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Degradation of Antiflammin 2 in Aqueous Solution
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Degradation of Antiflammin 2 in Aqueous Solution To the Editor: Antiflammins are synthetic peptides derived from the region of highest amino acid sequence similarity between uteroglobin and lipocortin I. Antiflammin 2 (His-Asp-MetAsn-Lys-Val-Leu-Asp-Leu; AF2) has the same sequence as residues 247-255 of lipocortin I and has shown potent antiphospholipase A2 (PLA)2 activity in vitro against porcine pancreatic PLAz and is a potent anti-inflammatory agent without any of the known side effects of corticosteroids a n d or nonsteroidal anti-inflammatory agents (Miele et al., 1988; Facchiano et al., 1991). It has been suggested that PLA2 activation may initiate induction of endotoxin-induced uveitis (Rosenbaum et al., 1983). It has been further demonstrated that topical administration of AF2 can inhibit endotoxininduced uveitis in rats, which is a model for anterior uveitis in humans (Chan et al., 1991). Therefore, a clinical trial has been initiated exploring the topical administration of antiflammin 2 (AF2) t o patients who experience acute anterior uveitis. The drug is provided as 3 mg of AF2 in 1 mL of Dacriose ophthalmic irrigating solution. To develop a suitable dosage form for a pharmaceutical agent, it is necessary to determine chemical reactivity under stress conditions. As more peptides and proteins become therapeutic agents, the literature describing drug stability testing grows (Brange et al., 1992a,b). Reviews of degradation pathways commonly encountered with peptides and proteins are also available (Manning et al., 1989; Wang and Pearlman, 1993). However, there have been no investigations on the chemical stability of AF2. This note describes the kinetics of degradation of AF2 as a function of pH and temperature by reversed-phase high-performance liquid chromatography. Although the focus is on the determination of reaction rates, degradation product identification also provides mechanistic information about AF2 chemistry in aqueous solutions over a wide pH range. AF2, Asp-4-AF2, isoAsp-4-AF2, and Met-(0)-3-AF2 were obtained from BACHEM Inc. (Torrance, CA). HPLC-grade
trifluoroacetic acid (TFA)and acetonitrile were obtained from Sigma and Mallinkrodt, respectively. All solutions and buffers were prepared with distilled deionized water that was filtered with a 0.22 pm membrane (Aquatron system, Bellco) prior t o use. All other materials were of reagent grade. The HPLC system consisted of a pump (LC-lOAD, Shimadzu), a controller (SCL-lOA, Shimadzu), an autosampler (SIL-lOA, Shimadzu), a detector (SPD-lOA, Shimadzu), an integrator (CR-501, Shimadzu), and an ODS column (5 pm, 4.6 x 250 mm, Vydac). AF2 and its degradation products were eluted from the column and detected at 220 nm. The mobile phase was 0.1% TFA in water-acetonitrile (80:20). The flow rate was 1.0 mL! min and the injection volume was 20 pL . A standard curve was constructed for each series of determinations over a range of 5-55 pg/mL AF2. The initial concentration of each AF2 solution was designated 100%; all subsequent concentrations were expressed as a percentage of the initial concentration. The AF2 solutions were approximately 50 pg/mL. They were prepared by adding the appropriate buffer to the bulk drug in a silanized volumetric flask. Aliquots were then placed in silanized autosampler vials (Alltech), capped, and placed in storage a t the appropriate temperature. At periodic intervals, vials were placed in the HPLC autosampler and analyzed for AF2 content. The buffers used for each of the pHs studied were citrate (pH 3.00,4.10),acetate (pH 5.251, phosphate (pH 6.16, 7.11, and 8.00),borate (pH 8.80), and bicarbonate (pH 10.00). The total ionic strength of each was adjusted to 0.6 M with NaC1. The buffers were all filtered through a 0.22 pm filter prior to addition to the volumetric flask. The HPLC chromatograms of AF'2 kept in solutions of differing pH at 40 "C for a specific time are shown in Figure 1. Three main degradation products were detected between pH 5 and 10, as shown in Figure 1. Two of the main degradation products were identified in this study by comparison to standards. The deamidated asparagine products
I
1
3
1
3
dh I
?
