Radiooznačeni peptidi v nuklearni medicini

Nuklearna medicina pri obravnavi onkoloških bolnikov omogoča slikovni prikaz oziroma lokalizacijo tumorjev, ciljano zdravljenje ter spremljanje uspešnosti zdravljenja. Visoko specifične radiooznačene peptidne učinkovine lahko izkoristimo za ciljanje tumorskih celic, ki imajo na svoji površini prekomerno izražene receptorje za te učinkovine. Enako peptidno učinkovino lahko radiooznačimo tako z diagnostičnimi kot terapevtskimi radionuklidi, kar nam omogoča personaliziran pristop pri obravnavi bolnikov. V preglednem članku opisujemo dva sistema teranostičnih parov, ki jih v nuklearni medicini uporabljamo za diagnosticiranje ter zdravljenje tumorjev. Klasičen primer takšnih teranostičnih parov so radiooznačeni analogi somatostatina, ki se rutinsko uporabljajo v diagnostiki ter zdravljenju nevroendokrinih tumorjev (NET) s prekomerno izraženimi receptorji za somatostatin. Po drugi strani pa so radiooznačeni analogi minigastrina, ki se vežejo na receptorje za holecistokinin-2, primerni za diagnostiko medularnega raka ščitnice (MTC), za njihovo varno zdravljenje pa še potekajo klinična preskušanja

Development and evaluation of novel approaches for radiolabelling of haematopoietic stem cells

