Development of Two Fluorine-18 Labeled Prostate-Specific Membrane Antigen Inhibitors for Positron Emission Tomography Imaging of Prostate Cancer

Prostate cancer remains one of the most frequently diagnosed cancers in men. While its localized form is typically slowly advancing, the aggressive and metastasized forms are responsible for a significant amount of deaths in men in developed countries. Thus, more reliable methods of diagnosis are currently highly sought-after. Prostate Specific Membrane Antigen (PSMA) remains a highly-researched receptor of choice, which has been found to be overexpressed in majority of prostate cancers. Several PSMA-targeting inhibitors with suitable radioisotopes are already being utilized for PET imaging (18F, 68Ga) and treatment (177Lu) of prostate cancer, with new radiotracers possessing improved characteristics being highly sought-after. Fluoroglycosylation is a typical method of altering the properties of radiotracers, leading into more desirable tracer characteristics, such as increased renal excretion due to the compound’s increased hydrophilicity. This can be achieved with chemical reactions, such as oxime formation, where the molecule is conjugated with a fluorine-containing carbohydrate. The aim of this study was to develop two 18F-labeled PSMA inhibitors via oxime formation by utilizing two 18F-labeled carbohydrates: 5-[18F]fluoro-5-deoxyribose ([18F]9) and 2-[18F]fluoro-2-deoxy-D-glucose ([18F]12). The precursor 3 moiety was successfully synthesized by utilizing an amide coupling reaction (yield 56%), followed by acid-catalyzed deprotection. The purification of precursor 3 was achieved by high-performance liquid chromatography (HPLC) with a yield of 29%. The precursor moiety was conjugated with [18F]9 and [18F]12 via oxime formation to yield compounds [19F]4 (yield 39%) and [19F]5 (yield 39%), respectively. This was followed by the synthesis of their respective radioisotopes, [18F]4 and [18F]5. [18F]9 exhibited more favorable labeling characteristics with precursor 3 compared to those of [18F]12, likely due to its readily-available aldehydic form, and milder reaction conditions. Conjugation of [18F]9 with precursor 3 moiety was successfully achieved in 15 minutes at room temperature in the presence of 0.3 M anilinium acetate buffer, with a radiolabeling yield up to 91% (1.5 mM peptide concentration). Comparably, conjugation with [18F]12 was achieved in 30 minutes at 85 ℃ in the presence of aniline, with a radiolabeling yield of 57% (9.8 mM peptide concentration). Minor by-product formation was also evident with [18F]5 while the reaction appeared more specific with [18F]4. Purification of [18F]4 was achieved by HPLC, yielding the radiotracer with 98% radiochemical purity. Similarly, purification of [18F]5 was demonstrated with HLPC using a smaller batch, yielding the product with a radiochemical purity of 88%. Minor degradation of the oxime ether bond into free [18F]9 or [18F]12 was evident as a function of time in an acidic environment, especially with [18F]5. The lipophilicity of the compounds was also demonstrated by the shake-flask method. Both compounds were found to be highly hydrophilic, with LogD7.4 values of –2.8±0.3 and -3.1±0.2 for [18F]5 and [18F]4, respectively. Further experiments should be made to optimize the radiosynthesis protocols for higher activities, and to determine the minimum peptide concentration and reaction time needed for the oxime ether formation. Additionally, the molar activities of the compounds should be determined. Also, the IC50 inhibition potency of PSMA with [18F]4 should be evaluated prior to any in vivo trials to better evaluate its potential as a possible PSMA inhibitor

Efficient cartridge purification for producing high molar activity [18F]fluoro-glycoconjugates via oxime formation

Introduction 18F-fluoroglycosylation via oxime formation is a chemoselective and mild radiolabeling method for sensitive molecules. Glycosylation can also improve the bioavailability, in vivo kinetics, and stability of the compound in blood, as well as accelerate clearance of biomolecules. A typical synthesis procedure for 18F-fluoroglycosylation with [18F]FDG (2-deoxy-2-[18F]fluoro-d-glucose) and [18F]FDR (5-deoxy-5-[18F]fluoro-d-ribose) involves two HPLC (high performance liquid chromatography) purifications: one after 18F-fluorination of the carbohydrate to remove its labeling precursor, and a second one after the oxime formation step to remove the aminooxy precursor. The two HPLC purifications can be time consuming and complicate the adaptation of the synthetic strategy in nuclear medicine applications and automated synthesis. We have developed a procedure in which SPE (solid phase extraction) and resin purification methods replace both of the needed HPLC purification steps. Methods We used [18F]FDR and [18F]FDG as prosthetic groups to radiolabel two aminooxy-modified model molecules, a tetrazine and a PSMA (prostate specific membrane antigen) inhibitor. After fluorination, the excess carbohydrate precursor was removed by derivatizing it with 4,4′-dimethoxytrityl chloride (DMT-Cl). The DMT moiety increases the hydrophobicity of the unreacted precursor making the separation from the fluorinated precursor possible with simple C18 Sep-Pak cartridge. For removal of the aminooxy precursor, we used a commercially available aldehyde resin (AminoLink, Thermo Fisher Scientific). C18 Sep-Pak SPE cartridge was used to separate [18F]FDR and [18F]FDG from the 18F-fluoroglycoconjugate end product. Results [18F]FDR and [18F]FDG were efficiently purified from their precursors, free fluorine-18, and other impurities. The aldehyde resin quantitatively removed the unreacted aminooxy precursors after the oxime formation. The fluorine-18 labeled oxime end products were obtained with high radiochemical purity (>99%) and molar activity (>600 GBq μmol−1). Conclusions We have developed an efficient cartridge purification method for producing high molar activity 18F-glycoconjugates synthesized via oxime formation.Peer reviewe

