Development of an inducible platform for intercellular protein delivery.

A challenge to protein based therapies is the ability to produce biologically active proteins and their ensured delivery. Various approaches have been utilised including fusion of protein transduction domains with a protein or biomolecule of interest. A compounding issue is lack of specificity, efficiency and indeed whether the protein fusions are actually translocated into the cell and not merely an artefact of the fixation process. Here we present a novel platform, allowing the inducible export and uptake of a protein of interest. The system utilises a combination of the Tetracyline repressor system, combined with a fusion protein containing the N-terminal signal peptide from human chorionic gonadotropin beta-subunit, and a C-terminal poly-arginine domain for efficient uptake by target cells. This novel platform was validated using enhanced green fluorescent protein as the gene of interest. Doxycycline efficiently induced expression of the fusion protein. The human chorionic gonadotropin beta-subunit facilitated the export of the fusion protein into the cell culture media. Finally, the fusion protein was able to efficiently enter into neighbouring cells (target cells), mediated by the poly-arginine cell penetrating peptide. Importantly we have addressed the issue of whether the observed uptake is an artefact of the fixation process or indeed genuine translocation. In addition this platform provides a number of potential applications in diverse areas such as stem cell biology, immune therapy and cancer targeting therapies.

Development of a radiolabeled beta-human chorionic gonadotropin.

beta-Human chorionic gonadotropin (beta-hCG) was successively labeled with [(67)Ga]-gallium chloride after conjugation with freshly prepared diethylenetriaminepentaacetic acid dianhydride (ccDTPA). After solid phase purification of the radiolabeled hormone, high performance liquid chromatography showed radiochemical purity higher than 95 % under optimized conditions (specific activity = 22-23 TBq mM(-1), labeling efficiency 80 %). Preliminary in vivo studies (ID g(-1), %) in male wild-type rats showed marked gonadal uptake of the tracer after 240 minutes in agreement with the biodistribution studies and reported beta-hCG receptors. Target to blood ratios were 5.1 and 15.2 after 3 and 24 hours, respectively, while target to muscle ratios were 35 and 40 after 3 and 24 hours, respectively.

Continuing the debate on empty follicle syndrome: can it be associated with normal bioavailability of beta-human chorionic gonadotrophin on the day of oocyte recovery?

This paper describes our experience with four ovarian stimulation in-vitro fertilization (IVF) cycles in which we failed to retrieve oocytes despite normal bioavailability of beta-human chorionic gonadotrophin (beta-HCG) in patients' blood 35 h after HCG administration. In three cases, the oocyte recovery procedure was interrupted, a second dose of HCG was administered and 24 h later mature oocytes were collected from two of the patients. In the first case, the three metaphase II oocytes collected fertilized after intracytoplasmic sperm injection (ICSI) and two cleaved grade three embryos were transferred but pregnancy did not ensue. In the second case, six out of eight metaphase II oocytes fertilized and cleaved following ICSI, leading to transfer of one grade two and two grade three embryos. This resulted in a clinical pregnancy which at the time of this report is ongoing. A similar rescue protocol was used for the third case who had empty follicle syndrome (EFS) in her previous treatment cycle but only cumulus-corona complexes were aspirated. Five additional patients who had EFS before instituting pregnancy diagnostic test screening have had further treatment cycles in which oocytes were collected but pregnancy did not ensue. We conclude that normal bioavailability of beta-HCG on the day of oocyte recovery does not exclude the diagnosis of EFS. EFS does not predict a reduced fertility potential in future cycles, although it may recur due to a biological abnormality in the availability of mature oocytes that are retrievable. In such patients, oocyte donation may offer the chance of achieving a pregnancy.

Human chorionic gonadotropin: pharmacokinetics of subcutaneous administration.

The objective of the present study was to evaluate the pharmacokinetics of human chorionic gonadotropin (hCG) following different regimens of subcutaneous and intramuscular single-dose administration. Two hypogonadotropic hypogonadal volunteers received hCG injections without prior ovarian stimulation. The regimens included a single dose of 10,000 IU hCG either subcutaneously or intramuscularly, or 5000 IU hCG intramuscularly. Serum beta-hCG concentrations were measured periodically up to 13 days after hCG administration. Each of the three regimens exhibit a similar pharmacokinetic profile and the highest serum beta-hCG concentrations were achieved with a dose of 10,000 IU administered subcutaneously. Seven days after hCG administration beta-hCG was detectable only after subcutaneous or intramuscular administration of 10,000 IU, but not after a single intramuscular injection of 5000 IU. From the preliminary results of the study it is suggested that a single intramuscular dose of 5000 IU hCG might be sufficient to trigger ovulation, but for luteal-phase support a higher dose may be needed. Subcutaneous administration of hCG for the induction of ovulation or luteal-phase support in gonadotropin-induced cycles is feasible and might offer a better tolerance and cost-effectiveness of infertility treatments, leading to their further simplification.