---6 8
10 15
20 25 0
A
5
10 15
B
20 25 0
5
10
15
L
20 25 0
C
5
10
15
20 25
D
Figure 1-RP-HPLC chromatograms of AF2 solution prepared in 0.005 M citrate (pH 3.00),0.005 M acetate (pH 5.25), 0.005 M phosphate (pH 8.00),and 0.005 M bicarbonate (pH 10.00) buffers. The total ionic strength was maintained at 0.6 M with sodium chloride. (A) pH 3.00, 29 days; (B) pH 5.25, 40 days; (C) pH 8.00,36 days; (D) pH 10.00,22 days. Peak 1, AF2; peak 2, Met-(0)-3-AF2; peak 3, isoAsp-4-AF2; peak 4, unknown; peak 5, Asp-4-AF2; peak 6, unknown; peak 7, asparlylsuccinimide-4-AF2 (tentative assignment); peak 8, unknown.
1762 / Journal of Pharmaceutical Sciences Vol. 83, No. 12, December 1994
0022-3549/94/3183-1762$04.50/0
0 1994, American Chemical Society and American Pharmaceutical Association
100
-1
-
-2
-
-3
-
pH3.00
pH4.10 pH5.25
0 Q
h
.
0 pH6.16
A A
pH7.11 pH8.00 X pH8.80 0 oH10.00
k
\
4-
. .
10
l
I
4
2
0
-
-
1
.
8
6
. i
10
'1
0
0
h r (
i
0
x
cd
9 0
2
-
-6
-
-71 0.0028
-
I
0.0029
=
I
-
0.0030
I
-
0.0031
I
0.0032
-
1
0.0033
l/r (OK)
time (days) Figure 2-First-order plot for the degradation of AF2 in 0.005 M citrate (pH 3.00 and 4.10), 0. acetate (pH 5.25), 0.005 M phosphate (pH 6.16,7.11, 8.00),0.005 M borate (pH 8.8),and 0.005 M bicarbonate (pH 10.00) buffers at 70 "C.
0-
-5
I
1
I
I
4
6
8
10
PH Figure 3-pH-rate profile for the degradation of AF2 in 0.005 M citrate (pH 3.00 and 4.10), 0.005 M acetate (pH 5.25), 0.005M phosphate (pH 6.16,7.1 1, 8.00), 0.005 M borate (pH 8.8),and 0.005 M bicarbonate (pH 10.00)buffers at 70" C.
Asp-4-AF2 and isohp-4-AF2 were identified as peaks 5 and 3, respectively. The methionine sulfoxide (Met-(0)-3-AF2)was identified as peak 2, a minor degradation product. Because the deamidation reaction is one of the most common chemical pathways of peptide degradation in the neutral to alkaline pH range (Geiger and Clarke, 1987; Patel and Borchardt, 1990a; and Patel and Borchardt, 1990b) and because this reaction has been extensively studied, peak 7 has tentatively been assigned as that corresponding to the cyclic imide. Degradation of AF'2 showed a marked dependence on pH and
Figure 4-Arrhenius plot of In (rate constant) vs 1/T (K)for the degradation of AF2 in pH 6.16phosphate buffer (0.005 M,p = 0.6 M).
temperature as is observed with general deamidation reactions (Patel and Borchardt, 1990a). Figure 1 also suggests that AF2 may degrade by several pathways. Degradation products 4, 6 , and 8 appeared at acidic pH, degradation product 7 was formed predominantly at pH 4-8, and degradation products 3 and 5 (the Asn degradation products) were the major products at pH 7-10, while degradation product 2 (the methionine sulfoxide product) was a minor product at pH 6-8. The kinetics of degradation for AF2 was also studied. Figure 2 shows a semilogarithmic plot of the residual percentage concentrations of AF2 versus time at several pH values a t 70 "C. It was found that the AF2 degradation rates were dependent on pH and that the observed degradation reaction rates approximately followed pseudo-first-order kinetics. The observed rate constants ( k o b s )were obtained from the slopes of the semilog plots of concentration versus time by statistical regression analysis. In all cases, the correlation coefficients were 0.99 or better (data not shown). The pH-rate profile for the degradation of AF2 was obtained by plotting the value of log Kobs versus pH (Figure 3). From Figure 3, it appears that AF'2 was most stable between pH 5.25 and 8.00. Figure 4 shows the Arrhenius plot for the degradation studies a t different temperatures ranging from 40 to 70 "C and a t a constant pH of 6.16. A linear relationship was observed with a correlation coefficient greater than 0.99. The slope gives an activation energy for the overall reaction of 21.58 kcaYmol at pH 6.16. In conclusion, we determined the degradation kinetics of AF'2 as a function of pH and temperature by reversed-phase high-performance liquid chromatography and identified two of the main degradation products of the peptide. The degradation followed pseudo-first-order kinetics and maximum stability was achieved by adjusting the pH of the solution to 5.25-8.00.
