NFIC regulates ribosomal biology and ER stress in pancreatic acinar cells and restrains PDAC initiation.

Pancreatic acinar cells rely on PTF1 and other transcription factors to deploy their transcriptional program. We identify NFIC as a NR5A2 interactor and regulator of acinar differentiation. NFIC binding sites are enriched in NR5A2 ChIP-Sequencing peaks. Nfic knockout mice have a smaller, histologically normal, pancreas with reduced acinar gene expression. NFIC binds and regulates the promoters of acinar genes and those involved in RNA/protein metabolism, and Nfic knockout pancreata show defective ribosomal RNA maturation. NFIC dampens the endoplasmic reticulum stress program through binding to gene promoters and is required for resolution of Tunicamycin-mediated stress. NFIC is down-regulated during caerulein pancreatitis and is required for recovery after damage. Normal human pancreata with low levels of NFIC transcripts display reduced expression of genes down-regulated in Nfic knockout mice. NFIC expression is down-regulated in mouse and human pancreatic ductal adenocarcinoma. Consistently, Nfic knockout mice develop a higher number of mutant Kras-driven pre-neoplastic lesions.

The PRSS3P2 and TRY7 deletion copy number variant modifies risk for chronic pancreatitis.

BACKGROUND: PRSS1 and PRSS2 constitute the only functional copies of a tandemly-arranged five-trypsinogen-gene cluster (i.e., PRSS1, PRSS3P1, PRSS3P2, TRY7 and PRSS2) on chromosome 7q35. Variants in PRSS1 and PRSS2, including missense and copy number variants (CNVs), have been reported to predispose to or protect against chronic pancreatitis (CP). We wondered whether a common trypsinogen pseudogene deletion CNV (that removes two of the three trypsinogen pseudogenes, PRSS3P2 and TRY7) might be associated with CP causation/predisposition. METHODS: We analyzed the common PRSS3P2 and TRY7 deletion CNV in a total of 1536 CP patients and 3506 controls from France, Germany, India and Japan by means of quantitative fluorescent multiplex polymerase chain reaction. RESULTS: We demonstrated that the deletion CNV variant was associated with a protective effect against CP in the French, German and Japanese cohorts whilst a trend toward the same association was noted in the Indian cohort. Meta-analysis under a dominant model yielded a pooled odds ratio (OR) of 0.68 (95% confidence interval (CI) 0.52-0.89; p = 0.005) whereas an allele-based meta-analysis yielded a pooled OR of 0.84 (95% CI 0.77-0.92; p = 0.0001). This protective effect is explicable by reference to the recent finding that the still functional PRSS3P2/TRY7 pseudogene enhancers upregulate pancreatic PRSS2 expression. CONCLUSIONS: The common PRSS3P2 and TRY7 deletion CNV was associated with a reduced risk for CP. This finding provides additional support for the emerging view that dysregulated PRSS2 expression represents a discrete mechanism underlying CP predisposition or protection.

A GATA6-centred gene regulatory network involving HNFs and ΔNp63 controls plasticity and immune escape in pancreatic cancer.

OBJECTIVE: Molecular taxonomy of tumours is the foundation of personalised medicine and is becoming of paramount importance for therapeutic purposes. Four transcriptomics-based classification systems of pancreatic ductal adenocarcinoma (PDAC) exist, which consistently identified a subtype of highly aggressive PDACs with basal-like features, including ΔNp63 expression and loss of the epithelial master regulator GATA6. We investigated the precise molecular events driving PDAC progression and the emergence of the basal programme. DESIGN: We combined the analysis of patient-derived transcriptomics datasets and tissue samples with mechanistic experiments using a novel dual-recombinase mouse model for Gata6 deletion at late stages of KRasG12D-driven pancreatic tumorigenesis (Gata6LateKO). RESULTS: This comprehensive human-to-mouse approach showed that GATA6 loss is necessary, but not sufficient, for the expression of ΔNp63 and the basal programme in patients and in mice. The concomitant loss of HNF1A and HNF4A, likely through epigenetic silencing, is required for the full phenotype switch. Moreover, Gata6 deletion in mice dramatically increased the metastatic rate, with a propensity for lung metastases. Through RNA-Seq analysis of primary cells isolated from mouse tumours, we show that Gata6 inhibits tumour cell plasticity and immune evasion, consistent with patient-derived data, suggesting that GATA6 works as a barrier for acquiring the fully developed basal and metastatic phenotype. CONCLUSIONS: Our work provides both a mechanistic molecular link between the basal phenotype and metastasis and a valuable preclinical tool to investigate the most aggressive subtype of PDAC. These data, therefore, are important for understanding the pathobiological features underlying the heterogeneity of pancreatic cancer in both mice and human.

