Detection of mucopolysaccharidosis type II by measurement of iduronate-2-sulfatase in dried blood spots and plasma samples

BACKGROUND: Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder related to a deficiency in the enzyme iduronate-2-sulfatase (IDS). Clinical trials of enzyme replacement therapy are in progress, but effective treatment will require screening assays to enable early detection and diagnosis of MPS II. Our study evaluated the diagnostic accuracy of IDS protein and enzyme activity measurements in dried blood spots and plasma. METHODS: We collected dried-blood-spot and plasma samples from unaffected control individuals and from MPS II patients. We measured IDS protein concentration with a 2-step time-delayed dissociation-enhanced lanthanide fluorescence immunoassay. To measure enzyme activity, we immobilized anti-IDS antibody on microtiter plates to capture the enzyme and measured its activity with the fluorogenic substrate 4-methylumbelliferyl sulfate. RESULTS: Dried-blood-spot samples from MPS II patients showed an almost total absence of IDS activity (0-0.075 micromol x h(-1) x L(-1)) compared with control blood spots (0.5-4.7 micromol x h(-1) x L(-1)) and control plasma (0.17-8.1 micromol x h(-1) x L(-1)). A dried-blood-spot sample from only 1 of 12 MPS II patients had detectable concentrations of IDS protein (24.8 microg/L), but no IDS protein was detected in plasma from MPS II patients. Ranges for IDS protein in control samples were 25.8-153 microg/L for blood spots and 22.8-349.4 microg/L for plasma. CONCLUSION: Measurement of the IDS protein concentration and enzyme activity (as measured by a simple fluorogenic assay with an immune capture technique) enables identification of the majority of MPS II patient samples from both dried blood spots and plasma samples.     

Correction of Neurological Disease of Mucopolysaccharidosis IIIB in Adult Mice by rAAV9 Trans-Blood�CBrain Barrier Gene Delivery

The greatest challenge in developing therapies for mucopolysaccharidosis (MPS) IIIB is to achieve efficient central nervous system (CNS) delivery across the blood–brain barrier (BBB). In this study, we used the novel ability of adeno-associated virus serotype 9 (AAV9) to cross the BBB from the vasculature to achieve long-term global CNS, and widespread somatic restoration of α-N-acetylglucosaminidase (NAGLU) activity. A single intravenous (IV) injection of rAAV9-CMV-hNAGLU, without extraneous treatment to disrupt the BBB, restored NAGLU activity to normal or above normal levels in adult MPS IIIB mice, leading to the correction of lysosomal storage pathology in the CNS and periphery, and correction of astrocytosis and neurodegeneration. The IV delivered rAAV9 vector also transduced abundant neurons in the myenteric and submucosal plexus, suggesting peripheral nervous system (PNS) targeting. While CNS entry did not depend on osmotic disruption of the BBB, it was significantly enhanced by pretreatment with an IV infusion of mannitol. Most important, we demonstrate that a single systemic rAAV9-NAGLU gene delivery provides long-term (>18 months) neurological benefits in MPS IIIB mice, resulting in significant improvement in behavioral performance, and extension of survival. These data suggest promising clinical potential using the trans-BBB neurotropic rAAV9 vector for treating MPS IIIB and other neurogenetic diseases.     

Homogeneous antibody–angiopep 2 conjugates for effective brain targeting

Antibody-based therapy has shown great success in the treatment of many diseases, including cancers. While antibodies and antibody–drug conjugates (ADCs) have also been evaluated for central nervous system (CNS) disorders as well as brain tumors, their therapeutic efficacy can be substantially limited due to low permeability across the blood–brain barrier (BBB). Thus, improving BBB permeability of therapeutic antibodies is critical in establishing this drug class as a reliable clinical option for CNS diseases. Here, we report that, compared with a conventional heterogeneous conjugation, homogeneous conjugation of the synthetic BBB shuttle peptide angiopep-2 (Ang2) to a monoclonal antibody (mAb) provides improved binding affinity for brain microvascular endothelial cells in vitro and accumulation into normal brain tissues in vivo. In a mouse model, we also demonstrate that the homogeneous anti-EGFR mAb–Ang2 conjugate administered intravenously efficiently accumulates in intracranial tumors. These findings suggest that homogeneous conjugation of BBB shuttle peptides such as Ang2 is a promising approach to enhancing the therapeutic efficacy of antibody agents for CNS diseases.

