A Novel In-Vitro Release Method for Liposomal Doxorubicin

A Novel In-Vitro Release Method for Liposomal Doxorubicin

Introduction Azaya Therapeutics has discovered a novel in vitro release method that delivers meaningful scientific data for liposomal or nanotechnology drug products. This approach is able to distinguish subtle differences in the manufactured drug product through an evaluation of the drug release rate. Companies developing liposome or nanotechnology products are aware that evaluating the drug release characteristics involve expensive in vivo studies or specialized in vitro release methods. Current in vitro release analytical methodology has failed to deliver meaningful results. As a result, regulatory agencies are requiring sponsors to perform additional in vivo clinical studies to prove bioequivalence for major process changes including site changes. Drug development for liposomal and nanotechnology drug products can be optimized without the need of high cost in vivo studies. Furthermore, site transfer for the manufacture of the nanotechnology drug product can be successfully qualified with the use of this in vitro release method. This breakthrough is a significant milestone for liposomal and nanotechnology drug development. Drug product quality can be verified with this simple, in vitro release test during development and stability. Azaya has enhanced the quality of Azaya Therapeutics products through this newly developed technique and is continuing to look for new and innovative breakthrough discoveries. Scientific Method Development Previous methodology for liposomal in vitro release studies involved mixing the product with an ammonium chloride solution maintained at 52ºC in a water bath. Samples were measured every hour up to four hours followed by immediate separation on a size exclusion column. This older methodology failed to prove any significant difference between various batches of manufactured liposomal doxorubicin. Three problems accounted for the issues observed with this older technology. First, the release rate of doxorubicin was too high. Second, the minimal number of time points caused high variability in the release rate slope on the same lot of material tested. Third, the size exclusion separation of the liposomes from doxorubicin displayed too much variability. Other issues such as pipet volume, column uptake and column packing material variances caused separations to be inconsistent. These known issues within the current methodology, provided the impetus to Azaya to develop an in vitro release method that is superior. To put this new method to the test, a control drug product was measured against deliberate changes to the manufacturing process and formulation. These changes were chosen based upon scientific evidence posted in the Ortho Citizen’s Petition for DOXIL. The Citizen’s Petition displays pharmacokinetic data that the


following elements of the drug product must be controlled in order to develop a quality liposomal doxorubicin drug product.    

Sulfate Content Drug to Lipid Ratio Degree of Pegylation Stability of the liposome (i.e. minimal degradation of the lipids into lysophosphocholine)

The parameters listed above were evaluated to prove the reliability of the in vitro release method and those results are listed below. In Vitro Release Method Results Evaluation of sulfate content A batch of ATI-0918 was manufactured with 100mM ammonium sulfate hydration solution instead of the normal 250mM ammonium sulfate solution. The in vitro release was evaluated and compared to the control to ensure that the difference in sulfate can be detected by the method using statistical analysis equivalence tests. The results for release rate slope are presented in Table 1 below. Figure 1 displays one-way analysis of variance results for the comparison of the control against the test batch. The test batch is not statistically equivalent to the control.

Table 1. In Vitro Release Rate comparison of a 100mM sulfate content batch Control Lot 121001 Slope µg/hr 284 292 298 325 322 300 Average 304 RSD 5.4

100mM Ammonium sulfate hydration Lot ATN 34-42 Slope µg/hr 483 479 486 494 505 476 Average 487 %RSD 2.2


Figure 1. Oneway ANOVA of In Vitro Release Rate Slope by Lot for 100mM Ammonium sulfate hydration

Drug to Lipid Ratio Manufactured batches of ATI-0918 with a low drug to lipid ratio and a high drug to lipid ratio were made. These batches were evaluated for in vitro release rate and compared to the control using statistical analysis equivalence tests. The results for release rate slope are presented in Tables 2 and 3 below. Figures 2 and 3 display one-way analysis of variance results for the comparisons of the control against the test batches. The test batches are not statistically equivalent to the control.

Table 2. In Vitro Release Rate comparison for a Low Drug to Lipid Ratio (2:24) batch Control 2:24 Lot 121001 Slope µg/hr Slope µg/hr 284 347 292 351 298 347 325 353 322 364 300 344 Average 304 Average 351 RSD 5.4 %RSD 2.1


Figure 2. Oneway ANOVA of In Vitro Release Rate Slope by Lot for Low Drug to Lipid Ratio (2:24)

Table 3. In Vitro Release Rate comparison for a High Drug to Lipid Ratio (2:8) batch Control 2:8 Slope µg/hr Slope µg/hr 284 177 292 200 298 195 325 198 322 188 300 189 Average 304 Average 191 RSD 5.4 %RSD 4.4


Figure 3. Oneway ANOVA of In Vitro Release Rate Slope by Lot for High Drug to Lipid Ratio (2:8)

Liposome Stability During the course of studies lysophosphocholine has been observed increasing at each stability time point. Liposomes were manufactured with a mixture of lysophosphocholine (LPC) to simulate an aged product. The in vitro release rate was subsequently analyzed and compared to the control product using statistical analysis equivalence tests. The results for release rate slope are presented in Table 4 below. Figure 4 displays one-way analysis of variance results for the comparison of the control against the test batch. The test batch is not statistically equivalent to the control. Table 4. In Vitro Release Rate of the control compared to a LPC batch Control 2:8 Slope µg/hr Slope µg/hr 284 292 292 288 298 298 325 279 322 262 300 263 Average 304 Average 280 RSD 5.4 %RSD 5.5


Figure 4. Oneway ANOVA of In Vitro Release Rate Slope by Lot for Liposome Stability