Comparative evaluation of corneal incision enlargement after intraocular lens delivery of new preloaded and manual implantation systems.

PURPOSE: To determine whether total corneal incision enlargement after implantation of an intraocular lens with a new preloaded delivery system is comparable to a standard-of-care manual delivery system using an in vitro human cadaver eye model, despite having a smaller initial incision size. METHODS: Human cadaver phakic whole eye globes were used for these studies (n = 16 per group). Each pair of eyes was randomly assigned to a new preloaded delivery system (UltraSertTM) or a manual delivery system (MONARCH® III D). The surgical procedure included creating a 2.2- and 2.4-mm corneal incision for the preloaded and manual delivery systems, respectively, measuring intraocular pressure and pre-implantation corneal incision size, delivering the intraocular lens into the anterior chamber, and measuring the post-implantation incision size. RESULTS: The final corneal incision enlargement after intraocular lens delivery using the preloaded delivery system was 2.33 ± 0.04 mm, compared to 2.54 ± 0.05 mm after intraocular lens delivery with the manual delivery system. The mean corneal incision enlargement was comparable between the two systems, being 0.13 ± 0.04 mm using the preloaded delivery system and 0.14 ± 0.05 mm using the manual delivery system (p = 0.432). CONCLUSION: In a human cadaver eye model, the preloaded delivery system demonstrated an intraocular lens delivery performance on cornea incision enlargement was noninferior to the manual, standard-of-care intraocular lens delivery system despite a smaller initial incision size. TRANSLATIONAL RELEVANCE: Smaller incision sizes for cataract surgery improve patient outcomes via faster visual and wound recovery and decreased risk of complications such as postoperative inflammation and surgically induced astigmatism.

Protease-Sensitive Pancreatic Lipase Variants Are Associated With Early Onset Chronic Pancreatitis.

OBJECTIVES: Premature activation of the digestive protease trypsin within the pancreatic parenchyma is a critical factor in the pathogenesis of pancreatitis. Alterations in genes that affect intrapancreatic trypsin activity are associated with chronic pancreatitis (CP). Recently, carboxyl ester lipase emerged as a trypsin-independent risk gene. Here, we evaluated pancreatic lipase (PNLIP) as a potential novel susceptibility gene for CP. METHODS: We analyzed all 13 PNLIP exons in 429 nonalcoholic patients with CP and 600 control subjects from Germany, in 632 patients and 957 controls from France, and in 223 patients and 1,070 controls from Japan by DNA sequencing. Additionally, we analyzed selected exons in further 545 patients with CP and 1,849 controls originating from Germany, United States, and India. We assessed the cellular secretion, lipase activity, and proteolytic stability of recombinant PNLIP variants. RESULTS: In the German discovery cohort, 8/429 (1.9%) patients and 2/600 (0.3%) controls carried a PNLIP missense variant (P = 0.02, odds ratio [OR] = 5.7, 95% confidence interval [CI] = 1.1-38.9). Variants detected in patients were prone to proteolytic degradation by trypsin and chymotrypsin. In the French replication cohort, protease-sensitive variants were also enriched in patients with early-onset CP (5/632 [0.8%]) vs controls (1/957 [0.1%]) (P = 0.04, OR = 7.6, 95% CI = 0.9-172.9). In contrast, we detected no protease-sensitive variants in the non-European populations. In the combined European data, protease-sensitive variants were found in 13/1,163 cases (1.1%) and in 3/3,000 controls (0.1%) (OR = 11.3, 95% CI = 3.0-49.9, P < 0.0001). CONCLUSIONS: Our data indicate that protease-sensitive PNLIP variants are novel genetic risk factors for the development of CP.

Identification of a functional enhancer variant within the chronic pancreatitis-associated SPINK1 c.101A>G (p.Asn34Ser)-containing haplotype.

