BIOPHARM (Czechia)

With an increasing focus on sustainable technologies in the pharmaceutical industry, milling provides a solvent-free approach to improve drug dissolution. Milling of drugs with an excipient offers additional opportunities to achieve supersaturation kinetics. Therefore, this work aims to present insights of co-milling fenofibrate and apremilast, two good glass formers with low and high glass transition temperatures (Tgs) respectively. Drugs were co-milled with croscarmellose sodium for various process durations followed by thermal analysis, investigation of crystallinity, surface area and dissolution. The dissolution enhancement of the low-Tg glass former fenofibrate highly correlated with the process-induced increase in surface area of co-milled systems (R2 = 0.96). In contrast, the high-Tg glass former apremilast lost its crystalline order gradually after ≥ 10 min of co-milling, and favourable supersaturation kinetics during biorelevant dissolution testing were observed. Interestingly, the melting point of co-milled apremilast decreased and linearly correlated with the highest measured drug concentration (cmax) during in vitro dissolution (onset temperature R2 = 0.98; peak temperature R2 = 0.96). The melting point depression remained stable after 90 days for apremilast, whereas fenofibrate co-milled for 20 min or more showed an increase in melting point upon storage. This study demonstrated that co-milling with croscarmellose sodium is ideally suited to good glass formers with a high Tg. The melting point depression is thereby proposed as an easily accessible critical quality attribute to estimate likely dissolution performance of drugs in dry co-milled formulations.

Avian sarcoma and leukosis virus (ASLV), diversified into seven phylogenetically relativesubgroups (A, B, C, D, E, J, and K), present as either exogenous or endogenous viruses in domesticchicken. [...]

A polymer probe based on N-(2-hydroxypropyl)methacrylamide copolymers labelled with a fluorescent dye Dy-633 or Cy-7 and decorated with targeting oligopeptides GE-7 or GE-11, specific targeting ligands binding to epidermal growth factor receptor (EGFR) highly expressed on surface of tumour cells, was designed, synthesised and characterised. Specific accumulation of the polymer probe in the tumour mass is a prerequisite for successful fluorescence-guided endoscopic surgery as the fluorescence signal from the malignant cells enables more precise resection of the tumour without damaging the healthy tissue. Flow cytometry and confocal microscopy was used to assess the binding efficacy of the oligopeptide conjugates to EGFR on the cell membranes of the malignant cells. The results showed that the highest binding efficacy was achieved with polymers bearing the GE-11 targeting oligopeptide in human EGFR-positive hypopharyngeal carcinoma cells (FaDu) and in breast adenocarcinoma cells (MDA-MB-231). Similarly, the polymer probes targeted by the GE-11 oligopeptidewere found in vivo as highly effective in tumour accumulation, as determined from fluorescence imaging. Indeed, the ex vivo cross-section of the tumours showed significant tumour border fluorescence proving the potential of the studied polymer probes. Moreover, the presence of the active targeting moiety on the polymer-drug conjugate should enable the use of such a conjugate as a targeted polymer system for treatment of solid tumours. Replacement of the fluorescent probe with a cytostatic drug provides a targeted polymer nanocancerostatic for advanced treatment of neoplastic diseases, thus the polymer probes have multiple functions.

The ongoing progress in primordial germ cell derivation and cultivation is opening new ways in reproductive biotechnology. This study tested whether functional sperm cells can be matured from genetically manipulated primordial germ cells after transplantation in adult testes and used to restore fertility. We show that spermatogenesis can be restored after mCherry-expressing or GFP-expressing primordial germ cells are transplantated into the testes of sterilized G0 roosters and that mCherry-positive or GFP-positive non-chimeric transgenic G1 offspring can be efficiently produced. Compared with the existing approaches to primordial germ cell replacement, this new technique eliminates the germ line chimerism of G0 roosters and is, therefore, faster, more efficient and requires fewer animals. Furthermore, this is the only animal model, where the fate of primordial germ cells in infertile recipients can be studied.