Homogeneous conjugation of angiopep-2 to a monoclonal antibody improves binding affinity for brain microvascular endothelial cells and accumulation into brain tissues and tumors across the BBB.     

Several rAAV Vectors Efficiently Cross the Blood–brain Barrier and Transduce Neurons and Astrocytes in the Neonatal Mouse Central Nervous System

Noninvasive systemic gene delivery to the central nervous system (CNS) has largely been impeded by the blood–brain barrier (BBB). Recent studies documented widespread CNS gene transfer after intravascular delivery of recombinant adeno-associated virus 9 (rAAV9). To investigate alternative and possibly more potent rAAV vectors for systemic gene delivery across the BBB, we systematically evaluated the CNS gene transfer properties of nine different rAAVEGFP vectors after intravascular infusion in neonatal mice. Several rAAVs efficiently transduce neurons, motor neurons, astrocytes, and Purkinje cells; among them, rAAVrh.10 is at least as efficient as rAAV9 in many of the regions examined. Importantly, intravenously delivered rAAVs did not cause abnormal microgliosis in the CNS. The rAAVs that achieve stable widespread gene transfer in the CNS are exceptionally useful platforms for the development of therapeutic approaches for neurological disorders affecting large regions of the CNS as well as convenient biological tools for neuroscience research.     

GHB (gamma-hydroxybutyrate) carrier-mediated transport across the blood-brain barrier

gamma-Hydroxybutyrate (sodium oxybate, GHB) is an approved therapeutic agent for cataplexy with narcolepsy. GHB is widely abused as an anabolic agent, euphoriant, and date rape drug. Recreational abuse or overdose of GHB (or its precursors gamma-butyrolactone or 1,4-butanediol) results in dose-dependent central nervous system (CNS) effects (respiratory depression, unconsciousness, coma, and death) as well as tolerance and withdrawal. An understanding of the CNS transport mechanisms of GHB may provide insight into overdose treatment approaches. The hypothesis that GHB undergoes carrier-mediated transport across the BBB was tested using a rat in situ brain perfusion technique. Various pharmacological agents were used to probe the pharmacological characteristics of the transporter. GHB exhibited carrier-mediated transport across the BBB consistent with a high-capacity, low-affinity transporter; averaged brain region parameters were V(max) = 709 +/- 214 nmol/min/g, K(m) = 11.0 +/- 3.56 mM, and CL(ns) = 0.019 +/- 0.003 cm(3)/min/g. Short-chain monocarboxylic acids (pyruvic, lactic, and beta-hydroxybutyric), medium-chain fatty acids (hexanoic and valproic), and organic anions (probenecid, benzoic, salicylic, and alpha-cyano-4-hydroxycinnamic acid) significantly inhibited GHB influx by 35 to 90%. Dicarboxylic acids (succinic and glutaric) and gamma-aminobutyric acid did not inhibit GHB BBB transport. Mutual inhibition was observed between GHB and benzoic acid, a well known substrate of the monocarboxylate transporter MCT1. These results are suggestive of GHB crossing the BBB via an MCT isoform. These novel findings of GHB BBB transport suggest potential therapeutic approaches in the treatment of GHB overdoses. We are currently conducting "proof-of-concept" studies involving the use of GHB brain transport inhibitors during GHB toxicity.     

A2A adenosine receptor modulates drug efflux transporter P-glycoprotein at the blood-brain barrier

The blood-brain barrier (BBB) protects the brain from toxic substances within the peripheral circulation. It maintains brain homeostasis and is a hurdle for drug delivery to the CNS to treat neurodegenerative diseases, including Alzheimer’s disease and brain tumors. The drug efflux transporter P-glycoprotein (P-gp) is highly expressed on brain endothelial cells and blocks the entry of most drugs delivered to the brain. Here, we show that activation of the A2A adenosine receptor (AR) with an FDA-approved A2A AR agonist (Lexiscan) rapidly and potently decreased P-gp expression and function in a time-dependent and reversible manner. We demonstrate that downmodulation of P-gp expression and function coincided with chemotherapeutic drug accumulation in brains of WT mice and in primary mouse and human brain endothelial cells, which serve as in vitro BBB models. Lexiscan also potently downregulated the expression of BCRP1, an efflux transporter that is highly expressed in the CNS vasculature and other tissues. Finally, we determined that multiple pathways, including MMP9 cleavage and ubiquitinylation, mediated P-gp downmodulation. Based on these data, we propose that A2A AR activation on BBB endothelial cells offers a therapeutic window that can be fine-tuned for drug delivery to the brain and has potential as a CNS drug-delivery technology.     