Zdravljenje z matičnimi celicami je danes v izjemnem vzponu na različnih področjih medicine, predvsem v kardiologiji, nevrologiji in onkologiji. Zaradi omejenih možnosti spremljanja presajenih celic in vivo je spremljanje in ovrednotenje uspešnosti zdravljenja, kljub množici slikovnih in drugih diagnostičnih ter laboratorijskih metod, ki so danes na razpolago, še vedno težavno. Označevanje matičnih celic z različnimi radioizotopi, oz. radiofarmaki, predstavlja primerno nuklearno medicinsko metodo za sledenje celicam in vivo po aplikaciji bolniku. Scintigram – slikovni prikaz prostorske porazdelitve označenih celic v telesu po presaditvi – lahko uporabimo tudi za kvantitativno oceno absolutnega ali relativnega števila celic v interesnem področju, npr. organu, kot je srce. Na področju nuklearno medicinske slikovne diagnostike se za neselektivno označevanje celic (levkocitov, trombocitov) danes rutinsko uporabljajo radiofarmaki, označeni z indijem-111 (8-hidroksikinolin (oksin) in 2-hidroksi-2,4,6-cikloheptatrienon (tropolonat)) in tehnecijem-99m (heksametilpropilenamin oksim oz. HMPAO) ali eksametazim. Oba izotopa ob razpadu izsevata žarke gama s primerno energijo sevanja ter imata ustrezen razpolovni čas za uporabo v medicini. Pri obeh izotopih se v postopku označevanja celic izkorišča lipofilnost kompleksa uporabljanega radiofarmaka, s katerim lahko neselektivno označimo vse celice, ki pridejo v stik. Z indijem-111 označen oksin ([111In][In(oksinat)3]) je bil prvi ligand, ki so ga v nuklearno medicinski diagnostiki uporabljali za označevanje levkocitov. Oksin in tropolonat tvorita z indijem-111 kompleksa v razmerju 3:1, ki sta nevtralna in visoko lipofilna, kar omogoča prehajanje kompleksa preko celične membrane. V celici pride do delne ali popolne disociacije kompleksov in vezave indija-111 na intracelularne beljakovine. S tehnecijem-99m označen HMPAO ([99mTc][Tc(HMPAO)]) je radiofarmak, ki je bil razvit z namenom spremljanja pretoka krvi skozi možgane. Kompleks [99mTc][Tc(HMPAO)] izkazuje visoko lipofilnost, je brez naboja in ima dovolj majhno molekulsko maso, da lahko prehaja preko možgansko krvne bariere. Mehanizem danes izkoriščamo tudi v klinični praksi za radiooznačevanje levkocitov. Po pasivni difuziji primarnega kompleksa skozi celično membrano, se kompleks pod vplivom celičnih encimov pretvori v sekundarni kompleks, ki zaradi nižje lipofilnosti ne more prehajati nazaj in ostane vezan v celici. Za razliko od indija-111 in tehnecija-99m, so galij-68, baker-64 in cirkonij-89 pozitronski sevalci, ki pri razpadu izsevajo pozitrone, kar izkoriščamo pri PET (pozitronska emisijska tomografija) slikovni diagnostiki. 10 krat večja občutljivost, v primerjavi s SPECT (enofotonska emisijska računalniška tomografija), boljša resolucija in lažja kvantifikacija PET omogočajo spremljanje manjših sprememb ter natančnejše spremljanje manjšega števila radiooznačenih celic. 68Ge/68Ga radionuklidni generator omogoča pridobivanje galija-68 na mestu uporabe, kar kljub kratki razpolovni dobi (68 min) ob uporabi avtomatiziranih sinteznih modulov omogoča pripravo z galijem-68 označenih radiofarmakov ustrezne kakovosti za rutinsko klinično uporabo. Baker-64 in cirkonij-89 imata daljši razpolovni dobi (12,7 h in 78,4 h) in sta zaradi možnosti daljšega sledenja označenih molekul v daljšem časovnem obdobju odlična kandidata za radiooznačevanje celic. Vsi našteti radionuklidi, enako kot indij-111, spadajo v skupino koordinacijiskih radiokovin, imajo podobne kemijske lastnosti kot indij-111 in podobno tvorijo komplekse. Vsi omogočajo tvorbo kompleksov z oksinom in tropolonatom, ki jih nato lahko uporabljamo za neselektivno radiooznačevanje celic. Doktorsko delo je razdeljeno na tri vsebinsko povezane sklope. V prvem sklopu obravnavamo glavni namen in cilj doktorskega dela, razvoj in ovrednotenje radiooznačevanja krvotvornih matičnih celic (CD34+). Krvotvorne matične celice (CD34+), pridobljene s stimulacijo kostnega mozga, za katere so v kliničnih študijah dokazali uporabnost za zdravljenje ishemičnih stanj (npr. ishemične bolezni srca) pri ljudeh, ki so neodzivni na standardno farmakološko zdravljenje ali zdravljenje z revaskularizacijo, smo označili z [99mTc](HMPAO)], ki danes v klinični praksi predstavlja zlati standard za radiooznačevanje levkocitov. Ovrednotili smo vpliv radiooznačevanja na fiziološko stanje celic (vpliv na viabilnost celic, citotoksičnost, izplavljanje aktivnosti iz celic). Radiooznačevanje krvotvornih matičnih celic (CD34+) z [99mTc][(HMPAO)] nima vpliva na funkcionalnost označenih celic in po aplikaciji omogoča sledenje in spremljanje vgnezditve celic na želeno mesto z uporabo SPECT. S pridobljenimi podatki lahko z uporabo nuklearno medicinske (NM) slikovne diagnostike pomembno prispevamo k oceni uspešnosti zdravljenja z matičnimi celicami in morebitnim potrebnim spremembam zdravljenja. Uporaba celic, radiooznačenih s pozitronskimi sevalci, je kljub prednostim PET slikovne diagnostike (boljša občutljivost in resolucija, lažja kvantifikacija) omejena. Vzroki so povezani z dosegljivostjo izotopov ki izsevajo pozitrone, ter možnostmi avtomatizirane priprave radiofarmakov ustrezne kakovosti v volumnih dovolj majhnih za radiooznačevanje celic. V drugem sklopu doktorskega dela smo preučili in ovrednotili uporabo avtomatiziranega modula z lastno zaščito, namenjenega popolnoma avtomatizirani pripravi radiofarmakov označenih z galijem-68 in/ali drugimi PET in beta sevalci. Uporaba takšnega modula je nujna za zmanjšanje sevalne obremenitve osebja, ki pripravlja radiofarmake, ter hkrati omogoča pripravo radiofarmakov primerne kakovosti in primernih volumnov. V tretjem sklopu doktorskega dela smo razvili in optimizirali metodo koncentriranja pozitronskih radioizotopov (galij-68, baker-64, cirkonij-89) na anionsko izmenjevalni koloni, radiooznačevanje oksina in tropolonata na koloni ter elucije nastalih kompleksov v fiziološkem mediju, v dovolj majhnih volumnih (manj kot 2 ml) in primernih za radiooznačevanje celic. Razvita metoda bi lahko v prihodnosti omogočala avtomatizirano pripavo radiofarmakov za radiooznačevanje celic. S pripravljenimi radiofarmaki ustrezne kakovosti smo radiooznačili eritrocite in ovrednotili označevanje ter izplavljanje radioaktivnosti iz celic. Na podlagi rezultatov označevanja eritrocitov smo z [64Cu][Cu(tropolonat)2] in [64Cu][Cu(oksinat)2] radiooznačili levkocite in ovrednotili označevanje. Potencialno primernost [64Cu][Cu(tropolonat)2] in [64Cu][Cu(oksinat)2] za nadaljnjo uprabo v klinični praksi smo potrdili tudi na živalskem modelu (BALB/c miši) vnetja in okužbe.Treatment with stem cells is in an exceptional ascent in various