References and Notes 1. Brange, J., Havelund, S., and Hou aard, P. (1992a) Chemical stability of insulin. 2. Formation o f Higher Molecular Weight
Journal of Pharmaceutical Sciences / 1763 Vol. 83, No. 12, December 1994
2.
3.
4.
5. 6. 7.
8.
9.
10.
Transformation Products during Storage of Pharmaceutical Preparations. Pharm. Res. 9, 727-734. Brange, J., Langkjaer, L., Havelund, S., and Volund, A. (1992b) Chemical Stability of Insulin. 1.Hydrolytic Degradation During Storage of Pharmaceutical Preparations. Pharm. Res. 9, 715726. Chan, C. C . , Ni, M., Miele, L., Cordella-Miele, E., Ferrick, M., Mukherjee, A. B., and Nussenblatt, R. B. (1991) Effect of Antiflammins on Endotoxin-Induced Uveitis in Rats. Archives of Ophthalmology 109, 278-281. Facchiano, A., Cordella-Miele, E., Miele, L., and Mukherjee, A. B. (1991) Inhibition of Pancreatic Phospholipase A2 Activity by Uteroglobin and Antiflammin Peptides: Possible Mechanism of Action. Life Sci. 48,453-464. Geiger, T. and Clarke, S. Deamidation, Isomerization, and Racemization at Asparaginyl and Aspartyl Residues in Peptides. (1987) J . Biol. Chem. 262, 785-794 . Manning, M. C., Patel, K. and Borchardt, R. T. (1989) Stability of Protein Pharmaceuticals. Pharm. Res. 6, 903-917. Miele, L., Cordella-Miele, E., Facchiano, A. and Mukherjee, A. B. (1988) Novel Antiinflammatory Peptides from the Region of Highest Similarity between Uteroglobin and Lipocortin I. Nature 335, 726-730. Patel, K. and Borchardt, R. T. (1990a) Chemical Pathways of Peptide Degradation. 11. Kinetics of Deamidation of an Asparaginyl Residue in a Model Hexapeptide. Pharm. Res. 7, 703711. Patel, K. and Borchardt, R. T. (1990b) Chemical Pathways of Peptide Degradation. 111. Effect of Primary Sequence on the Pathways of Deamidation of Asparaginyl Residues in Hexapeptides. Pharm. Res. 7, 787-793. Rosenbaum, J. T., Hendricks, P. A., Shiverly, J. E., and McDougall, I. R. (1983) Distribution of Radiolabelled Endotoxin with Particular Reference to the Eye. J . Nucl. Med. 24, 29-33.
1764 / Joumal of Pharmaceutical Sciences Vol. 83, No. 12, December 1994
11. Wang, Y. J. and Pearlman, R., Eds. (1993) Stability and Characterization of Protein and Peptide Drugs, Plenum Press, New York.
Acknowledgments The authors would like to acknowledge Allan Bokser (Genta) for preliminary analytical method development.
JANETL. WOLFE'~~, GRACEE. LEES, GOPALK. PorriS, AND JOSEPHF. GALLELLIS *Department of Pharmaceutical Sciences University of Tennessee, Memphis Memphis, TN 38163 *Warren G. Magnuson Clinical Center Pharmacy Pharmaceutical Development Section National Institutes of Health Bethesda. MD 20892 Received July 26, 1994. Accepted for publication September 22, 1994.