The haplotype harboring the SPINK1 c.101A>G (p.Asn34Ser) variant (also known as rs17107315:T>C) represents the most important heritable risk factor for idiopathic chronic pancreatitis identified to date. The causal variant contained within this risk haplotype has however remained stubbornly elusive. Herein, we set out to resolve this enigma by employing a hypothesis-driven approach. First, we searched for variants in strong linkage disequilibrium (LD) with rs17107315:T>C using HaploReg v4.1. Second, we identified two candidate SNPs by visual inspection of sequences spanning all 25 SNPs found to be in LD with rs17107315:T>C, guided by prior knowledge of pancreas-specific transcription factors and their cognate binding sites. Third, employing a novel cis-regulatory module (CRM)-guided approach to further filter the two candidate SNPs yielded a solitary candidate causal variant. Finally, combining data from phylogenetic conservation and chromatin accessibility, cotransfection transactivation experiments, and population genetic studies, we suggest that rs142703147:C>A, which disrupts a PTF1L-binding site within an evolutionarily conserved HNF1A-PTF1L CRM located ∼4 kb upstream of the SPINK1 promoter, contributes to the aforementioned chronic pancreatitis risk haplotype. Further studies are required not only to improve the characterization of this functional SNP but also to identify other functional components that might contribute to this high-risk haplotype.

In vitro delivery performance assessment of a new preloaded intraocular lens delivery system.

PURPOSE: To compare the delivery performance of a new preloaded intraocular lens (IOL) delivery system (Ultrasert [system U]) with that of 2 commercially available preloaded systems (iSert [system S] and Tecnis iTec [system T]), and a manual system (Monarch III D). SETTING: Alcon Laboratories, Fort Worth, Texas, USA. DESIGN: Experimental study. METHODS: Freshly excised porcine eyes were randomly assigned to 5 groups of 10 eyes each as follows: preloaded systems U, S, T 2.2 mm incision, T 2.4 mm incision, and manual system. Corneal incision size was measured before and after delivery. The rate of successful in-the-bag IOL delivery, IOL adherence to the plunger tip, trapped trailing haptic, and presence of nozzle tip splitting were assessed. Statistical analysis was performed to determine the significance of differences between groups. RESULTS: All systems successfully placed the IOL in the capsular bag. System U had the least corneal incision enlargement (mean 0.07 mm ± 0.05 [SD]) and the smallest final incision size (mean 2.31 ± 0.06 mm) (P < .05). An associated split in the nozzle tip was observed for system S devices (P < .05). Furthermore, in more than 50% of deliveries with system T, the IOL remained adhered to the plunger tip after exiting the nozzle (P < .05). Systems S and T each had 1 occurrence of a trapped trailing haptic during IOL delivery. CONCLUSION: The new design of the system U with a depth-guard nozzle tip allowed for the smallest incision enlargement with an absence of nozzle tip splitting and uneventful IOL delivery compared with the other preloaded systems tested in this study. FINANCIAL DISCLOSURE: Drs. Tjia and Lane are consultants to Alcon Laboratories, Inc. Drs. Wang, Wolfe, Paliwal, and Miss Chernosky are employees of Alcon Laboratories, Inc.

No Association Between CEL-HYB Hybrid Allele and Chronic Pancreatitis in Asian Populations.

A hybrid allele between the carboxyl ester lipase gene (CEL) and its pseudogene, CELP (called CEL-HYB), generated by nonallelic homologous recombination between CEL intron 10 and CELP intron 10', was found to increase susceptibility to chronic pancreatitis in a case-control study of patients of European ancestry. We attempted to replicate this finding in 3 independent cohorts from China, Japan, and India, but failed to detect the CEL-HYB allele in any of these populations. The CEL-HYB allele might therefore be an ethnic-specific risk factor for chronic pancreatitis. An alternative hybrid allele (CEL-HYB2) was identified in all 3 Asian populations (1.7% combined carrier frequency), but was not associated with chronic pancreatitis.

Association Analysis of PRSS1-PRSS2 and CLDN2-MORC4 Variants in Nonalcoholic Chronic Pancreatitis Using Tropical Calcific Pancreatitis as Model.

