Eugenia Nicolov

Delivering enhanced testosterone replacement therapy through nanochannels.

Primary or secondary hypogonadism results in a range of signs and symptoms that compromise quality of life and requires life-long testosterone replacement therapy. In this study, an implantable nanochannel system is investigated as an alternative delivery strategy for the long-term sustained and constant release of testosterone. In vitro release tests are performed using a dissolution set up, with testosterone and testosterone:2-hydroxypropyl-β-cyclodextrin (TES:HPCD) 1:1 and 1:2 molar ratio complexes release from the implantable nanochannel system and quantify by HPLC. 1:2 TES:HPCD complex stably achieve 10-15 times higher testosterone solubility with 25-30 times higher in vitro release. Bioactivity of delivered testosterone is verified by LNCaP/LUC cell luminescence. In vivo evaluation of testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH) levels by liquid chromatography mass spectrometry (LC/MS) and multiplex assay is performed in castrated Sprague-Dawley rats over 30 d. Animals are treated with the nanochannel implants or degradable testosterone pellets. The 1:2 TES:HPCD nanochannel implant exhibits sustained and clinically relevant in vivo release kinetics and attains physiologically stable plasma levels of testosterone, LH, and FSH. In conclusion, it is demonstrated that by providing long-term steady release 1:2 TES:HPCD nanochannel implants may represent a major breakthrough for the treatment of male hypogonadism.

The Nanochannel Delivery System for Constant Testosterone Replacement Therapy

INTRODUCTION The goal of testosterone replacement is to provide long-term physiological supplementation at sufficient levels to mitigate the symptoms of hypogonadism. AIM The objective of this work is to determine if the implantable nanochannel delivery system (nDS) can present an alternative delivery strategy for the long-term sustained and constant release of testosterone. METHODS A formulation of common testosterone esters (F1) was developed to enable nanochannel delivery of the low water soluble hormone. In vivo evaluation of testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels by liquid chromatography/mass spectrometry and a multiplex assay, respectively, in castrated Sprague-Dawley rats implanted with nDS-F1 implants or polymeric pellets was performed over a 6-month period. The percent of testosterone concentrations observed that fell within the normal range of testosterone levels for each animal was calculated and used to compare the study groups. MAIN OUTCOME MEASURES Sustain release of testosterone in vivo for over 6 months. RESULTS The subcutaneous release of F1 from nDS implants exhibited sustained in vivo release kinetics and attained stable clinically relevant plasma testosterone levels. Plasma LH and FSH levels were significantly diminished in nDS-F1 implant-treated animals, confirming biological activity of the released testosterone. CONCLUSIONS In conclusion, we demonstrate that nDS-F1 implants represents a novel approach for the treatment of male hypogonadism. Further studies will be performed in view of translating the technology to clinical use.

Docetaxel/2-Hydroxypropyl β -Cyclodextrin Inclusion Complex Increases Docetaxel Solubility and Release from a Nanochannel Drug Delivery System

Breast cancer remains the second leading cause of cancer deaths for women in the U.S. The need for new and alternative strategies to treat this cancer is imperative. Here we show the optimization of our nanochannel delivery system (nDS) for constant and sustained delivery of docetaxel (DTX) for thetreatment of triple negative breast cancer. DTX is a highly hydrophobic drug, making it difficult to reach the therapeutic levels when released in aqueous solutions from our implantable delivery system. To overcome this challenge and test the release of DTX from nDS, we prepared DTX/2-hydroxypropyl β-cyclodextrin (DTX/HPCD) inclusion complexes in different molar ratios. The 1:10 DTX/HPCD complex achieved 5 times higher solubility than the 1:2 complex and 3 times higher in vitrorelease of DTX than with free DTX. When released in SCID/Beige mice from nanochannel system, the DTX/HPCD complex showed reduced tumor growth, comparable to the standard bolus injections of DTX, indicating that the structural stability and biological activity of DTX were retained in the complex, after its diffusion through the nanochannel system.