Degradation of Antiflammin 2 in Aqueous Solution To the Editor: Antiflammins are synthetic peptides derived from the region of highest amino acid sequence similarity between uteroglobin and lipocortin I. Antiflammin 2 (His-Asp-MetAsn-Lys-Val-Leu-Asp-Leu; AF2) has the same sequence as residues 247-255 of lipocortin I and has shown potent antiphospholipase A2 (PLA)2 activity in vitro against porcine pancreatic PLAz and is a potent anti-inflammatory agent without any of the known side effects of corticosteroids a n d or nonsteroidal anti-inflammatory agents (Miele et al., 1988; Facchiano et al., 1991). It has been suggested that PLA2 activation may initiate induction of endotoxin-induced uveitis (Rosenbaum et al., 1983). It has been further demonstrated that topical administration of AF2 can inhibit endotoxininduced uveitis in rats, which is a model for anterior uveitis in humans (Chan et al., 1991). Therefore, a clinical trial has been initiated exploring the topical administration of antiflammin 2 (AF2) t o patients who experience acute anterior uveitis. The drug is provided as 3 mg of AF2 in 1 mL of Dacriose ophthalmic irrigating solution. To develop a suitable dosage form for a pharmaceutical agent, it is necessary to determine chemical reactivity under stress conditions. As more peptides and proteins become therapeutic agents, the literature describing drug stability testing grows (Brange et al., 1992a,b). Reviews of degradation pathways commonly encountered with peptides and proteins are also available (Manning et al., 1989; Wang and Pearlman, 1993). However, there have been no investigations on the chemical stability of AF2. This note describes the kinetics of degradation of AF2 as a function of pH and temperature by reversed-phase high-performance liquid chromatography. Although the focus is on the determination of reaction rates, degradation product identification also provides mechanistic information about AF2 chemistry in aqueous solutions over a wide pH range. AF2, Asp-4-AF2, isoAsp-4-AF2, and Met-(0)-3-AF2 were obtained from BACHEM Inc. (Torrance, CA). HPLC-grade
trifluoroacetic acid (TFA)and acetonitrile were obtained from Sigma and Mallinkrodt, respectively. All solutions and buffers were prepared with distilled deionized water that was filtered with a 0.22 pm membrane (Aquatron system, Bellco) prior t o use. All other materials were of reagent grade. The HPLC system consisted of a pump (LC-lOAD, Shimadzu), a controller (SCL-lOA, Shimadzu), an autosampler (SIL-lOA, Shimadzu), a detector (SPD-lOA, Shimadzu), an integrator (CR-501, Shimadzu), and an ODS column (5 pm, 4.6 x 250 mm, Vydac). AF2 and its degradation products were eluted from the column and detected at 220 nm. The mobile phase was 0.1% TFA in water-acetonitrile (80:20). The flow rate was 1.0 mL! min and the injection volume was 20 pL . A standard curve was constructed for each series of determinations over a range of 5-55 pg/mL AF2. The initial concentration of each AF2 solution was designated 100%; all subsequent concentrations were expressed as a percentage of the initial concentration. The AF2 solutions were approximately 50 pg/mL. They were prepared by adding the appropriate buffer to the bulk drug in a silanized volumetric flask. Aliquots were then placed in silanized autosampler vials (Alltech), capped, and placed in storage a t the appropriate temperature. At periodic intervals, vials were placed in the HPLC autosampler and analyzed for AF2 content. The buffers used for each of the pHs studied were citrate (pH 3.00,4.10),acetate (pH 5.251, phosphate (pH 6.16, 7.11, and 8.00),borate (pH 8.80), and bicarbonate (pH 10.00). The total ionic strength of each was adjusted to 0.6 M with NaC1. The buffers were all filtered through a 0.22 pm filter prior to addition to the volumetric flask. The HPLC chromatograms of AF'2 kept in solutions of differing pH at 40 "C for a specific time are shown in Figure 1. Three main degradation products were detected between pH 5 and 10, as shown in Figure 1. Two of the main degradation products were identified in this study by comparison to standards. The deamidated asparagine products
I
1
3
1
3
dh I
?