OBJECTIVE: Association of PRSS1-PRSS2 (rs10273639) and CLDN2-MORC4 (rs12688220 and rs7057398) variants with alcohol-related chronic pancreatitis (CP) is established but with nonalcoholic CP is unclear. We addressed this inconsistency using tropical calcific pancreatitis (TCP) as model. METHODS: We sequenced 5'-UTR of PRSS1 and genotyped CLDN2-MORC4 variants in 555 patients with TCP and 801 controls and performed association analysis. Gene-gene interaction between PRSS1 and CLDN2-MORC4 variants and with p.Asn34Ser SPINK1 and p.Leu26Val CTSB was also evaluated. RESULTS: We observed significant association of rs10273639/rs4726576 in PRSS1-PRSS2 (odds ratio [OR] = 0.72; P = 3.50 × 10) and CLDN2-MORC4 variants, rs12688220 (OR = 1.54; P = 1.22 × 10) and rs7057398 (OR = 1.50; P = 1.22 × 10) with TCP. Patients carrying p.Asn34Ser SPINK1 were significantly younger than those with rs4726576 risk genotype (30.0 vs 38.0 years; P = 0.015) and those carrying both were even younger (22.0 years; P = 0.001). Presence of risk allele at rs12688220 in patients carrying p.Asn34Ser SPINK1 delayed the age of onset (32.0 vs 24.0 years; P = 0.013). CONCLUSIONS: Our study establishes strong association of PRSS1-PRSS2 and CLDN2-MORC4 variants with TCP and thus with nonalcoholic CP. These variants independently interact with p.Asn34Ser SPINK1 and influence the age of onset in TCP. However, latter results need to be replicated in other cohorts.

Genetic and phenotypic heterogeneity in tropical calcific pancreatitis.

Tropical calcific pancreatitis (TCP) is a form of chronic non-alcoholic pancreatitis initially reported in the developing parts of the tropical world. The clinical phenotype of TCP has undergone marked changes since its first description in 1968. The disease is now seen in relatively older people with less severe symptoms. In addition, there are varying reports on the proportion of cases presenting with imaging abnormalities like calcification, ductal dilation, and glandular atrophy. Significant progress has also been made in understanding the etiopathology of TCP. The role of malnutrition and cassava toxicity in its pathogenesis is disproven and few studies have focused on the role of micronutrient deficiency and oxidative stress in the etiopathogenesis of TCP. Emerging evidence support an important role for genetic risk factors in TCP. Several studies have shown that, rather than mutations in trypsinogens, variants in serine protease inhibitor kazal type 1, cathepsin B, chymotrypsin C, cystic fibrosis transmembrane regulator, and carboxypeptidase A1, predict risk of TCP. These studies also provided evidence of mutational heterogeneity between TCP and chronic pancreatitis in Western populations. The current review summarizes recent advances that have implications in the understanding of the pathophysiology and thus, heterogeneity in genotype-phenotype correlations in TCP.

Skin extracellular matrix stimulation following injection of a hyaluronic acid-based dermal filler in a rat model.

BACKGROUND: Hyaluronic acid-based dermal fillers have gained rapid acceptance for treating facial wrinkles and deep tissue folds. Although their space-filling properties are well understood, this study evaluates the cellular and molecular changes in skin, as a secondary effect, following injection of a commercially available, 24-mg/ml, cross-linked hyaluronic acid-based filler (HYC-24L+) in a rodent model. METHODS: Sprague-Dawley rats, aged 2 to 4 months, were injected intradermally with 20 µl of HYC-24L+ using a linear threading technique and followed to 12 weeks after injection. Untreated skin and saline injection were used as study controls. Enzyme-linked immunosorbent assay and reverse-transcriptase polymerase chain reaction methods were used to investigate changes in the expression of several extracellular matrix proteins and genes over time. RESULTS: HYC-24L+ significantly increased the protein expression levels of collagen types I and III in rat dermal tissue for up to 12 weeks. The ratio of collagen type III to type I protein, however, remained unchanged, suggesting maintenance of collagen homeostasis. A significant increase in dermal elastin after HYC-24L+ injection was also observed. Gene expression analysis confirmed that several genes associated with extracellular matrix production and assembly were also transiently up-regulated, and that these changes temporally preceded those observed at the protein level. CONCLUSION: In addition to its well-understood space-filling function, as a secondary effect, the authors demonstrate that HYC-24L+ stimulates the production of several extracellular matrix components, including dermal collagen and elastin.

A review of the metabolism of 1,4-butanediol diglycidyl ether-crosslinked hyaluronic acid dermal fillers.

BACKGROUND: Cosmetic procedures are growing ever more common, and the use of soft tissue fillers is increasing. Practicing physicians need to be aware of the biological behavior of these products in tissue to enable them to respond to any safety concerns that their patients raise. OBJECTIVES: To provide an overview of the metabolism of 1,4-butanediol diglycidyl ether (BDDE)-crosslinked hyaluronic acid (HA) dermal fillers and to examine the safety of the resulting byproducts. METHODS: A review of available evidence was conducted. RESULTS: After reaction with HA, the epoxide groups of BDDE are neutralized, and only trace amounts of unreacted BDDE remain in the product (<2 parts per million). When crosslinked HA, uncrosslinked HA, and unreacted BDDE degrade, they break down into harmless byproducts or into byproducts that are identical to substances already found in the skin. CONCLUSION: Clinical and biocompatibility data from longer than 15 years support the favorable clinical safety profile of BDDE-crosslinked HA and its degradation products. Given the strength of the empirical evidence, physicians should be confident in offering these products to their patients.