Porous Silicon as a Platform for Radiation Theranostics Together with a Novel RIB-Based Radiolanthanoid

Mesoporous silicon (PSi) is biocompatible and tailorable material with high potential in drug delivery applications. Here, we report of an evaluation of PSi as a carrier platform for theranostics by delivering a radioactive ion beam- (RIB-) based radioactive lanthanoid into tumors in a mouse model of prostate carcinoma. Thermally hydrocarbonized porous silicon (THCPSi) wafers were implanted with Dy-159 at the facility for radioactive ion beams ISOLDE located at CERN, and the resulting [Dy-159]THCPSi was postprocessed into particles. The particles were intratumorally injected into mice bearing prostate cancer xenografts. The stability of the particles was studied in vivo, followed by ex vivo biodistribution and autoradiographic studies. We showed that the process of producing radionuclide-implanted PSi particles is feasible and that the [Dy-159]THCPSi particles stay stable and local inside the tumor over seven days. Upon release of Dy-159 from the particles, the main site of accumulation is in the skeleton, which is in agreement with previous studies on the biodistribution of dysprosium. We conclude that THCPSi particles are a suitable platform together with RIB-based radiolanthanoids for theranostic purposes as they are retained after administration inside the tumor and the radiolanthanoid remains embedded in the THCPSi.Peer reviewe

Engineered antibody-functionalized porous silicon nanoparticles for therapeutic targeting of pro-survival pathway in endogenous neuroblasts after stroke

Generation of new neurons by utilizing the regenerative potential of adult neural stem cells (NSCs) and neuroblasts is an emerging therapeutic strategy to treat various neurodegenerative diseases, including neuronal loss after stroke. Committed to neuronal lineages, neuroblasts are differentiated from NSCs and have a lower proliferation rate. In stroke the proliferation of the neuroblasts in the neurogenic areas is increased, but the limiting factor for regeneration is the poor survival of migrating neuroblasts. Survival of neuroblasts can be promoted by small molecules; however, new drug delivery methods are needed to specifically target these cells. Herein, to achieve specific targeting, we have engineered biofunctionalized porous silicon nanoparticles (PSi NPs) conjugated with a specific antibody against polysialylated neural cell adhesion molecule (PSA-NCAM). The PSi NPs loaded with a small molecule drug, SC-79, were able to increase the activity of the Akt signaling pathway in doublecortin positive neuroblasts both in cultured cells and in vivo in the rat brain. This study opens up new possibilities to target drug effects to migrating neuroblasts and facilitate differentiation, maturation and survival of developing neurons. The conjugated PSi NPs are a novel tool for future studies to develop new therapeutic strategies aiming at regenerating functional neurocircuitry after stoke

Engineered antibody-functionalized porous silicon nanoparticles for therapeutic targeting of pro-survival pathway in endogenous neuroblasts after stroke

Generation of new neurons by utilizing the regenerative potential of adult neural stem cells (NSCs) and neuroblasts is an emerging therapeutic strategy to treat various neurodegenerative diseases, including neuronal loss after stroke. Committed to neuronal lineages, neuroblasts are differentiated from NSCs and have a lower proliferation rate. In stroke the proliferation of the neuroblasts in the neurogenic areas is increased, but the limiting factor for regeneration is the poor survival of migrating neuroblasts. Survival of neuroblasts can be promoted by small molecules; however, new drug delivery methods are needed to specifically target these cells. Herein, to achieve specific targeting, we have engineered biofunctionalized porous silicon nanoparticles (PSi NPs) conjugated with a specific antibody against polysialylated neural cell adhesion molecule (PSA-NCAM). The PSi NPs loaded with a small molecule drug, SC-79, were able to increase the activity of the Akt signaling pathway in doublecortin positive neuroblasts both in cultured cells and in vivo in the rat brain. This study opens up new possibilities to target drug effects to migrating neuroblasts and facilitate differentiation, maturation and survival of developing neurons. The conjugated PSi NPs are a novel tool for future studies to develop new therapeutic strategies aiming at regenerating functional neurocircuitry after stoke.Peer reviewe

