Cardiovascular Safety Pharmacology Study of a Single … · 2015. 12. 3. · SRI Study No. M748- 10...

Cardiovascular Safety Pharmacology Study of a Single Intravenous Dose of CUMI in Conscious Beagle Dogs SRI Study No. M748-10

Appendix B

ANALYTICAL CHEMISTRY

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Cardiovascular Safety Pharmacology Study of a Single Intravenous Dose of CUMI in Conscious Beagle Dogs SRI Study No. M748-10

Appendix B-1

CERTIFICATE OF ANALYSIS

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Certificate of Analysis

Chemical Name:

Catalog Number:

Molecular Formula:

Molecular Weight:

Structure:

2-(4-(4-(2-methoxyphenyI)piperazine-1 -yl)butyl)4-methyl-1,2,4-triazine-3,5(2H,4H)-dioneAB0011

C19H27N5O3

373.45 _ _

lU/Vu

Quantity: 500 mg

Plate: EM Science Silica gel 60 F254 GlassEluent: 5% MeOH in chloroformVisualization:254 nM

Column:2.1X50mm 3.5uM C18Injection volume: 3 ul

Mobile Phase: gradient from 5% to 100%organic in 3 min

Detection: 210, 230. 254 nm

APCIconsistent with structure m/2=374 (M+1

1 spot. Rf=0.41

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Cardiovascular Safety Pharmacology Study of a Single Intravenous Dose of CUMI in Conscious Beagle Dogs

SRI Study No. M748-10

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I. INTRODUCTION

Dose verification analyses were performed on formulations containing the test article, CUMI, in the selected vehicle, 5% ethanol in saline. The concentration, homogeneity, and stability of the test article in the vehicle were determined by high performance liquid chromatography (HPLC). In addition, the test article chromatographic purity was analyzed using the same HPLC method. The methodology, procedures, and final results are summarized in this report.

II. EXPERIMENTAL

A. Reference Standard

The test article (AB-090505R) was used as the reference standard in the dose analyses.

B. Analytical Method

Instrument: Hewlett-Packard Model 1100 series liquid chromatography system

Column: Phenomenex Luna C18 (2), 5µm, 4.6 x 250 mm Mobile Phase: A: 25 mM aqueous ammonium formate, 60% B: Acetonitrile (ACN), 40% Flow rate: 1.2 ml/min Run time: 10 min Injection Volume: 75 µl Detection: 254 nm Column Temp.: 25oC Data System: HP Chemstation Software; version A.10.02

C. Procedures

1. Preparation of Standard Solutions

Standard Stock Solutions: A test article sample was weighed and dissolved in 60% water: 40% ACN (the diluent) as standard stock solution Sa. To ensure the accuracy of the standard stock solution, a second standard stock solution (Sb) was



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prepared in the same way as that for Sa. Concentrations of the standard stock solutions were ~0.1 mg/ml.

Calibration Standard Solutions: With each experiment, five or six calibration standard solutions at different concentrations were prepared from the stock solution using the diluent. Concentrations of the calibration standards were between 1 and 10 µg/ml. The calibration standards were chromatographed to demonstrate the linearity of the calibration curve over the concentration range.

For the test article purity evaluation, standard solutions of 0.1 mg/ml were prepared and analyzed.

2. Preparation of the Sample Solution from the Dose Formulation

For dose verification, two or three aliquots were sampled for each dose concentration. Aliquots were accurately transferred into separate volumetric flasks or a selected container and diluted to the desired concentration with the diluent.

For homogeneity testing, triplicate aliquots were sampled from the lowest and the highest concentration of formulations. Aliquots were taken from the top, middle, and bottom of a fully mixed solution to demonstrate homogeneity.

For stability determination, two or three aliquots were sampled and prepared.

D. Analysis

1. System Suitability

A single standard solution was chromatographed six consecutive times to determine the system repeatability [reported as percent relative standard deviation (% RSD) of the peak area or response factor]. The % RSD should be ≤3.0% for the six injections.

The response factors of the calibration standards and the standards used during the run (e.g., bracketing standards) were calculated. The % RSD of these response factors should be ≤ 10.0%.

The response factor of the accuracy check standard Sb was compared with the response factor of the standard stock solution Sa. The percent accuracy of the two, when compared, should be within 100.0 ± 5.0%.

System suitability tests were completed for each experiment for the study. The experiments for the study are referred to Homogeneity and Stability (t=0) on 05/17/10, Dose Verification (also Stability t=0,t=4 hr at room temperature, t=4 days at refrigerator and t=4 hrs at room temperature after 4 days refrigerated)



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on 05/21/10, Dose Verification on 06/04/10, 06/11/10 and 06/25/10, and Test Article Purity on 07/07/10.