---6 8
10 15
20 25 0
A
5
10 15
B
20 25 0
5
10
15
L
20 25 0
C
5
10
15
20 25
D
Figure 1-RP-HPLC chromatograms of AF2 solution prepared in 0.005 M citrate (pH 3.00),0.005 M acetate (pH 5.25), 0.005 M phosphate (pH 8.00),and 0.005 M bicarbonate (pH 10.00) buffers. The total ionic strength was maintained at 0.6 M with sodium chloride. (A) pH 3.00, 29 days; (B) pH 5.25, 40 days; (C) pH 8.00,36 days; (D) pH 10.00,22 days. Peak 1, AF2; peak 2, Met-(0)-3-AF2; peak 3, isoAsp-4-AF2; peak 4, unknown; peak 5, Asp-4-AF2; peak 6, unknown; peak 7, asparlylsuccinimide-4-AF2 (tentative assignment); peak 8, unknown.
1762 / Journal of Pharmaceutical Sciences Vol. 83, No. 12, December 1994
0022-3549/94/3183-1762$04.50/0
0 1994, American Chemical Society and American Pharmaceutical Association
100
-1
-
-2
-
-3
-
pH3.00
pH4.10 pH5.25
0 Q
h
.
0 pH6.16
A A
pH7.11 pH8.00 X pH8.80 0 oH10.00
k
\
4-
. .
10
l
I
4
2
0
-
-
1
.
8
6
. i
10
'1
0
0
h r (
i
0
x
cd
9 0
2
-
-6
-
-71 0.0028
-
I
0.0029
=
I
-
0.0030
I
-
0.0031
I
0.0032
-
1
0.0033
l/r (OK)
time (days) Figure 2-First-order plot for the degradation of AF2 in 0.005 M citrate (pH 3.00 and 4.10), 0. acetate (pH 5.25), 0.005 M phosphate (pH 6.16,7.11, 8.00),0.005 M borate (pH 8.8),and 0.005 M bicarbonate (pH 10.00) buffers at 70 "C.
0-
-5
I
1
I
I
4
6
8
10
PH Figure 3-pH-rate profile for the degradation of AF2 in 0.005 M citrate (pH 3.00 and 4.10), 0.005 M acetate (pH 5.25), 0.005M phosphate (pH 6.16,7.1 1, 8.00), 0.005 M borate (pH 8.8),and 0.005 M bicarbonate (pH 10.00)buffers at 70" C.
Asp-4-AF2 and isohp-4-AF2 were identified as peaks 5 and 3, respectively. The methionine sulfoxide (Met-(0)-3-AF2)was identified as peak 2, a minor degradation product. Because the deamidation reaction is one of the most common chemical pathways of peptide degradation in the neutral to alkaline pH range (Geiger and Clarke, 1987; Patel and Borchardt, 1990a; and Patel and Borchardt, 1990b) and because this reaction has been extensively studied, peak 7 has tentatively been assigned as that corresponding to the cyclic imide. Degradation of AF'2 showed a marked dependence on pH and
Figure 4-Arrhenius plot of In (rate constant) vs 1/T (K)for the degradation of AF2 in pH 6.16phosphate buffer (0.005 M,p = 0.6 M).
temperature as is observed with general deamidation reactions (Patel and Borchardt, 1990a). Figure 1 also suggests that AF2 may degrade by several pathways. Degradation products 4, 6 , and 8 appeared at acidic pH, degradation product 7 was formed predominantly at pH 4-8, and degradation products 3 and 5 (the Asn degradation products) were the major products at pH 7-10, while degradation product 2 (the methionine sulfoxide product) was a minor product at pH 6-8. The kinetics of degradation for AF2 was also studied. Figure 2 shows a semilogarithmic plot of the residual percentage concentrations of AF2 versus time at several pH values a t 70 "C. It was found that the AF2 degradation rates were dependent on pH and that the observed degradation reaction rates approximately followed pseudo-first-order kinetics. The observed rate constants ( k o b s )were obtained from the slopes of the semilog plots of concentration versus time by statistical regression analysis. In all cases, the correlation coefficients were 0.99 or better (data not shown). The pH-rate profile for the degradation of AF2 was obtained by plotting the value of log Kobs versus pH (Figure 3). From Figure 3, it appears that AF'2 was most stable between pH 5.25 and 8.00. Figure 4 shows the Arrhenius plot for the degradation studies a t different temperatures ranging from 40 to 70 "C and a t a constant pH of 6.16. A linear relationship was observed with a correlation coefficient greater than 0.99. The slope gives an activation energy for the overall reaction of 21.58 kcaYmol at pH 6.16. In conclusion, we determined the degradation kinetics of AF'2 as a function of pH and temperature by reversed-phase high-performance liquid chromatography and identified two of the main degradation products of the peptide. The degradation followed pseudo-first-order kinetics and maximum stability was achieved by adjusting the pH of the solution to 5.25-8.00.