Whole body correction of mucopolysaccharidosis IIIA by intracerebrospinal fluid gene therapy

For most lysosomal storage diseases (LSDs) affecting the CNS, there is currently no cure. The BBB, which limits the bioavailability of drugs administered systemically, and the short half-life of lysosomal enzymes, hamper the development of effective therapies. Mucopolysaccharidosis type IIIA (MPS IIIA) is an autosomic recessive LSD caused by a deficiency in sulfamidase, a sulfatase involved in the stepwise degradation of glycosaminoglycan (GAG) heparan sulfate. Here, we demonstrate that intracerebrospinal fluid (intra-CSF) administration of serotype 9 adenoassociated viral vectors (AAV9s) encoding sulfamidase corrects both CNS and somatic pathology in MPS IIIA mice. Following vector administration, enzymatic activity increased throughout the brain and in serum, leading to whole body correction of GAG accumulation and lysosomal pathology, normalization of behavioral deficits, and prolonged survival. To test this strategy in a larger animal, we treated beagle dogs using intracisternal or intracerebroventricular delivery. Administration of sulfamidase-encoding AAV9 resulted in transgenic expression throughout the CNS and liver and increased sulfamidase activity in CSF. High-titer serum antibodies against AAV9 only partially blocked CSF-mediated gene transfer to the brains of dogs. Consistently, anti-AAV antibody titers were lower in CSF than in serum collected from healthy and MPS IIIA–affected children. These results support the clinical translation of this approach for the treatment of MPS IIIA and other LSDs with CNS involvement.     

Detection of Hunter syndrome (mucopolysaccharidosis type II) in Taiwanese: biochemical and linkage studies of the iduronate-2-sulfatase gene defects in MPS II patients and carriers

BACKGROUND: Hunter syndrome (mucopolysaccharidosis type II) is an X-linked recessive lysosomal storage disease caused by a defect of the iduronate-2-sulfatase (IDS) gene. The result is impaired IDS enzyme function. METHODS: To characterize the biochemical and molecular defects in IDS-deficient patients and their families, we measured IDS enzyme activity by fluorimetric enzyme assay and identified the IDS gene mutations in 14 unrelated Taiwanese patients with varying clinical phenotypes. In addition, haplotype analysis was also performed. RESULTS: Three novel (IVS2+1G>C, 1055del12, and G489D) and 7 previously reported (N63K, P228L, K347E, R468Q, R468W, I485R, and 1241delAG) mutations were found. Together R468Q and R468W account for 42.8% mutations found in our patients. Haplotype analysis using IDS flanking markers DXS1113 and DXS1123 revealed that the unrelated R468Q alleles were independent in origin whereas the unrelated R468W alleles are probably of the same origin. The R468Q mutation in patient 1150 and I485R mutation in patient 710 occurred de novo in male meioses. Once the mutation in a family was identified, restriction analysis was also performed for rapid diagnosis of female carriers in 8 families. Leukocyte IDS measurement revealed significantly wide range of IDS activity in normal controls and MPS II carriers (19.2 - 70.6 vs. 8.4 - 26.6 nmol/h/mg cell protein). The average leukocyte IDS activity of normal controls (n=43) was 43.9+/-13.3 nmol/h/mg protein, whereas patients with MPS II (n=14) had <5% of mean normal IDS activity (0.9+/-0.6 nmol/h/mg protein), and carriers (n=13) had a mean activity of 17.5 (+/-5.7) nmol/h/mg protein. The mean leukocyte IDS activity in female carriers was less than a half of the normal level. CONCLUSION: Due to a small overlapping range of normal and carriers, the level of enzyme activity cannot be used alone for carrier detection.     

Charting the course across the blood-brain barrier

The blood-brain barrier (BBB) presents a significant obstacle to delivery of targeted therapies to brain tumors. In this issue of the JCI, Staquicini and colleagues apply an in vivo phage-displayed library of random peptides to identify differentially expressed peptides that can be used to transport targeted agents across the intact BBB. The authors uncover a non-canonical, peptide-mediated iron-mimicry mechanism to induce transport of the transferrin/transferrin receptor complex across the BBB. They then demonstrate the ability of phage-targeting approaches to deliver therapeutic cargo and molecular imaging reporters across the BBB in an intracranial glioblastoma mouse model.     