Expression of miR-32, Ki-67, Btg2, and Pten in Transgenic Mouse Prostate Tissue

Prostate cancer is the most common type of cancer found in men. An advanced form of the cancer – castration-resistant prostate cancer – is responsible for over 28 000 deaths in the United States every year with no current curative treatments available. As a result, new biomarkers and treatments are desperately needed. Putative biomarkers include, for example, certain microRNAs – small non-coding RNAs that are involved in the regulation of gene expression – or tumor suppressor genes that are responsive for cell cycle regulation. The objective of the thesis was to investigate the expression of several biomarkers in transgenic mouse prostate tissue. The purpose was to establish protocols for the in situ hybridization of miR-32, and immunohistochemical staining methods for Ki-67, Btg2, and Pten – and to investigate their expression in transgenic mouse prostate tissue. During this thesis, a successful protocol for the immunohistochemistry of Ki-67, Btg2, and Pten with PAXgene fixation was established. A working protocol for the in situ hybridization of miR-32 was also established, but with inconsistent staining. A low level of miR-32 expression was detected in the dorsolateral lobe of both wild type (WT) and transgenic (TG) prostates. Ki-67 was shown to have a low expression in both WT and TG prostates. Btg2 was expressed mainly in the basal cells of both WT and TG prostates, including the urethra and ductus deferens. Similarily, Pten expression was observed in the basal cells of both WT and TG urethra and, additionally, in the prostate. All biomarkers had close to identical expression in both WT and TG prostates. In conclusion, the protocol established here for the immunohistochemistry of Ki-67, Btg2, and Pten is sufficient for further use with commercial PAXgene fixation. However, the immunohistochemistry here should be repeated with fresh tissue sections, in order to ensure consistent staining of all sections. Further optimization should be attempted with in situ hybridization before moving into samples with confirmed prostatic lesions. In the future, the protocol established here for Ki-67, Btg2, and Pten can be used in mouse samples with confirmed prostatic lesions in order to help evaluate their role in prostate carcinogenesis, and in the formation of castration resistance.Eturauhassyöpä on miesten yleisin syöpä. Kastraatioresistentti eturauhassyöpä on eturauhassyövän edistynyt muoto, johon ei ole löydetty parantavaa hoitokeinoa. Kastraatioresistentti eturauhassyöpä on vastuussa yli 28 000 kuolemasta vuosittain USA:ssa. Tästä johtuen uusia biomarkkereita ja hoitokeinoja etsitään jatkuvasti. Mahdollisia biomarkkereita voivat olla esimerkiksi mikroRNA:t, jotka ovat geenien ilmentymistä sääteleviä lyhyitä ei-koodaavia RNA:ita. Biomarkkereihin voivat kuulua myös eri tuumorisuppressorigeenit, jotka ovat osa solusyklin säätelyä. Opinnäytetyön tavoitteena oli tutkia useiden eri biomarkkerien ilmentymistä transgeenisen hiiren eturauhaskudoksessa. Opinnäytetyön tarkoituksena oli pystyttää in situ hybridisaatio -menetelmä miR-32:n määritykseen sekä menetelmä Ki-67:n, Btg2:n ja Pten:n immunohistokemiallista värjäystä varten ja tutkia pystytettyjen menetelmien perusteella näiden biomarkkerien ilmentymistä. Ki-67:n, Btg2:n ja Pten:n määritystä varten pystytettiin yleispätevä immunohistokemiallinen värjäysmenetelmä PAXgene-fiksauksella. Lisäksi pystytettiin menetelmä miR-32:n määritykseen in situ hybridisaation avulla, mutta saatu värjäystulos oli epätasaista. In situ hybridisaation tulosten perusteella miR-32 ilmentymistä oli havaittavissa villityypin (WT) ja trasgeenisen (TG) hiiren dorsolateraalisessa eturauhaskudoksessa. Ki-67 ilmentyi pienissä määrin WT- ja TG-hiirten dorsaalissa eturauhaskudoksessa. Btg2 ilmentyi lähinnä WT- ja TG-hiirten eturauhasen, virtsaputken ja siemenjohtimen basaalisoluissa. Pten ilmentyi WT- ja TG-hiirten virtsaputken basaalisoluissa ja heikosti eturauhaskudoksessa. WT- ja TG-hiirten ilmentymisessä ei havaittu suurta eroa minkään biomarkkerin yhteydessä. Opinnäytetyössä pystytetty immunohistokemiallinen värjäysmenetelmä on tarpeeksi luotettava jatkotutkimuksia varten. In situ hybridisaation tulosten parantamiseksi tulisi suorittaa lisää menetelmän optimointia. Lisäksi saadut immunohistokemialliset värjäykset tulisi toistaa tuoreemmilla kudosleikkeillä, jotta saataisiin selvitettyä syy mahdollisiin värjäyseroihin kudosleikkeiden välillä. Tulevaisuudessa opinnäytetyössä pystytettyjä menetelmiä voidaan käyttää hyödyksi eturauhassyöpää sairastavien hiirilinjojen tutkimisessa. Tutkimalla ilmentymistä näissä hiirilinjoissa voidaan arvioida näiden biomarkkerien roolia eturauhassyövän synnyssä sekä kastraatioresistentin eturauhassyövän muodostuksessa