References and Notes 1. Brange, J., Havelund, S., and Hou aard, P. (1992a) Chemical stability of insulin. 2. Formation o f Higher Molecular Weight
Journal of Pharmaceutical Sciences / 1763 Vol. 83, No. 12, December 1994
2.
3.
4.
5. 6. 7.
8.
9.
10.
Transformation Products during Storage of Pharmaceutical Preparations. Pharm. Res. 9, 727-734. Brange, J., Langkjaer, L., Havelund, S., and Volund, A. (1992b) Chemical Stability of Insulin. 1.Hydrolytic Degradation During Storage of Pharmaceutical Preparations. Pharm. Res. 9, 715726. Chan, C. C . , Ni, M., Miele, L., Cordella-Miele, E., Ferrick, M., Mukherjee, A. B., and Nussenblatt, R. B. (1991) Effect of Antiflammins on Endotoxin-Induced Uveitis in Rats. Archives of Ophthalmology 109, 278-281. Facchiano, A., Cordella-Miele, E., Miele, L., and Mukherjee, A. B. (1991) Inhibition of Pancreatic Phospholipase A2 Activity by Uteroglobin and Antiflammin Peptides: Possible Mechanism of Action. Life Sci. 48,453-464. Geiger, T. and Clarke, S. Deamidation, Isomerization, and Racemization at Asparaginyl and Aspartyl Residues in Peptides. (1987) J . Biol. Chem. 262, 785-794 . Manning, M. C., Patel, K. and Borchardt, R. T. (1989) Stability of Protein Pharmaceuticals. Pharm. Res. 6, 903-917. Miele, L., Cordella-Miele, E., Facchiano, A. and Mukherjee, A. B. (1988) Novel Antiinflammatory Peptides from the Region of Highest Similarity between Uteroglobin and Lipocortin I. Nature 335, 726-730. Patel, K. and Borchardt, R. T. (1990a) Chemical Pathways of Peptide Degradation. 11. Kinetics of Deamidation of an Asparaginyl Residue in a Model Hexapeptide. Pharm. Res. 7, 703711. Patel, K. and Borchardt, R. T. (1990b) Chemical Pathways of Peptide Degradation. 111. Effect of Primary Sequence on the Pathways of Deamidation of Asparaginyl Residues in Hexapeptides. Pharm. Res. 7, 787-793. Rosenbaum, J. T., Hendricks, P. A., Shiverly, J. E., and McDougall, I. R. (1983) Distribution of Radiolabelled Endotoxin with Particular Reference to the Eye. J . Nucl. Med. 24, 29-33.
1764 / Joumal of Pharmaceutical Sciences Vol. 83, No. 12, December 1994
11. Wang, Y. J. and Pearlman, R., Eds. (1993) Stability and Characterization of Protein and Peptide Drugs, Plenum Press, New York.
Acknowledgments The authors would like to acknowledge Allan Bokser (Genta) for preliminary analytical method development.
JANETL. WOLFE'~~, GRACEE. LEES, GOPALK. PorriS, AND JOSEPHF. GALLELLIS *Department of Pharmaceutical Sciences University of Tennessee, Memphis Memphis, TN 38163 *Warren G. Magnuson Clinical Center Pharmacy Pharmaceutical Development Section National Institutes of Health Bethesda. MD 20892 Received July 26, 1994. Accepted for publication September 22, 1994.