injury, and suppress the detrimental effects of acute inflammation. In this study, we have used Sinomenine (Sino), a potent anti-inflammatory and antioxidant drug conjugated to hydroxyl terminated generation-4 PAMAM dendrimer (D-Sino) as a potential therapy for attenuating early inflammation in TBI. D-Sino conjugates were synthesized using highly robust copper-catalyzed click reaction with high purity. D-Sino conjugates enhanced the intracellular availability of Sino due to their rapid cellular uptake, significantly attenuated early/acute inflammation by suppressing pro-inflammatory cytokines (TNF-α, IL-1ß, CCL-3 and IL-6), and reduced oxidative stress (iNOS and NO) in LPS activated murine macrophages (RAW 264.7) by inhibiting NF-κB activation and its nuclear translocation (the root cause for inflammation inception) significantly more as compared to the free drug. Upon systemic administration in a rabbit model of pediatric TBI, D-Sino conjugates specifically targeted activated microglia/macrophages at the site of injury in the brain. Single dose of D-Sino attenuated inflammation in the injured brain areas by suppressing inflammatory cytokines expression whereas free Sino treatment did not demonstrate a significant effect. Together, these results suggest that D-Sino conjugate may open up new avenues for increasing the therapeutic window in the treatment of early inflammation and for improving the efficacy of the drug in TBI. Moreover, this treatment can work in conjunction with current clinical practices such as therapeutic hypothermia and pharmacologically induced coma for many indications associated with TBI, where acute inflammation plays a critical role in disease progression.

Dense hydroxyl polyethylene glycol dendrimer targets activated glia in multiple CNS disorders.

Poor transport of neuropharmaceutics through central nervous system (CNS) barriers limits the development of effective treatments for CNS disorders. We present the facile synthesis of a novel neuroinflammation-targeting polyethylene glycol-based dendrimer (PEGOL-60) using an efficient click chemistry approach. PEGOL-60 reduces synthetic burden by achieving high hydroxyl surface density at low generation, which plays a key role in brain penetration and glia targeting of dendrimers in CNS disorders. Systemically administered PEGOL-60 crosses impaired CNS barriers and specifically targets activated microglia/macrophages at the injured site in diverse animal models for cerebral palsy, glioblastoma, and age-related macular degeneration, demonstrating its potential to overcome impaired blood-brain, blood-tumor-brain, and blood-retinal barriers and target key cells in the CNS. PEGOL-60 also exhibits powerful intrinsic anti-oxidant and anti-inflammatory effects in inflamed microglia in vitro. Therefore, PEGOL-60 is an effective vehicle to specifically deliver therapies to sites of CNS injury for enhanced therapeutic outcomes in a range of neuroinflammatory diseases.

Abemaciclib in Combination with Single-Agent Options in Patients with Stage IV Non-Small Cell Lung Cancer: A Phase Ib Study.

Purpose: Abemaciclib, a dual inhibitor of cyclin-dependent kinases 4 and 6, has demonstrated preclinical activity in non-small cell lung cancer (NSCLC). A multicenter, nonrandomized, open-label phase Ib study was conducted to test safety, MTD, pharmacokinetics, and preliminary antitumor activity of abemaciclib in combination with other therapies for treatment in patients with metastatic NSCLC.Patients and Methods: An initial dose escalation phase was used to determine the MTD of twice-daily oral abemaciclib (150, 200 mg) plus pemetrexed, gemcitabine, or ramucirumab, followed by an expansion phase for each drug combination. Pemetrexed and gemcitabine were administered according to label. The abemaciclib plus ramucirumab study examined two dosing schedules.Results: The three study parts enrolled 86 patients; all received ≥1 dose of combination therapy. Across arms, the most common treatment-emergent adverse events were fatigue, diarrhea, neutropenia, decreased appetite, and nausea. The trial did not identify an abemaciclib MTD for the combination with pemetrexed or gemcitabine but did so for the combination of abemaciclib with days 1 and 8 ramucirumab (8 mg/kg). Plasma sample analysis showed that abemaciclib did not influence the pharmacokinetics of the combination agents and the combination agents did not affect abemaciclib exposure. The disease control rate was 57% for patients treated with abemaciclib-pemetrexed, 25% for abemaciclib-gemcitabine, and 54% for abemaciclib-ramucirumab. Median progression-free survival was 5.55, 1.58, and 4.83 months, respectively.Conclusions: Abemaciclib demonstrated an acceptable safety profile when dosed on a continuous twice-daily schedule in combination with pemetrexed, gemcitabine, or ramucirumab. Abemaciclib exposures remained consistent with those observed in single-agent studies. Clin Cancer Res; 24(22); 5543-51. ©2018 AACR.

Scalable synthesis and validation of PAMAM dendrimer-N-acetyl cysteine conjugate for potential translation.

