Biopharmaceuticals

Example 7

Synthetic urine is prepared by dissolving 14.1 g of NaCl, 2.8 g KCl, 17.3 g of urea, 19 ml ammonia water (25%), 0.60 g CaCl₂ and 0.43 g MgSO₄ in 0.02 mole/L of HCl. The final pH of synthetic urine is adjusted to 6.04 by using HCl and ammonia water.

40 mg Sigma creatinine is dissolved in 10 ml of synthetic urine solution. 3 mg of human albumin is dissolved in 10 ml of synthetic urine solution to prepare the micro albumin solution.

4 mg Sigma hemin is dissolved in 20 ml of synthetic urine, 20 μL Hemin solution is used as a receptor for urine albumin detection at different creatinine concentration.

A desired volume of the biological sample (synthetic urine) is taken and dispensed on the electrode of the biosensor device and the corresponding cyclic voltammogram is obtained by the CHI-Electrochemical workstation using the potential window, that varies from 0 V to −1 V with scan rate of 0.1 V/sec.

The albumin content in the urine sample binds hemin thereby demonstrates a linear decrease in peak redox current with urine albumin concentration as shown in FIG. 15(a) for different creatinine concentrations. If the concentration of albumin in urine sample is increased, then the albumin increasingly binds with hemin thereby reducing the free hemin concentration on the electrode resulting in the decrease in peak redox current of free hemin. FIG. 16 shows the urine albumin concentrations, urine creatinine concentrations and calculated ACR for different samples.

The values of concentrations of the urine albumin (mg/L) and creatinine for different samples is shown in Table 4.

Example 49

The functional activity of compounds was determined in a cell line where p70S6K is constitutively activated. Test article was dissolved in DMSO to make a 10 μM stock. PathScan® Phospho-S6 Ribosomal Protein (Ser235/236) Sandwich ELISA Kit was purchased from Cell Signaling Technology. A549 lung cancer cell line, was purchased from American Type Culture Collection. A549 cells were grown in F-12K Medium supplemented with 10% FBS. 100 μg/mL penicillin and 100 μg/mL streptomycin were added to the culture media. Cultures were maintained at 37° C. in a humidified atmosphere of 5% CO₂ and 95% air. 2.0×10⁵ cells were seeded in each well of 12-well tissue culture plates for overnight. Cells were treated with DMSO or test article (starting at 100 μM, 10-dose with 3 fold dilution) for 3 hours. The cells were washed once with ice cold PBS and lysed with 1× cell lysis buffer. Cell lysates were collected and samples were added to the appropriate wells of the ELISA plate. Plate was incubated for overnight at 4° C. 100 μL of reconstituted Phospho-S6 Ribosomal Protein (Ser235/236) Detection Antibody was added to each well and the plate was incubated at 37° C. for 1 hour. Wells were washed and 100 μl of reconstituted HRP-Linked secondary antibody was added to each well. The plate was incubated for 30 minutes at 37° C. Wash procedure was repeated and 100 μL of TMB Substrate was added to each well. The plate was incubated for 10 minutes at 37° C. 100 μL of STOP Solution was added to each well and the absorbance was read at 460 nm using Envision 2104 Multilabel Reader (PerkinElmer, Santa Clara, CA). IC₅₀ curves were plotted and IC₅₀ values were calculated using the GraphPad Prism 4 program based on a sigmoidal dose-response equation.

Unless otherwise noted, compounds that were tested had an IC₅₀ of less than 50 μM in the S6K Binding assay. A=less than 0.05 μM; B=greater than 0.05 μM and less than 0.5 μM; C=greater than 0.5 μM and less than 10 μM;

Example 19

To confirm bioactivity of 3 and 7, experiments were performed with the HH cell line, a mature T cell line derived from peripheral blood of a patient with aggressive cutaneous T cell leukemia/lymphoma (ATCC® CRL-2105™) which been demonstrated to only express the IL-2Rβ/γ. One of the earliest events in cytokine mediated activation of lymphocytes such as CD8+ T cells and NK cells is Janus Associated Kinase mediated phosphorylation and activation of Signal transducer and activator of transcription (pSTAT5). Thus, pSTAT5 was used to measure biological activity of 3 and 7 alongside 12. 3 demonstrated clear bioactivity in IL-2Rβ/γ expressing HH cells (EC₅₀: 773 ng/ml) that was approximately 3.5 fold lower than 12 (EC₅₀: 233 ng/ml). Additionally, 7 induced bioactivity (EC₅₀: 756 ng/ml) very similar to 3, demonstrating that 7 retains bioactivity after being released from prodrug 5 even after accelerated (stress) conditions.