This review discusses various techniques of storage of avian genetic resources. Semen cryopreservation still seems to represent one of the most effective and feasible methods for preservation of genetic resources. Various cryoprotective agents, cooling rates and semen packaging methods are discussed. The use of ovarian tissue, embryonic cells, such as primordial germ cells (PGCs) or blastodermal cells, is the most promising approach because both male and female genetic information can be preserved. A relatively innovative technique; transplantation of cryopreserved testicular cells into adult animals with subsequent production of donor-derived progeny is also mentioned. This transplantation method may contribute to the preservation of endangered avian species and to maintaining their genetic variability.

1. The aim of this study was to evaluate the ability of frozen-thawed testicular cells transplanted into infertile cocks to restore spermatogenesis and to compare two cryoprotectants (CPA) (dimethylsulfoxide (DMSO) and Biofreeze).2. A total of 24 infertile White Leghorn (WL) cocks were transplanted with cryopreserved testicular cells from fertile adult donor cocks. Both genetically close and phylogenetically distant chicken breeds were used as donor cocks.3. Twelve out of 24 WL recipient cocks with cryopreserved testicular cells restored spermatogenesis within 2 months after the transplantation. Six out of 12 recipient cocks with restored spermatogenesis successfully produced progeny expressing the donor phenotype.4. There was no difference between the CPA in cell viability after thawing or in the number of offspring produced from cryopreserved testicular tissue.5. The present work represents the first report of production of a donor-derived healthy progeny following frozen-thawed testicular cell transplantation in adult birds. The described results may contribute to preservation of endangered avian species and to maintaining their genetic variability.

Transplantation of male germ cells into sterilized recipients in chicken opened a new approach for chicken transgenesis as well as for preservation of endangered bird species. We describe the post transplantation re-population of the recipient seminiferous epithelium up to the production of heterologous sperm in about 50% of transplanted males. However, it is important to precisely identify basic germinal cell populations that trigger renewal cascade of the whole process of the spermatogenesis in the testes of cockerels. Detection of the nuclear DNA content by fluorescence-activated cell sorting (FACS) and expression of specific genes in single cell suspensions of testicular tissues seems to be suitable way for selection of proper cells. An improvement in the approach to germ cell transplantation between fowl males seems to be remarkably influenced by using an enriched subpopulation of c-Kit positive spermatogonia as well as SSEA-1 positive testicular cells as donor cells. Other chicken spermatogonia specific gene markers such as gfra, stra 8 or dazl can also be used for basic spermatogonial cells identification. Chicken recombinant GFRA1 receptor protein has been prepared and was used for obtaining mouse specific polyclonal antibodies. The presence of GFRA1 protein on the cell surface was confirmed by mmunocytochemical and immunohistochemical analyses. Particular identification of cellular and molecular markers unique to the germ cells together with efficient transplantation model will make testicular stem cells an indispensable tool not only for biotechnological application but can also contribute to endangered species preservation programs.

1. The aim of the study was to investigate the possibility of preparing adult fowl testes for the production of exogenous germ-lines by eradication of recipient spermatogenesis using gamma-radiation. 2. A comparison between several radiation therapy treatments (based on 60Co isotope) of male testes was conducted using gamma-rays of 18, 22 and 26 Gy in a single dose or repeated doses of 5 × 8 Gy over a 15-d period. Sperm concentration and motility were determined after each treatment. 3. Altered spermatogenesis was observed after a single treatment dose of 18 Gy, while single doses of 26 Gy were followed by reduced sperm numbers (from 22 × 109 to 31 × 106 sperm/ml) within 60 to 100 d after treatment. After a single treatment of 26 Gy sperm motility was reduced by 50%. In contrast, a fractionated treatment (5 × 8 Gy) with gamma-rays halted spermatogenesis 39 d after the distribution of the first 8 Gy dose. 4. Observations of the seminiferous tubules by electron microscopy performed 12 months after this treatment confirmed that moderate doses of gamma-rays (8 Gy) distributed repeatedly (5 ×) over a limited period (15 d) sterilise adult fowl testes but maintain morphologically normal somatic (Leydig and Sertoli) cell populations.