PEG at the Liposome Surface Batches of ATI-0918 were manufactured with 1% PEG and 9% PEG and compared against the control (which contains 5% PEG). The in vitro release rate is expected to change primarily due to the liposome stability and permeability of doxorubicin through the liposome bilayer. The in vitro release rate was analyzed and compared to the control product using statistical analysis equivalence tests. The results for release rate slope are presented in Tables 5 and 6 below. Figures 5 and 6 display one-way analysis of variance results for the comparisons of the control against the test batches. The test batches are not statistically equivalent to the control. Table 5. In Vitro Release Rate comparison for a 1% PEG batch Control 1% PEG Lot 121001 Lot ATN 34-46 Slope µg/hr Slope µg/hr 284 441 292 443 298 431 325 434 322 448 300 441 Average 304 Average 440 RSD 5.4 %RSD 1.5


Figure 5. Oneway ANOVA of In Vitro Release Rate Slope by Lot for 1% PEG batch

Table 6. In Vitro Release Rate comparison for a 9% PEG batch Control 9% PEG Lot 121001 Lot ATN 34-48 Slope µg/hr Slope µg/hr 284 439 292 437 298 450 325 440 322 458 300 438 Average 304 Average 444 RSD 5.4 %RSD 1.9 Figure 6. Oneway ANOVA of In Vitro Release Rate Slope by Lot for 9% PEG batch


Comparison of ATI-0918 with the Reference Drug The clinical material batch of ATI-0918 (Lot 0918-121001) was compared against CAELYX (lot TAE124). The in vitro release rate was analyzed and compared using statistical analysis equivalence tests. Results are presented in Figure 7 below. The In Vitro release rates are equivalent for both test samples. In fact there is no statistically significant difference observed between the two samples.

Figure 7. Comparison of the In Vitro Release Rate of ATI0918 (Lot 0918-121001) with CAELYX (Lot TAE124) In Vitro Release Comparison of DOXIL/CAELYX to ATI-0918 1.0hr to 8.0hr / average n=6 3000 y = 297.06x + 718.92 2500

µg released

y = 303.54x + 666.69 2000

1500

1000

500 0.50

1.50

0918-121001

2.50

3.50 hours

CAELYX TAE124

4.50

5.50

Linear (0918-121001)

6.50

7.50

8.50

Linear (CAELYX TAE124)


Figure 8. Comparison of the In Vitro Release Rate of ATI0918 (Lot 0918-121001) Against Manufactured Batches with Variances designed by the Formulation Process In-Vitro Release Comparison of All batches 6500 34-42 (100mM) 6000

34-48 (9%PEG) 34-46 (1%PEG)

5500

y = 487.12x + 1,542.54 R² = 0.92

29-47 (24:2, Lipid:Drug) 5000

y = 443.68x + 1,113.85 R² = 0.93

121001_occasion 1 34-60 (LPC)

4500

y = 439.69x + 919.48 R² = 0.93

34-38 (8:2, Lipid:Drug)

y = 350.94x + 817.33 R² = 0.93

3500

y = 303.54x + 666.69 R² = 0.94

3000

y = 280.40x + 734.44 R² = 0.94

µg released

4000

2500 2000 y = 191.10x + 641.72 R² = 0.90

1500 1000 500 0.50

1.50

2.50

3.50

4.50

5.50

6.50

7.50

8.50

hours

Figure 8 above displays an overlay of the in vitro release rate of each batch compared to the clinical trial lot. It is apparent that the release rate of each batch is significantly different. The closet match to the control batch is the batch that was manufactured with lysophosphocholine to simulate aged product. Even though that particular batch has a similar release profile, Table 4 and Figure 4 show that these batches are not statistically equivalent. This is very valuable information for the quality of the final drug product.


Conclusion The results presented indicate that the in vitro release method can discern batch to batch differences in pegylated liposomal doxorubicin (PLD) drug product. Utilizing this information from the in vitro release method places a high degree of confidence in the comparability of a developed drug, ATI-0918 and the innovator product, DOXIL/CAELYX. It is important to note that the Citizen’s Petition points out that chemical characterization is not sufficient to display similarities between drug products. The Citizen’s petition also points out that the degree of pegylation could not be discerned with analytical technology. This in vitro method has the capability to overcome that challenge. The current FDA regulatory guidance requires that any technical transfer of the manufacturing process of the PLD drug product will have to prove bioequivalence in a human clinical trial. This poses a problem for companies developing a generic drug product. Yet, by utilizing the in vitro method and comparing the release rates against a control, equivalence can be determined with a high degree of confidence obviating the need of an additional clinical trial. This in vitro method is highly valuable to the liposomal drug development world and regulatory bodies alike. The information learned here can help develop a more robust and reproducible technology in a less expensive manner.

In the case of Azaya Therapeutics drug development program; ATI-0918 has been developed to be equivalent to DOXIL/CAELYX. The in vitro release rates of the clinical trial material of ATI0918 are statistically equivalent to CAELYX (lot TAE124). This is especially important for Azaya Therapeutics drug product development and marks a significant milestone in the drug development program. ATI-0918, Lot 0918-121001, is being used in a bioequivalence human clinical trial in a crossover study against DOXIL/CAELYX. It is expected that the PK data will display bioequivalence since the in vitro method provides information that the liposome release rates are equivalent. Some information such as the analytical method procedure was not disclosed for protection of intellectual property. If you would like to request additional information please contact us. Attn: Richard Underbrink Phone: 210-341-6600 ext. 219 Email: [email protected]