GS-1590

Analogues of vitamin D3 are extensively used in the treatment of various illnesses, such as osteoporosis, inflammatory skin diseases, and cancer. Functional testing of new vitamin D3 analogues and formulations for improved systemic and topical administration is supported by sensitive screening methods that allow a comparative evaluation of drug properties. As a new tool in functional screening of vitamin D3 analogues, we describe a genomically integratable sensor for sensitive drug detection. This system facilitates assessment of the pharmacokinetic and pharmadynamic properties of vitamin D3 analogues. The tri-cistronic genetic sensor encodes a drug-sensoring protein, a reporter protein expressed from an activated sensor-responsive promoter, and a resistance marker.

The three expression cassettes, inserted in a head-to-tail orientation in a Sleeping Beauty DNA transposon vector, are efficiently inserted as a single genetic entity into the genome of cells of interest in a reaction catalyzed by the hyperactive SB100X transposase. The applicability of the sensor for screening purposes is demonstrated by the functional comparison of potent synthetic analogues of vitamin D3 designed for the treatment of psoriasis and cancer. In clones of human keratinocytes carrying from a single to numerous insertions of the vitamin D3 sensor, a sensitive sensor read-out is detected upon exposure to even low concentrations of vitamin D3 analogues. In comparative studies, the sensor unveils superior potency of new candidate drugs in comparison with analogues that are currently in clinical use.

Our findings demonstrate the use of the genetic sensor as a tool in first-line evaluation of new vitamin D3 analogues and pave the way for new types of drug delivery studies in sensor-transgenic animals.

Background and objective  1,25‐dihydroxyvitamin D3[1α,25(OH)2D3, calcitriol], and its less calcaemic synthetic analogues have therapeutic potential in several diseases, including hyperparathyroidism (HPT). We have suggested that non‐1α‐hydroxylated (nonactive) vitamin D analogues may present an alternative in tumour cells expressing 25‐hydroxyvitamin D3 1α‐hydroxylase (1α‐hydroxylase). The aim of this study was to investigate biological effects of a non‐1α‐hydroxylated vitamin D analogue in normal and tumour parathyroid cells.Patients and methods  Effects of vitamin D analogues and ketoconazole on parathyroid hormone (PTH) secretion (radioimmunoassay) and PTH mRNA expression (reverse transcription‐polymerase chain reaction) were studied in primary bovine parathyroid cells. Proliferation of tumour cells isolated from HPT patients was determined by thymidine incorporationResults  EB1285, non‐1α‐hydroxylated precursor of the vitamin D analogue EB1089, suppressed PTH secretion and PTH mRNA level as well as increased expression of 25‐hydroxyvitamin D3‐24‐hydroxylase (24‐hydroxylase) in bovine parathyroid cells. EB1285 also inhibited cell proliferation of parathyroid tumour cells from primary (pHPT) and secondary HPT (sHPT) patients. Combined treatment with the cytochrome P450‐dependent enzyme inhibitor ketoconazole and EB1285 or with active vitamin D compounds potentiated the suppressive effect on PTH secretion from bovine parathyroid cells. Ketaconazole alone displayed PTH suppression and increased 24‐hydroxylase expression.Conclusion  The results support the idea that a non‐1α‐hydroxylated vitamin D analogue may elicit vitamin D receptor (VDR) effects in 1α‐hydroxylase expressing parathyroid tumour cells. Further studies are warranted to elucidate whether precursor vitamin D analogues as well as inhibitors of 24‐hydroxylase present therapeutic alternatives in patients suffering from HPT.