fields of medicine, including cardiology, neurology and oncology. The evaluation of the success of the treatment is still difficult due to the limited possibilities of the in vivo monitoring of transplanted cells, despite the multitude of imaging, diagnostic and laboratory methods nowadays available. The radiollabeling of stem cells with various isotopes bound in complexes (radiopharmaceuticals) represents an appropriate method that enables in vivo cell tracking after administration. Scintigram (scan), which is a visual representation of the spatial distribution of labelled cells in the body after the transplantation, can be used for quantitative estimation of the absolute or relative number of cells in the region of interest, e.g. an organ like heart, as well. In the field of nuclear medicine imaging, radiopharmaceuticals labelled with indium-111 (oxine, tropolone) and technetium-99m (HMPAO) are routinely used to label cells (leukocytes, platelets). Both isotopes are gamma emitters, with appropriate energy and half-life for use in medicine. Both isotopes form highly lipophilic complexes with ligands, by which all the cells that come into contact with, can be non-selectively radiolabelled. 111In-labelled oxine (8-hydroxyquinoline) was the first ligand to be used in the radiolabelling of leukocytes in the field of nuclear medicine imaging. Both, oxine and the tropolone, form a 3:1 complex with indium-111 that are neutral and highly lipophilic, which allows the diffusion of the complex across the cell membrane. In the cell, partial or complete dissociation of the complexes and the binding of indium-111 to intracellular proteins occur. Technetium-99m labelled hexamethylpropyleneamine oxime (HMPAO) or exametazim is a radiopharmaceutical that was developed to monitor changes in regional cerebral blood flow. The [99mTc][Tc(HMPAO)] complex exhibits high lipophilicity, has no charge, and has a molecular weight low enough to pass through the blood brain barrier. The described mechanism is being exploited in routine nuclear medicine imaging practice for cell (leukocytes) radiolabelling. After the passive diffusion of the primary complex across the cell membrane, the cell enzymes transform the complex into a secondary complex with lower lipophilicity and thus remain in the cell. Galium-68, copper-64 and zirconium-89 are positron emitters, used in PET (Positron Emission Tomography) nuclear imaging diagnostics. Compared to SPECT (Single Photon Emission Computed Tomography), PET is characterized by higher sensitivity (10-times higher), better resolution and easier quantification, allowing the detection of smaller lesions and more accurate tracking of a small number of radiolabelled cells. The 68Ge/68Ga radionuclide generator allows the gallium-68 to be produced at the site of use. Use of the automated synthesis modules enables the production of tracers labelled with gallium-68 of appropriate quality for routine clinical use, despite its short half-life (68 min). Copper-64 and zirconium-89 have longer half-lives (12.7 h, 78.4 h) that could allow longer tracking of radiolabelled cells, and represent excellent candidates for the synthesis of radiopharmaceuticals for radiolabelling cells. All three isotopes, like indium-111, belong to a group of radiometals. They have similar chemistry as indium-111, and form similar complexes, enabling the formation of complexes with oxine (8-hydroxyquinoline) and tropolone (2-hydroxy-2,4,6-cycloheptatrienone) that can be further used for non-selective radiolabelling of cells. The doctoral thesis is comprised of three content-related sections. In the first section, covering the main goal of the doctoral thesis, development and evaluation of radiolabelling of haematopoietic stem cells (CD34+) were conducted. We radiolabeled haematopoietic stem cells (CD34+) with [99mTc][Tc(HMPAO)], a technique that today represents the gold standard for radiolabelling of WBC in daily clinical practice. The treatment with haematopoietic stem cells (CD34+) is clinically proven to be useful in the treatment of ischemic conditions, e.g. ischemic heart disease, in patients who are not responding to standard pharmacological treatment or treatment with revascularization. CD34+ cells obtained by bone marrow stimulation were labelled with [99mTc][Tc(HMPAO)] and the effect of radiolabelling on the physiological state (effect on cell viability, cytotoxicity, efflux of radiopharmaceuticals from cells) was evaluated. No influence of cell labelling on the physiological status of the cells was observed. Radiolabelling of CD34+ cells with [99mTc][Tc(HMPAO)] enables short term cell tracking of radiolabelled cells an engraftment evaluation using SPECT imaging. With the obtained data, we can significantly contribute to the assessment of the effectiveness of stem cell therapy and possible changes in treatment. Up to now the preparation of cell radiolabelling tracers of appropriate quality and small enough volumes for PET, with its superiority over SPECT (better sensitivity, quantification and resolution) is still a major limitation for wider use. Reasons are generally connected with possibilities of automation of the tracer preparation and depending on the radionuclide, properties of PET tracer itself, enabling cell radiolabelling. Our goal of preparing PET radiopharmaceuticals for cell radiolabelling is explained in two sections. In the second section of the thesis, we studied and evaluated the use of a self-shielded radiosynthesis module for fully automated preparation of radiotracers labelled with gallium-68 and/or other PET and beta emitters. The use of such a module is necessary in order to reduce the radiation load of the personnel preparing the radiopharmaceuticals, while at the same time allowing the preparation of radiopharmaceuticals of appropriate quality and in suitable volumes. In the third section we developed and optimized the method for the concentration of PET radioisotopes (gallium-68, copper-64, zirconium-89), subsequent radiolabelling of oxine and tropolone on the anion exchange column and the elution of the complexes formed on the column in the physiological medium in volumes small enough for radiolabelling of cells and potentially suitable for the automation. As a proof of concept, we radiolabelled erythrocytes with prepared radiopharmaceuticals and evaluated efflux of the radioactivity from the cells, followed by radiolabelling of leukocytes with the most promising compounds (64Cu-labelled tropolone and oxine). The suitability for potential use in clinical practice was confirmed in an animal model (BALB/c mice) of inflammation and infection