Prodrug based optimal drug delivery via membrane transporter/receptor

The carrier-mediated absorption of drugs and prodrugs across epithelial and endothelial barriers is emerging as a novel trend in biotherapeutics. This review examines the important advances in this field in the past decade. The feasibility of drug absorption of the parent drug or the appropriately modified prodrug via these transporters is discussed in detail. Several successful examples of synthesis of prodrugs recognised by the targeted transporters are described. The applicability of this approach in translocating drugs across the almost impenetrable blood-brain barrier (BBB) has also been examined.     

Effective Intravenous Therapy for Neurodegenerative Disease With a Therapeutic Enzyme and a Peptide That Mediates Delivery to the Brain

The blood–brain barrier (BBB) presents a major challenge to effective treatment of neurological disorders, including lysosomal storage diseases (LSDs), which frequently present with life-shortening and untreatable neurodegeneration. There is considerable interest in methods for intravenous delivery of lysosomal proteins across the BBB but for the most part, levels achievable in the brain of mouse models are modest and increased lifespan remains to be demonstrated. In this study, we have investigated delivery across the BBB using a mouse model of late-infantile neuronal ceroid lipofuscinosis (LINCL), a neurodegenerative LSD caused by loss of tripeptidyl peptidase I (TPP1). We have achieved supraphysiological levels of TPP1 throughout the brain of LINCL mice by intravenous (IV) coadministration of recombinant TPP1 with a 36-residue peptide that contains polylysine and a low-density lipoprotein receptor binding sequence from apolipoprotein E. Importantly, IV administration of TPP1 with the peptide significantly reduces brain lysosomal storage, increases lifespan and improves neurological function. This simple “mix and inject” method is immediately applicable towards evaluation of enzyme replacement therapy to the brain in preclinical models and further exploration of its clinical potential is warranted.     

A novel mutation of IDS gene in a Chinese patient with mucopolysaccharidosis II by next-generation sequencing

BackgroundMucopolysaccharidosis (MPS) is induced by the absence or malfunctioning of lysosomal enzymes. MPS I and MPS II are similar in phenotypes but they are different in genotypes, which are caused by the deficiencies of alpha-L-iduronidase gene (IDUA) and iduronate 2-sulfatase gene (IDS) respectively. In this work, a 5-year-old Chinese young male with manifestations of MPS in a family with unaffected parents was described.Methods12 kb of all the targeted exon sequences plus flanking sequences chromosomal DNA of IDS and IDUA genes from the proband and 20 other case-unrelated controls were captured and sequenced by using next-generation sequencing technology.ResultsOne single-nucleotide deletion variant (c.1270delG) resulting in frameshift and premature truncation of I2S enzyme was detected, out of 20 controls, only in the proband, and which was further verified by Sanger sequencing. The proband's mother was also proved carrying c.1270delG by Sanger method but not for his father.ConclusionsThe novel variant (c.1270delG) is a candidate disease-causing mutation predicted to affect the normal structure and function of the enzyme. Target sequence capture and next-generation sequencing technology can be effective for the gene testing of MPS II disorder.     

Brain transplantation of genetically modified bone marrow stromal cells corrects CNS pathology and cognitive function in MPS VII mice

Current therapies for lysosomal storage diseases (LSDs), enzyme replacement therapy and bone marrow transplantation are effective for visceral organ pathology of LSD, but their effectiveness for brain involvement in LSDs is still a subject of controversy. As an alternative approach, we transplanted genetically modified bone marrow stromal (BMS) cells to lateral ventricle of newborn mucopolysaccharidosis VII (MPS VII) mice. MPS VII is one of LSDs and caused by deficiency of beta-glucuronidase (GUSB), resulting in accumulation of glycosaminoglycans (GAGs) in brain. At 2 weeks after transplantation, the GUSB enzyme-positive cells were identified in olfactory bulb, striatum and cerebral cortex, and the enzymatic activities in various brain areas increased. The GAGs contents in brain were reduced to near normal level at 4 weeks after transplantation. Although GUSB activity declined to homozygous level after 8 weeks, the reduction of GAGs persisted for 16 weeks. Microscopic examination indicated that the lysosomal distention was not found in treated animal brain. Cognitive function in MPS VII animals as evaluated by Morris Water Maze test in treated mice showed a marked improvement over nontreated animals. Brain transplantation of genetically modified BMS cells appears to be a promising approach to treat diffuse CNS involvement of LSDs.     