Dendrimer-N-acetyl cysteine (D-NAC) conjugate has shown significant promise in multiple preclinical models of brain injury and is undergoing clinical translation. D-NAC is a generation-4 hydroxyl-polyamidoamine dendrimer conjugate where N-acetyl cysteine (NAC) is covalently bound through disulfide linkages on the surface of the dendrimer. It has shown remarkable potential to selectively target and deliver NAC to activated microglia and astrocytes at the site of brain injury in several animal models, producing remarkable improvements in neurological outcomes at a fraction of the free drug dose. Here we present a highly efficient, scalable, greener, well-defined route to the synthesis of D-NAC, and validate the structure, stability and activity to define the benchmarks for this compound. This newly developed synthetic route has significantly reduced the synthesis time from three weeks to one week, uses industry-friendly solvents/reagents, and involves simple purification procedures, potentially enabling efficient scale up.

Dendrimer-N-acetyl-L-cysteine modulates monophagocytic response in adrenoleukodystrophy.

OBJECTIVE: X-linked adrenoleukodystrophy (ALD) is a neurodegenerative disorder due to mutations in the peroxisomal very long-chain fatty acyl-CoA transporter, ABCD1, with limited therapeutic options. ALD may manifest in a slowly progressive adrenomyeloneuropathy (AMN) phenotype, or switch to rapid inflammatory demyelinating cerebral disease (cALD), in which microglia have been shown to play a pathophysiological role. The aim of this study was to determine the role of patient phenotype in the immune response of ex vivo monophagocytic cells to stimulation, and to evaluate the efficacy of polyamidoamine dendrimer conjugated to the antioxidant precursor N-acetyl-cysteine (NAC) in modulating this immune response. METHODS: Human monophagocytic cells were derived from fresh whole blood, from healthy (n = 4), heterozygote carrier (n = 4), AMN (n = 7), and cALD (n = 4) patients. Cells were exposed to very long-chain fatty acids (VLCFAs; C24:0 and C26:0) and treated with dendrimer-NAC (D-NAC). RESULTS: Ex vivo exposure to VLCFAs significantly increased tumor necrosis factor α (TNFα) and glutamate secretion from cALD patient macrophages. Additionally, a significant reduction in total intracellular glutathione was observed in cALD patient cells. D-NAC treatment dose-dependently reduced TNFα and glutamate secretion and replenished total intracellular glutathione levels in cALD patient macrophages, more efficiently than NAC. Similarly, D-NAC treatment decreased glutamate secretion in AMN patient cells. INTERPRETATION: ALD phenotypes display unique inflammatory profiles in response to VLCFA stimulation, and therefore ex vivo monophagocytic cells may provide a novel test bed for therapeutic agents. Based on our findings, D-NAC may be a viable therapeutic strategy for the treatment of cALD. Ann Neurol 2018;84:452-462.

Subconjunctival dendrimer-drug therapy for the treatment of dry eye in a rabbit model of induced autoimmune dacryoadenitis.

PURPOSE: To investigate the efficacy of a single subconjunctival injection of dendrimer-dexamethasone conjugate in a rabbit model of induced autoimmune dacryoadenitis (AID). METHODS: Dendrimer biodistribution after subconjunctival injection in AID animals was evaluated using Cy5-labelled dendrimer (D-Cy5) and confocal microscopy. Diseased animals were treated with free dexamethasone (Free-Dex), dendrimer-dexamethasone (D-Dex), or saline via a single subconjunctival injection. The efficacy was evaluated using various clinical evaluations, such as Schirmer's test, tear breakup time (TBUT), and fluorescein and rose Bengal staining. Histopathology was evaluated by H&E staining and immunostaining. Levels of inflammatory cytokines and aquaporin proteins in the LGs were determined by real-time PCR. RESULTS: Subconjunctivally administered dendrimers selectively localized in the inflamed LGs, and were taken up by the infiltrating cells. At two weeks post single dose-treatment, the D-Dex group showed improved clinical evaluations. No significant changes were observed in other groups. H&E staining demonstrated less inflammatory cell infiltration and fewer atrophic acini in D-Dex group, compared to those treated with saline or Free-Dex. Immunohistochemistry demonstrated that the intensity of CD-18 (+) and RTLA (+) was weaker in LGs in the D-Dex group than in other treatment groups. Pro-inflammatory gene expression levels of MMP9, IL6, IL8, and TNFα were significantly decreased in the D-Dex group compared to the Free-Dex and saline group. CONCLUSIONS: The dendrimer exhibits pathology-dependent biodistribution in the inflamed LGs. Subconjunctivally administered D-Dex suppressed LG inflammation, leading to partial recovery of LG function with clinical improvement in induced AID. Sjögren's patients may benefit from this targeted nanomedicine approach.