Enzyme-linked immunosorbent assay for the quantitative determination of Denosumab (Prolia®) in serum and plasma. Assay Genie Denosumab ELISA has been especially developed for the quantitative analysis of Denosumab in serum and plasma samples between the Cmin and Cmax range of concentrations indicated in the pharmacokinetics section of the technical manual.

Solid phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich principle. Standards and samples (serum or plasma) are incubated in the microtitre plate coated with the reactant specific for Denosumab (Prolia®). Following incubation wells are washed and then horse radish peroxidase (HRP) is added and binds to Denosumab. After incubation, the wells are washed, and the bound enzymatic activity is detected by addition of chromogen-substrate. The colour developed is proportional to the amount of Denosumab (Prolia®) in the sample or standard. Results of samples can be determined directly using the standard curve.

Denosumab (Prolia®) has a mechanism of action unlike other osteoporosis treatments. Administered every six months as a subcutaneous injection, Denosumab (Prolia®) is the only therapy that targets RANK Ligand, an essential regulator of osteoclasts (the cells that break down bone). Through highly specific inhibition of RANK ligand, Denosumab (Prolia®) decreases bone resorption, and improves bone density at all measured skeletal sites. Denosumab (Prolia®) is reimbursed for women and men aged 70 or over with a Bone Mineral Density (BMD) T-score of -2.5 or less.

Denosumab (Prolia®) is used for the treatment of osteoporosis in postmenopausal women to reduce the risk of vertebral, non-vertebral and hip fractures and to increase bone mass in men with osteoporosis at increased risk of fracture.

Denosumab (Prolia®) is a twice-yearly injection under the skin, usually administered by a general practitioner or practice nurse, and works by targeting the cells that break down bone (osteoclasts) thereby making bone less susceptible to osteoporosis-related fractures.

Denosumab (Prolia®) binds to RANKL, a transmembrane or soluble protein essential for the formation, function, and survival of osteoclasts, the cells responsible for bone resorption. Prolia prevents RANKL from activating its receptor, RANK, on the surface of osteoclasts and their precursors. Prevention of the RANKL/RANK interaction inhibits osteoclast formation, function, and survival, thereby decreasing bone resorption and increasing bone mass and strength in both cortical and trabecular bone.

In clinical studies, treatment with 60 mg of Prolia resulted in reduction in the bone resorption marker serum type 1 C-telopeptide (CTX) by approximately 85% by 3 days, with maximal reductions occurring by 1 month. CTX levels were below the limit of assay quantitation (0.049 ng/mL) in 39-68% of subjects 1-3 months after dosing of Prolia. At the end of each dosing interval, CTX reductions were partially attenuated from a maximal reduction of > 87% to <45% (range: 45% to 80%), as serum denosumab levels diminished, reflecting the reversibility of the effects of Prolia on bone remodelling. These effects were sustained with continued treatment. Upon reinitiation, the degree of inhibition of CTX by Prolia was similar to that observed in patients initiating Prolia treatment. Consistent with the physiological coupling of bone formation and resorption in skeletal remodelling, subsequent reductions in bone formation markers (i.e., osteocalcin and procollagen type 1 N-terminal peptide [PlNP]) were observed starting 1 month after the first dose of Prolia.

After discontinuation of Prolia therapy, markers of bone resorption increased to levels 40-60% above pre-treatment values but returned to baseline levels within 12 months. In a study conducted in healthy male and female volunteers (n = 73, age range: 18 to 64 years) following a single subcutaneously administered Prolia dose of 60 mg after fasting (at least for 12 hours), the mean maximum denosumab concentration (Cmax) was 6.75 mcg/mL (standard deviation [SD] = 1.89 mcg/mL). The median time to maximum denosumab concentration (Tmax) was 10 days (range: 3 to 21 days).

After Cmax, serum denosumab concentrations declined over a period of 4 to 5 months with a mean half-life of 25.4 days (SD = 8.5 days; n=46). No accumulation or change in denosumab pharmacokinetics with time was observed upon multiple dosing of 60 mg subcutaneously administered once every 6 months. Prolia pharmacokinetics were not affected by the formation of binding antibodies. A population pharmacokinetic analysis was performed to evaluate the effects of demographic characteristics.

Prolia® is a trademark of Amgen Inc.