Constitucionalidad en materia de fraude.

The stability evaluation of a selected group of compounds was done in three different experiments aiming to answer the following questions: i) are the compounds stable at physiological conditions, ii) are the compounds stable in the formulation solutions and iii) what would be the ideal storage conditions for the compounds. In order to answer these questions, three sets of experiments were designed. The first one consists of dissolving the compounds in the above- described formulations followed by six times dilution in PBS (pH = 7.4) and incubation at 37 ˚C for 24 hours. Samples were periodically injected into an HPLC instrument and the real-time decomposition of the compound was monitored. Compounds BW-AQ-112, BW-AQ-124, and BW-AQ-131 appeared to be relatively stable (Table 1.5.8). The compounds showed about 5.5 %, 4.9 %, and 0.8 % respective decomposition at simulated physiological conditions. The lead compound BW-AQ-101 showed about 8.3 % decomposition at the same condition after 24 hours,

however, only 1.6 % after two hours incubation. These results are indicative for the general stability of the structures and their therapeutic potential. Unfortunately compound BW-AQ-126 showed significant decomposition of over 38 % (Table 1.5.8) at close to physiological conditions. The lack of stability if this compound eliminates it from further consideration for a drug candidate. Nevertheless, the other four compounds that were tested showed good stability and promise to be taken for further pre-clinical evaluations.

Table 1.17 Stability studies in solution at 37 ˚C

* 0 min. readings indicate the initial purity of the compounds.

BW-AQ-101 BW-AQ-112 BW-AQ-124 BW-AQ-131 BW-AQ-126 0 min. 97.2 % 98.6 % 99.1 % 87.7 % 95.3 % 120 min. 95.6 % 98.4 % 97.6 % 87.2 % 91.2 % 24 h. 88.9 % 93.0 % 94.2 % 86.9 % 56.4 %

The purpose of the second stability study that was performed was to evaluate the storage capability of the active compounds in the established formulation solutions for an extended period of time (up to eight weeks). This long-term stability test was done on BW-AQ-101, BW-AQ-112, BW-AQ-113, and BW-AQ-124 based on their activities and physicochemical properties. These compounds were dissolved in the most appropriate formulations (discussed in section 1.5.2) and stored under three different conditions: room temperature, 4 ˚C, and -20 ˚C. None of the compounds exhibited significant decomposition at -20 ˚C after eight weeks (data shown in the appendix). Compounds BW-AQ-101 and BW-AQ-124 showed about 1 % and 2 % decomposition at 4 ˚C and the solutions of BW-AQ-112, BW-AQ-113 appeared completely stable at this temperature. Eight-week solution storage at room temperature resulted in minor decomposition of all compounds. While BW-AQ-101 and BW-AQ-112 showed only 3 % and 4 % decomposition, compounds BW-AQ-124 and BW-AQ-113 showed 9 % and 7 % decomposition respectively. The

eight-week study revealed that solution storage may be suitable for some of the rhein analogs at low temperatures (4 ˚C, and -20 ˚C), but cannot be utilized at room temperature.

The third storage stability evaluation of these compounds was done at solid state and under the following temperature conditions: room temperature, 4 ˚C, and -20 ˚C. The compounds showed no significant decomposition after 17 weeks storage in a solid form at 4 ˚C, and -20 ˚C (data presented in the appendix). Compounds BW-AQ-101 and BW-AQ-124 showed about 1 % decomposition when stored in a solid form at room temperature. Furthermore, BW-AQ-112 and BW-AQ-113 showed no significant evidence for decomposition when stored as solids at room temperature.

The above-described stability studies show that the active rhein analogs are relatively stable compounds and can be stored as solids for prolonged periods of time. Ideal storage conditions will require low temperatures (4 ˚C or -20 ˚C). Most compounds appear tolerable to storage at room temperature as well. Short-term storage in a solution form at -20 ˚C are plausible for these compounds, however, long term (over 8 weeks) solution storage at these conditions is not recommended.

1.5.3.1 Experimental section

All solutions for the stability determination of compounds BW-AQ-101 and BW-AQ-124 were prepared using ethanol/cremophor 4/1 (v/v) ration. All solutions for the stability determination of compounds BW-AQ-112 and BW-AQ-113 were prepared using isopropanl/solutol 4/1 (v/v) ration. 1 mM stock solutions of each compound were prepared in the appropriate formulation. 30-fold dilution in PBS was done to achieve 30 µM final concentration samples of the tested compounds. The prepared samples were then injected into the HPLC (Shimadzu UFLC SIL-20AHT) with Waters SinFire C18, 3.5 µm, 4.6 × 100 mm column. The

mobile phase was either acetonitrile/water (0.25 % TFA) (50/50, v/v) for BW-AQ-101 and BW- AQ-124, or acetonitrile/water (0.25 % TFA) (40/60, v/v) for BW-AQ-112 and BW-AQ-113 with flow rate of 1 mL/ min. The data was collected at 250 nm with threshold 50 and minimum detectable are of 7000 (< 1 %).

1.5.4 References

(1) Baka, E.; Comer, J. E. A.; Takacs-Novak, K. J.Pharmaceut. Biomed.2008, 46, 335.

(2) Volgyi, G.; Baka, E.; Box, K. J.; Comer, J. E. A.; Takacs-Novak, K. Anal. Chim. Acta2010, 673, 40.

(3) Glomme, A.; Marz, J.; Dressman, J. B. J. Pharm. Sci.2005, 94, 1.

(4) Box, K. J.; Volgyi, G.; Baka, E.; Stuart, M.; Takacs-Novak, K.; Comer, J. E. A. J. Pharm. Sci.2006, 95, 1298.

(5) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.2012, 64, 4.

(6) Tetko, I. V.; Gasteiger, J.; Todeschini, R.; Mauri, A.; Livingstone, D.; Ertl, P.; Palyulin, V.; Radchenko, E.; Zefirov, N. S.; Makarenko, A. S.; Tanchuk, V. Y.; Prokopenko, V. V. J. Comput. Aid. Mol. Design2005, 19, 453.

(7) Gelderblom, H.; Verweij, J.; Nooter, K.; Sparreboom, A. Eur. J. Cancer2001, 37, 1590.