Genetic correction of the fetal brain increases the lifespan of mice with the severe multisystemic disease mucopolysaccharidosis type VII.

Neurogenetic diseases typically have globally distributed lesions, and pathology usually develops early in life, requiring early diagnosis and treatment. We investigated the effects of transferring a corrective gene into the fetal brain before the onset of pathology in the mucopolysaccharidosis (MPS) type VII mouse, a model of a lysosomal storage disease. A single adeno-associated virus serotype 1 vector injection into the ventricle at 15.5 days of gestation resulted in widespread distribution and lifelong expression of the normal gene in the brain and spinal cord. The normal enzyme was distributed to neighboring cells (as expected) and completely prevented the development of storage lesions throughout the central nervous system (CNS). No vector transfer was found outside the CNS, including the gonads, but a small amount of enzyme was present in visceral tissues, consistent with transfer from cerebrospinal fluid to venous circulation. The enzyme was present peripherally in such low amounts that it did not result in the severe skeletal dysmorphology that occurs readily when systemic treatment is used in neonates. However, the survival probability of the treated animals was significantly increased. The results suggest that the nervous system disease may contribute to the overall physiologic health of the animal in this type of disease.     

Therapeutic Efficacy of Bone Marrow Transplant, Intracranial AAV-mediated Gene Therapy, or Both in the Mouse Model of MPS IIIB

Sanfilippo syndrome type B (MPS IIIB) is a lysosomal storage disease resulting from a deficiency of N-acetyl-glucosaminidase (NAGLU) activity. In an attempt to correct the disease in the murine model of MPS IIIB, neonatal mice were treated with intracranial AAV2/5-NAGLU (AAV), syngeneic bone marrow transplant (BMT), or both (AAV/BMT). All treatments resulted in some improvement in clinical phenotype. Adeno-associated viral (AAV) treatment resulted in improvements in lifespan, motor function, hearing, time to activity onset, and daytime activity level, but no reduction of lysosomal storage. BMT resulted in improved hearing by 9 months, and improved circadian measures, but had no effect on lifespan, motor function, or central nervous system (CNS) lysosomal storage. AAV/BMT treatment resulted in improvements in hearing, time to activity onset, motor function, and reduced CNS lysosomal storage, but had no effect on lifespan. Combination therapy compared to either therapy alone resulted in synergistic effects on hearing and CNS lysosomal inclusions but antagonistic effects on motor function and lifespan. AAV alone is more efficacious than BMT or AAV/BMT treatment for lifespan. BMT was the least efficacious treatment by all measures. CNS-directed AAV treatment alone appears to be the preferred treatment, combining the most efficacy with the least toxicity of the approaches assessed.     

Insulin in the brain: There and back again

Insulin performs unique functions within the CNS. Produced nearly exclusively by the pancreas, insulin crosses the blood-brain barrier (BBB) using a saturable transporter, affecting feeding and cognition through CNS mechanisms largely independent of glucose utilization. Whereas peripheral insulin acts primarily as a metabolic regulatory hormone, CNS insulin has an array of effects on brain that may more closely resemble the actions of the ancestral insulin molecule. Brain endothelial cells (BECs), the cells that form the vascular BBB and contain the transporter that translocates insulin from blood to brain, are themselves regulated by insulin. The insulin transporter is altered by physiological and pathological factors including hyperglycemia and the diabetic state. The latter can lead to BBB disruption. Pericytes, pluripotent cells in intimate contact with the BECs, protect the integrity of the BBB and its ability to transport insulin. Most of insulin's known actions within the CNS are mediated through two canonical pathways, the phosphoinositide-3 kinase (PI3)/Akt and Ras/mitogen activated kinase (MAPK) cascades. Resistance to insulin action within the CNS, sometimes referred to as diabetes mellitus type III, is associated with peripheral insulin resistance, but it is possible that variable hormonal resistance syndromes exist so that resistance at one tissue bed may be independent of that at others. CNS insulin resistance is associated with Alzheimer's disease, depression, and impaired baroreceptor gain in pregnancy. These aspects of CNS insulin action and the control of its entry by the BBB are likely only a small part of the story of insulin within the brain.     