Semaglutide delays 4-hour gastric emptying in women with polycystic ovary syndrome and obesity

Aim: To evaluate the effect of once-weekly subcutaneous semaglutide 1.0 mg on the late digestive period of gastric emptying (GE) after ingestion of a standardized solid test meal by using technetium scintigraphy, the reference method for this purpose. Methods: We conducted a single-blind, placebo-controlled trial in 20 obese women with polycystic ovary syndrome (PCOSmean [range] age 35 [32.3-40.8] years, body mass index 37 [30.7-39.8] kg/m$^2$) randomized to subcutaneous semaglutide 1.0 mg once weekly or placebo for 12 weeks. GE was assessed after ingestion of [$^{99mT}$c] colloid in a pancake labelled with radiopharmaceutical by scintigraphy using sequential static imaging and dynamic acquisition at baseline and at Week 13. Estimation of GE was obtained by repeated imaging of remaining [$^{99mT}$c] activity at fixed time intervals over the course of 4 hours after ingestion. Results: From baseline to the study end, semaglutide increased the estimated retention of gastric contents by 3.5% at 1 hour, 25.5% at 2 hours, 38.0% at 3 hours and 30.0% at 4 hours after ingestion of the radioactively labelled solid meal. Four hours after ingestion, semaglutide retained 37% of solid meal in the stomach compared to no gastric retention in the placebo group (P = 0.002). Time taken for half the radiolabelled meal to empty from the stomach was significantly longer in the semaglutide group than the placebo group (171 vs. 118 minP < 0.001). Conclusion: Semaglutide markedly delayed 4-hour GE in women with PCOS and obesity

Post-radiation xerostomia therapy with allogeneic mesenchymal stromal stem cells in patients with head and neck cancer

Background: Xerostomia is a common side effect of radiotherapy in patients with head and neck tumors that negatively affects quality of life. There is no known effective standard treatment for xerostomia. Here, we present the study protocol used to evaluate the safety and preliminary efficacy of allogeneic mesenchymal stromal stem cells (MSCs) derived from umbilical cord tissue. Methods: Ten oropharyngeal cancer patients with post-radiation xerostomia and no evidence of disease recurrence 2 or more years after (chemo)irradiation (intervention group) and 10 healthy volunteers (control group) will be enrolled in this nonrandomized, open-label, phase I exploratory study. MSCs from umbilical cord tissue will be inserted under ultrasound guidance into both parotid glands and both submandibular glands of the patients. Toxicity of the procedure will be assessed according to CTCAE v5.0 criteria at days 0, 1, 5, 28, and 120. Efficacy will be assessed by measuring salivary flow and analyzing its composition, scintigraphic evaluation of MSC grafting, retention, and migration, and questionnaires measuring subjective xerostomia and quality of life. In addition, the radiological, functional, and morphological characteristics of the salivary tissue will be assessed before, at 4 weeks, and at 4 months after the procedure. In the control group subjects, only salivary flow rate and salivary composition will be determined. Discussion: The use of allogeneic MSCs from umbilical cord tissue represents an innovative approach for the treatment of xerostomia after radiation. Due to the noninvasive collection procedure, flexibility of cryobanking, and biological advantages, xerostomia therapy using allogeneic MSCs from umbilical cord tissue may have an advantage over other similar therapies