2. Linearity

Each of the calibration standards was chromatographed, and a linear regression calibration curve was generated. The correlation coefficient (r) should be ≥0.990.

3. Test Article Chromatographic Purity

The chromatographic purity of the test article was measured both before and after the in-life study. The same HPLC method was used both in concentration and in purity analyses. The purity was calculated by peak area percentage. The results were used to evaluate the test article stability during the study period.

4. Dose Verification

Two or three samples from each dose level were analyzed. The verified concentration, reported as a percentage of the nominal concentration, should be within 100.0 ± 10.0% of the nominal concentration.

5. Homogeneity Determination

Triplicate samples from the lowest and highest concentrations of the formulations were analyzed. The individual values, reported as percentages of nominal, from each set of triplicate samples were averaged. The % RSD for each set should be ≤ 5.0%.

6. Stability Determination

The formulation stability was established before and concurrently with the in-life study. Formulations were kept at room temperature and in refrigerator for stability evaluation. Stability results should be within 100.0 ± 10.0% of the initial concentration.

E. Calculations

The concentration of the test article in the dose formulations was determined by HPLC using an external standard method. The linear regression curve obtained from the calibration standards was used to calculate the concentration of test article in each formulation. Results from each dose concentration level were averaged as a percentage of the nominal concentration. Homogeneity was measured as % RSD of the triplicate samplings at the lowest and highest concentration levels. Stability results were calculated using an approach similar to that for concentration determination. Chromatographic purity was calculated by normalized peak area percentage.



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III. RESULTS/DISCUSSION

A. System Suitability

System suitability results are summarized in Table B-1 for each analytical experiment for the study. All system suitability test results were within the protocol requirements.

The prepared calibration standard curves were also found to be linear, and the correlation coefficients (r) are included in Table B-1. An example of a calibration curve is shown in Figure B-1. Representative HPLC chromatograms of test article standard solution and formulation sample are shown in Figure B-2.

B. Test Article Purity

The chromatographic purity of the test article results are summarized in Table B-2. As shown in Table B-2, the purity value did not change during the study duration, suggesting that the test article was stable for the time period.

C. Dose Verification

Concentrations of the test article in the dose formulations are shown in Table B-3. Dose verification results met the protocol criteria of within 100.0 ± 10.0% of nominal concentration, except for the test on 05/21/10. The dose verification result of 89.9% on 5/21/10 was outside the protocol criterion of 100.0 ± 10.0% of the nominal concentration. A deviation was written, and the study director was notified. [Note: one of the results from the test on 05/17/10 (2.64 µg/ml) was outside of protocol criterion of 100.0 ± 10.0% of the nominal concentration, but because it was for the mock formulation used only to assess stability, no deviation was written.]

D. Homogeneity Determination

Homogeneity results for the formulation solutions are summarized in Table B-4. The results met the protocol requirements of % RSD ≤ 5.0%.

E. Stability Determination

Results of the stability study are summarized in Table B-5. Formulations at a concentration range of 2.64 to 132 µg/ml (nominal) were stable for at least 4 days under refrigeration and 4 hr at room temperature (16.0 to 25.5°C) after refrigeration for 4 days (3.0 to 4.5°C).

IV. CONCLUSIONS

The dose verification results indicate that the formulations had the claimed concentrations and that the formulations were also homogeneous, except for the formulation on 05/21/10 when the dose concentration was outside of protocol criterion of



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100.0 ± 10.0% of the nominal concentration. A deviation was written, and the study director was notified.

Formulations at a concentration range of 2.64 to 132 µg/ml (nominal) were stable for at least 4 days under refrigeration and 4 hr at room temperature (16.0 to 25.5°C) after refrigeration for 4 days (3.0 to 4.5°C). Chromatographic purity results supported the conclusion that the test article was stable during the study period.



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Figure B-1. A representative calibration curve used in the analysis.



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Figure B-2. Representative HPLC chromatograms of the test article standard dissolved with the diluent

(water: acetonitrile 60:40) at 7.9 µg/ml (top panel) and 132 µg/ml mock formulation diluted to 7.9 µg/ml with diluent (bottom panel). Peaks at ~4 min are from the test article, CUMI.



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Table B-1. System Suitability and Method Performance Results1 System Suitability (Protocol Criteria) Experiment Results Comments

Repeatability (% RSD ≤ 3.0%)

Homogeneity and Stability (t=0) on 05/17/10

0.1 Pass

Dose Verification (also Stability t=0, t=4 hr, t=4

days) on 05/21/10 0.2 Pass

Dose Verification on 06/4/10 0.0 Pass



Test Article Stability on 07/07/10 0.1 Pass

Reproducibility of the Standard.