3D Printed Vascularized Device for Subcutaneous Transplantation of Human Islets

Transplantation of pancreatic islets or stem cell derived insulin secreting cells is an attractive treatment strategy for diabetes. However, islet transplantation is associated with several challenges including function-loss associated with dispersion and limited vascularization as well as the need for continuous immunosuppression. To overcome these limitations, here we present a novel 3D printed and functionalized encapsulation system for subcutaneous engraftment of islets or islet like cells. The devices were 3D printed with polylactic acid and the surfaces treated and patterned to increase the hydrophilicity, cell attachment, and proliferation. Surface treated encapsulation systems were implanted with growth factor enriched platelet gel, which helped to create a vascularized environment before loading human islets. The device protected the encapsulated islets from acute hypoxia and kept them functional. The adaptability of the encapsulation system was demonstrated by refilling some of the experimental groups transcutaneously with additional islets.

Sustained zero-order delivery of GC-1 from a nanochannel membrane device alleviates metabolic syndrome

Background/Objectives:Our objective was to assess the sustained, low-dose and constant administration of the thyroid receptor-β (TRβ)-selective agonist GC-1 (sobetirome) from a novel nanochannel membrane device (NMD) for drug delivery. As it known to speed up metabolism, accomplish weight loss, improve cholesterol levels and possess anti-diabetic effects, GC-1 was steadily administered by our NMD, consisting of an implantable nanochannel membrane, as an alternative to conventional daily administration, which is subject to compliance issues in clinical settings.Subjects/Methods:Diet-induced obese C57BL/J6 male mice were fed a very high-fat diet (VHFD) and received NMD implants subcutaneously. Ten mice per group received capsules containing GC-1 or phosphate-buffered saline (control). Weight, lean and fat mass, as well as cholesterol, triglycerides, insulin and glucose, were monitored for 24 days. After treatment, plasma levels of thyroid-stimulating hormone (TSH) and thyroxine were compared. mRNA levels of a panel of thermogenic markers were examined using real-time PCR in white adipose tissue (WAT) and brown adipose tissue (BAT). Adipose tissue, liver and local inflammatory response to the implant were examined histologically. Pancreatic islet number and β-cell area were assessed.Results:GC-1 released from the NMD reversed VHFD-induced obesity and normalized serum cholesterol and glycemia. Significant reductions in body weight and fat mass were observed within 10 days, whereas reductions in serum cholesterol and glucose levels were seen within 7 days. The significant decrease in TSH was consistent with TRβ selectivity for GC-1. Levels of transcript for Ucp1 and thermogenic genes PGC1a, Cidea, Dio2 and Cox5a showed significant upregulation in WAT in NMD–GC-1-treated mice, but decreased in BAT. Although mice treated by NMD–GC-1 showed a similar number of pancreatic islets, they exhibited significant increase in β-cell area.Conclusions:Our data demonstrate that the NMD implant achieves steady administration of GC-1, offering an effective and tightly controlled molecular delivery system for treatment of obesity and metabolic disease, thereby addressing compliance.

Sustained Administration of Hormones Exploiting Nanoconfined Diffusion through Nanochannel Membranes

Implantable devices may provide a superior means for hormone delivery through maintaining serum levels within target therapeutic windows. Zero-order administration has been shown to reach an equilibrium with metabolic clearance, resulting in a constant serum concentration and bioavailability of released hormones. By exploiting surface-to-molecule interaction within nanochannel membranes, it is possible to achieve a long-term, constant diffusive release of agents from implantable reservoirs. In this study, we sought to demonstrate the controlled release of model hormones from a novel nanochannel system. We investigated the delivery of hormones through our nanochannel membrane over a period of 40 days. Levothyroxine, osteocalcin and testosterone were selected as representative hormones based on their different molecular properties and structures. The release mechanisms and transport behaviors of these hormones within 3, 5 and 40 nm channels were characterized. Results further supported the suitability of the nanochannels for sustained administration from implantable platforms.