Medicinal Chemistry Based Approaches and Nanotechnology‐Based Systems to Improve CNS Drug Targeting and Delivery

The central nervous system (CNS) is protected by various barriers, which regulate nervous tissue homeostasis and control the selective and specific uptake, efflux, and metabolism of endogenous and exogenous molecules. Among these barriers is the blood–brain barrier (BBB), a physical and physiological barrier that filters very efficiently and selectively the entry of compounds from the blood to the brain and protects nervous tissue from harmful substances and infectious agents present in the bloodstream. The BBB also prevents the entry of potential drugs. As a result, various drug targeting and delivery strategies are currently being developed to enhance the transport of drugs from the blood to the brain. Following a general introduction, we briefly overview in this review article the fundamental physiological properties of the BBB. Then, we describe current strategies to bypass the BBB (i.e., invasive methods, alternative approaches, and temporary opening) and to cross it (i.e., noninvasive approaches). This section is followed by a chapter addressing the chemical and technological solutions developed to cross the BBB. A special emphasis is given to prodrug‐targeting approaches and targeted nanotechnology‐based systems, two promising strategies for BBB targeting and delivery of drugs to the brain.     

Brazilian reference values for MPS II screening in dried blood spots — A fluorimetric assay

ObjectivesMucopolysaccharidosis II (MPS II), or Hunter Syndrome, is a lysosomal storage disorder that is caused by the deficiency or absence of iduronate-2-sulfatase (IDS) enzyme; in this disease, early diagnosis is essential to provide higher life expectancy for patients. This study validates a fluorimetric assay that is used to assess IDS enzyme activity using dried blood spot (DBS) samples and presents the reference interval for the Brazilian population.Design and methodsVenous blood sample was collected in heparin tubes for leukocyte extraction and DBS preparation. IDS activity in the leukocytes was analyzed, and the results were considered the gold standard reference for the categorization of volunteers as positive or negative controls (PC and NC, respectively). IDS activity in the DBS was analyzed using an adapted version of the leukocyte assay. Statistical analyses were performed using a ROC curve to determine cutoff values and using a parametric Student's t test to compare values between genders. To verify that the assay yielded consistent results, a Bland–Altman plot was prepared.ResultsLeukocyte IDS activity values ranged between 2.71 and 17.36 nmol/mg protein/h in the NC group and between 0 and 0.11 nmol/mg protein/h in the PC group. Based on the DBS assay, activities ranged between 1.83 and 16.86 μmol/L blood/h in the NC group and between 0.58 and 4.32 μmol/L blood/h in the PC group.ConclusionsReference values of IDS activity were determined in DBS with acceptable sensitivity and specificity. Therefore, the DBS assay described in this work may be a useful tool to screen MPS II patients in the Brazilian population.     

Effects of gamma- and hydroxypropyl-gamma-cyclodextrins on the transport of doxorubicin across an in vitro model of blood-brain barrier

Association between doxorubicin (DOX) and gamma-cyclodextrin (gamma-CD) or hydroxypropyl-gamma-CD (HP-gamma-CD) has been examined to increase the delivery of this antitumoral agent to the brain. The stoichiometry and the stability constant of gamma-CD or HP-gamma-CD and DOX complexes were determined in physiological medium by UV-visible spectroscopy. By using an in vitro model of the blood-brain barrier (BBB), endothelial permeability and toxicity toward the brain capillary endothelial cells of DOX, gamma-CD, and HP-gamma-CD were performed. For each CD, endothelial permeability was relatively low and a disruption of the BBB occurred at 20 microM, 20 mM, and 50 mM DOX, gamma-CD, and HP-gamma-CD, respectively. Increasing amounts of CDs were added to a fixed DOX concentration. Addition of gamma-CD or HP-gamma-CD, up to 15 and 35 mM, respectively, decreased the DOX delivery, probably due to the low complex penetration across the BBB and the decrease in free DOX concentration. Higher CD concentrations increased the DOX delivery to the brain, but this effect is due to a loss of BBB integrity. In contrast to what was observed on Caco-2 cell model with various drugs, CDs are not able to increase the delivery of DOX across our in vitro model of BBB.