Throughout the Run (% RSD ≤ 10.0%)

Homogeneity and Stability (t=0) on 05/17/10 0.4 Pass


days) on 05/21/10 1.3 Pass




Accuracy Check (Within 100.0 ±

5.0%)

Homogeneity and Stability (t=0) on 05/17/10) 99.2 Pass


days) on 05/21/10 97.7 Pass

Dose Verification on 06/4/10

101.3 Pass


99.9 Pass


97.3 Pass

Linearity (r ≥0.990)

Homogeneity and Stability (t=0) on 05/17/10

0.99998 Pass


days) on 05/21/10 0.99994 Pass




1 The standard stock solution and calibration standards were used for system suitability and method performance tests. Standard stock solutions (~0.1 mg/ml) were used for accuracy checks. Calibration standards (~1-10 µg/ml) were used for other tests.



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Table B-2. Test Article Purity

Experiment Date

Sample Concentration

(mg/ml) Purity

(Peak Area %) Comment

05/17/10 0.101 99.9 No change in purity value 07/07/10 0.104 99.9



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Table B-3. Dose Verification of the Test Article in the Vehicle1

Dose Level (µg/kg)

Dose Formulations (µg/ml)

Percentage of Nominal

(%)

Average Percentage

of Nominal (%) Nominal Conc. Actual Conc.

1.32 (05/17/10,

mock formulation)

2.64

1.740 65.91

66.02 1.744 66.05

1.743 66.03

66.0 (05/17/10)

(mock formulation)

132

123.601 93.64

93.4 122.991 93.17

123.092 93.25

1.3 (5/21/10,

mock formulation)

2.64

1.770 67.06

67.32 1.782 67.50

13.2 (05/21/10) 26.4

23.677 89.69 89.93

23.775 90.06

66.0

(5/21/10, mock

formulation)

132

119.664 90.65

91.0

120.650 91.40

6.6 (06/04/10)

13.2 11.785 89.28

90.0 11.978 90.74

3.3 (06/11/10) 6.6

6.387 96.78 96.7

6.383 96.71

3.3 (06/25/10) 6.6

6.421 97.28 97.3

6.417 97.23 1 Results (% nominal) reported are based on nonrounded numbers. All other values may have been rounded for report entry. This note applies to all the tables in this appendix. 2 Outside of the protocol criterion of 100.0 ± 10.0% of the nominal concentration, but no deviation occurred because this formulation was used only to verify homogeneity and stability and not dosed to animals. 3 Outside of the protocol criterion of 100.0 ± 10.0% of the nominal concentration. A deviation was written, and the study director was notified.



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Table B-4. Homogeneity of the Test Article in the Vehicle

Nominal Conc. (µg/ml)

Actual Conc. (µg/ml)

Percentage of Nominal (%)

Homogeneity (% RSD)

2.64 (Top) 1.740 65.91

0.1 2.64 (Middle) 1.744 66.05

2.64 (Bottom) 1.743 66.03

132 (Top) 123.601 93.64

0.3 132 (Middle) 122.991 93.17

132 (Bottom) 123.092 93.25



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Table B-5. Stability of the Formulation Based on the Initial Concentration

Time Point

Formulation Concentration (µg/ml)

Percentage of Nominal or Initial,%

Average Percentage of Nominal or Initial,%

Nominal or Initial

Concentration Actual

T=0 (05/17/10)

2.64

1.740 65.91

66.0² 1.744 66.05

1.743 66.03

132.0

123.601 93.64

93.4 122.991 93.17

123.092 93.25

T=0 (05/21/10) 26.4

23.677 89.69 89.9²

23.775 90.06

T=4 hr RT (05/21/10)1 23.726

23.646 99.66 99.4

23.522 99.14

T=4 days Refrigerated (05/21/10)

1.742 1.770 101.63

102.0 1.782 102.30

123.228 119.664 97.11

97.5 120.650 97.91

T=4 hr RT after 4 days

Refrigerated (05/21/10)

1.742 1.766 101.38

101.4 1.766 101.35

123.228 120.518 97.80

97.9 120.663 97.92

¹The room temperature (RT) range was from 16.0 to 25.5°C. The refrigerator temperature range was from 3.0 to 4.5°C.²Outside of protocol criterion of 100.0 ± 10.0% of the nominal concentration for dose verification, but the formulation was not dosed to animals. The stability over the four days passed.

Cardiovascular Safety Pharmacology Study of a Single … · 2015. 12. 3. · SRI Study No. M748- 10...

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