Variants in CPA1 are strongly associated with early onset chronic pancreatitis.

Chronic pancreatitis is an inflammatory disorder of the pancreas. We analyzed CPA1, encoding carboxypeptidase A1, in subjects with nonalcoholic chronic pancreatitis (cases) and controls in a German discovery set and three replication sets. Functionally impaired variants were present in 29/944 (3.1%) German cases and 5/3,938 (0.1%) controls (odds ratio (OR) = 24.9, P = 1.5 × 10(-16)). The association was strongest in subjects aged ≤ 10 years (9.7%; OR = 84.0, P = 4.1 × 10(-24)). In the replication sets, defective CPA1 variants were present in 8/600 (1.3%) cases and 9/2,432 (0.4%) controls from Europe (P = 0.01), 5/230 (2.2%) cases and 0/264 controls from India (P = 0.02) and 5/247 (2.0%) cases and 0/341 controls from Japan (P = 0.013). The mechanism by which CPA1 variants confer increased pancreatitis risk may involve misfolding-induced endoplasmic reticulum stress rather than elevated trypsin activity, as is seen with other genetic risk factors for this disease.

Diagnostic opportunities based on skin biomarkers.

Systemic as well as localized skin diseases modify the molecular composition of human skin. Changes in skin chemistry have been observed in diseases such as cancer, psoriasis, eczema, diabetes, and atherosclerosis. Skin chemistry, represented by an enormous wealth of disease biomarkers including lipids, structural proteins, inflammatory mediators, nucleic acids and small molecules, therefore, can serve as a "window to body's health". Various methods including tape-stripping, iontophoresis, microneedles and ultrasound, among others, are being developed to access skin biomarkers and understand skin's detailed molecular composition. This information provides opportunities to diagnose various diseases and their response to therapeutic treatments. This review provides an overview of such diagnostic and theranostic opportunities.

Comprehensive screening of chymotrypsin C (CTRC) gene in tropical calcific pancreatitis identifies novel variants.

OBJECTIVE: In a previous study, the authors have shown that rather than variants in trypsinogen gene(s), mutations in pancreatic secretory trypsin inhibitor (encoded by SPINK1) and cathepsin B (CTSB) are associated with tropical calcific pancreatitis (TCP). Recently, chymotrypsin C (CTRC) variants that diminish its activity or secretion were found to predict susceptibility to chronic pancreatitis (CP). The authors analysed CTRC variants in a large, ethnically matched case-control TCP cohort. DESIGN: The authors sequenced all eight exons and flanking regions in CTRC in 584 CP patients (497 TCP, 87 idiopathic CP) and 598 normal subjects and analysed the significance of association using χ(2) test. The authors also investigated interaction of CTRC variants with p.N34S SPINK1 and p.L26V CTSB mutations. RESULTS: The authors identified 14 variants in CTRC, of which non-synonymous variants were detected in 71/584 CP patients (12.2%) and 22/598 controls (3.7%; OR 3.62, 95% CI 2.21 to 5.93; p=6.2 × 10(-8)). Rather than the commonly reported p.K247_R254del variant in Caucasians, p.V235I was the most common mutation in Indian CP patients (28/575 (4.9%); OR 7.60, 95% CI 2.52 to 25.71; p=1.01 × 10(-5)). Another pathogenic variant, p.A73T was identified in 3.1% (18/584) patients compared with 0.3% (2/598) in controls (OR=9.48, 95% CI 2.19 to 41.03, p=2.5 × 10(-4)). The authors also observed significant association for the synonymous variant c.180C>T (p.(=)) with CP (OR 2.71, 95% CI 1.79 to 4.12, p=5.3 × 10(-7)). Two novel nonsense mutations, p.G242AfsX9 and p.W113X were also identified exclusively in CP patients. No interaction between CTRC variants and p.N34S SPINK1 or p.L26V CTSB mutations was observed. CONCLUSION: This study on a large cohort of TCP patients provides evidence of allelic heterogeneity and confirms that CTRC variants play a significant role in its pathogenesis.