A pharmacokinetic study of GC-1 delivery using a nanochannel membrane device

This study demonstrated a nanochannel membrane device (NMD) for controlled and sustained release of GC-1 in rats, in the context of the treatment of metabolic syndrome. Release profiles were established in vitro both with and without 5% labrasol for over 2 months. In vivo pharmacokinetic evaluation showed effective GC-1 plasma concentrations, which resulted in significant reductions in body weight after just one week of treatment when compared to the NMD releasing vehicle only (PBS). We also provided evidence that rats treated with NMD-GC-1 present sub-active thyroids and clear differences in the morphology of the epithelium and follicles as compared to the controls, while the heart showed changes in weight. Moreover, body temperatures remained stable throughout treatment, and glucose, pancreatic islet size, and liver histology appeared similar between the treated and control groups. Prolonged constant administration of GC-1 from the NMD proved to be a valid strategy to facilitate weight loss.

Local and sustained delivery of tamoxifen for the prevention of ER+ breast cancer using a nanochannel delivery platform

A high incidence (∼75%) of primary breast cancers are estrogen receptor positive (ER+), and a large fraction of these patients can pursue chemopreventive therapies. However, due to adverse side effects, only 5% to 20% of the women at high risk who could benefit from chemotherapeutics enroll in preventive treatment. There is a clear need for alternative preventive strategies that minimize side effects and improve enrollment and compliance. Selective estrogen receptor modulators, such as tamoxifen (TMX), have been shown to reduce ER+ breast cancer incidence by up to 50% among high-risk women. Importantly, along with raloxifene, it is one of only two FDA-approved drugs for breast cancer prevention. TMX has been in use for over 40 years and has a proven record in pre- and post-menopausal women. However, the drug is marred by side effects, the most common being symptoms of menopause. Further, women treated systemically and chronically with TMX were found to have an increased incidence of endometrial carcinoma. Although rare, this side effect, along with other serious adverse effects (such as blood clots, strokes, and cataracts), has resulted in a debate concerning TMX use in cancer prevention. As the key for breast cancer chemoprevention relies upon long-term delivery of drugs while circumventing side effects, we have developed a novel local delivery strategy for the constant and sustained administration of TMX. We maintain a long-term, local release of TMX in mammary tissues by utilizing a novel implantable nanochannel Delivery System (nDS). The nDS consists of a bioinert, implantable, and mechanically robust silicon membrane which houses an exact number of densely packed slit-nanochannels as small as 2.5 nm with tight tolerances on size, geometry, and surface properties. Providing steady levels of TMX at the mammary gland target through nDS delivery maximizes the therapeutic index while limiting the unwanted secondary effects, which will ultimately improve patient compliance. In this work we chemically induced tumorigenesis in Sprague-Dawley rats by N-methyl-N-nitrosourea (NMU) injection to promote development of estrogen-dependent tumors. We performed ovariectomies seven days after NMU injection to mimic post-menopausal biology. nDS implants loaded with either TMX or PEG400 (negative control) were inserted under the left abdominal mammary gland to determine effects of nDS-TMX on tumor growth and biomarkers. Utilizing LC/MS we were able to determine the amount of TMX released from the nDS. Rats were examined for palpable tumors to assess breast tumor incidence, latency to onset, and multiplicity. Our results show that the nDS implant enables the effective delivery of TMX in this breast tumor model. Further, this technology has the potential to rapidly provide long-term breast cancer protection with significant improvement in the quality of life of patients at high risk, thereby saving thousands of lives every year. Citation Format: Carly S. Filgueira, Eugenia Nicolov, Andrea Ballerini, R. Lyle Hood, Priya Jain, Giacomo Bruno, Alessandro Grattoni. Local and sustained delivery of tamoxifen for the prevention of ER+ breast cancer using a nanochannel delivery platform. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1320.