Sampling of disease biomarkers from skin for theranostic applications.

Dermatological diseases including psoriasis, eczema, infections, and cancer collectively constitute a large category of human conditions. The large area and ease of access of skin open excellent opportunities for theranostic applications, that is, diagnosis as well as therapy of the disease. Such applications can be based on evaluation of skin's molecular composition in terms of proteins, nucleic acids, and small molecules. Currently, however, such molecular information is not used in clinical practice. To bring this molecular information to routine clinical dermatology, it is essential to develop convenient and minimally invasive methods for rapid sampling molecules from skin. Here, we demonstrate an ultrasonic sampling technique that can recover a wide variety of biomolecules from skin in a minimally invasive manner. We show that ultrasound can retrieve nearly all major tissue constituents, including structural and functional proteins (cytokines, keratins, etc.), lipids (polar and non-polar lipids), and nucleic acids (DNA and RNA). Comparative analyses of skin's molecular constituents obtained by ultrasonic sampling and skin homogenate showed high resemblance between the two biomolecular profiles, enabling us to build a unique molecular signature of skin. Using different mouse models of dermatological conditions, the ultrasonic analysis for changes in the molecular composition of skin confirmed specific regulation of several established biomarkers.

Assessing the pathological relevance of SPINK1 promoter variants.

The SPINK1 gene, encoding the human pancreatic secretory trypsin inhibitor, is one of the major genes involved in predisposition to chronic pancreatitis (CP). In this study we have assessed the potential functional impact of 11 SPINK1 promoter variants by means of both luciferase reporter gene assay and electrophoretic mobility shift assay (EMSA), using human pancreatic COLO-357 cells as an expression system. The 11 promoter variants were found to be separable into three distinct categories on the basis of the reporter gene assay results viz loss-of-function, gain-of-function and functionally neutral. These findings, which were validated by EMSA, concurred with data from previous deletion studies and DNase I footprinting assays. Further, binding sites for two transcription factors, HNF1 and PTF1, were newly identified within the SPINK1 promoter by virtue of their being affected by specific variants. Combining the functional data with epidemiological data (derived by resequencing the SPINK1 promoter region in French, German and Indian CP patients and controls), then allowed us to make meaningful inferences as to each variant's likely contribution to CP. We conclude that only the three promoter variants associated with a loss-of-function (ie, -53C>T, -142T>C and -147A>G) are likely to be disease-predisposing alterations.

One-step acquisition of functional biomolecules from tissues.

Direct determination of functional biomolecular chemistry of clinically relevant tissues in vivo is a challenging task. Current approaches, based on tissue retrieval by biopsy and subsequent solubilization, are limited in terms of accurate representation of tissue constituents, reproducibility, and retention of functionality of solubilized tissue biomolecules. Using a pool of known surfactants, we designed and screened a large combinatorial library of surfactant formulations, which led to the discovery of rare synergistic formulations that greatly enhance tissue solubilization as well as preserve bioactivity of solubilized molecules, in particular proteins. By combining these formulations with a short ultrasound application, we developed a tissue sampling method--STAMP (Surfactant-based Tissue Acquisition for Molecular Profiling)--for rapid one-step determination of functional tissue chemistry in vivo. We specifically demonstrate STAMP-assisted profiling of a multitude of proteins, lipids, and genomic DNA in skin and mucosal tissues. Applications of this sampling methodology to rapid molecular diagnostics of cutaneous allergies and infectious diseases are also presented.

Ultrasound-enhanced drug transport and distribution in the brain.

Drug delivery in the brain is limited by slow drug diffusion in the brain tissue. This study tested the hypothesis that ultrasound can safely enhance the permeation of drugs in the brain. In vitro exposure to ultrasound at various frequencies (85 kHz, 174 kHz, and 1 MHz) enhanced the permeation of tritium-labeled molecules with molecular weight up to 70 kDa across porcine brain tissue. A maximum enhancement of 24-fold was observed at 85 kHz and 1,200 J/cm(2). In vivo exposure to 1-MHz ultrasound further demonstrated the ability of ultrasound to facilitate molecule distribution in the brain of a non-human primate. Finally, ultrasound under conditions similar to those used in vivo was shown to cause no damage to plasmid DNA, siRNA, adeno-associated virus, and fetal rat cortical neurons over a range of conditions. Altogether, these studies demonstrate that ultrasound can increase drug permeation in the brain in vitro and in vivo under conditions that did not cause detectable damage.