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Article history: Spantide II is an 11 amino acid peptide that has been shown to be a potential anti-
Received 4 February 2005 inflammatory agent. The stability and degradation profiles of Spantide II in aqueous solu-
Received in revised form 12 tions were evaluated with the long-term objective of developing topical formulations of this
September 2005 compound for various skin disorders. The stability profile of Spantide II at various tem-
Accepted 14 September 2005 perature and pH conditions was monitored by high performance liquid chromatography
Available online 2 November 2005 (HPLC) and the resulting degradation products were identified by liquid chromatography-
mass spectroscopy (LC-MS). Forced degradation of Spantide II was performed at extreme
Keywords: acidic (pH <2.0) and alkaline (pH >10.0) conditions and by addition of hydrogen peroxide
Spantide II (oxidizing agent). The degradation pattern of Spantide II followed pseudo first-order kinet-
Stability ics. The shelf life (T90% ) of Spantide II in aqueous ethanol (50%) was determined to be 230
Topical days at 25 ◦ C. Spantide II was susceptible to degradation at pH <2 and pH >5 and showed
Transdermal maximum stability at pH 3–5. The stability under various pH conditions indicates that
Peptide Spantide II was most stable at pH 3.0 with a half-life of 95 days at 60 ◦ C. Spantide II degrada-
tion was attributed to hydrolysis of peptide bonds [Pro2 -(pyridyl)Ala3 , (nicotinoyl)Lys1 -Pro2 ,
Pro4 -PheCl2 5 , Trp7 -Phe8 , Phe8 -Trp9 , Nle11 -NH2 ), racemization of the peptide fragments that
resulted from hydrolysis, cleavage and formation of (nicotinoyl)Lys1 -Pro2 diketopiperazine.
In the presence of an oxidizing agent, Pro2,4 residues degraded by ring opening to form
glutamyl-semialdehyde and by bond cleavage at Pro4 to form 2-pyrrolidone, while Phe5,8
degraded to form 2-hydroxyphenylalanine. Spantide II was found to be stable in aqueous
medium with T90% of 230 days. The major degradation pathways of Spantide II were identi-
fied as hydrolysis, racemization, cleavage and formation of diketopiperazine.
© 2005 Elsevier B.V. All rights reserved.
∗
Corresponding author. Tel.: +1 850 561 2790; fax: +1 850 599 3347.
E-mail address: mandip.sachdeva@famu.edu (M. Singh).
0928-0987/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2005.09.005
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 7 ( 2 0 0 6 ) 158–166 159
we demonstrated the anti-inflammatory effect of Spantide II grade) were purchased from Fisher Scientific (Atlanta, GA,
in an allergic contact dermatitis mouse model (Jaiani et al., USA). All other chemicals were standard reagent grade.
2002; Babu et al., 2004).
Due to the increased interest in proteins and peptides 2.2. High-performance liquid chromatography (HPLC)
as pharmaceutical therapies, it is necessary to have a broad assay
understanding of the chemical and physical stability of these
agents in order to design suitable formulations. Generally, the A HPLC system (Waters Corporation) along with a Vydac
amino acid residues on a peptide sequence are sensitive to var- reverse phase C18 (300 Å pore size silica) analytical column
ious degradation pathways such as hydrolysis, deamidation (5 m, 4.6 mm × 250 mm) were used for the analysis of Span-
and metal catalyzed oxidation (Manning et al., 1989; Reubsaet tide II. The HPLC system consisted of an autosampler (model
et al., 1998). In a previous study, we reported the preformu- 717 plus), two pumps (model 515), and an UV detector (model
lation stability of Spantide II as a function of pH, tempera- 996 PDA), all interfaced with EmpowerTM software. The mobile
ture, salt concentration and various dermatological vehicles phases used were 0.1% TFA in water (solvent A) and 0.1%
(Kikwai et al., 2004). However, the mechanism of degradation TFA in acetonitrile (solvent B). The mobile phase was filtered,
of Spantide II and the resulting degradation products under degassed by sonication prior to use and analysis was run at
various accelerated conditions have not been fully character- a gradient of 68%:32% (solvent A:B, respectively), which was
ized. The characterization of peptide degradation is of impor- reversed to 32%:68% (solvent A:B, respectively) over 30 min,
tance, since an unstable peptide product may worsen product with a flow rate of 1 ml/min. Spantide II content in the sam-
purity, potency and appearance. ples was determined using a PDA-UV detector set at 230 nm.
The purpose of this study was to further characterize the All analyses were performed at room temperature, and the
stability and degradation mechanisms of Spantide II in aque- retention time of Spantide II was 20.8 min.
ous solutions. The HPLC assay method used in the present
study was stability indicating, with a clear distinction between 2.3. Liquid chromatography-mass spectrometry
active peptide and the degradation products and further anal- (LC-MS)
ysis was done by LC-MS to characterize the degradation prod-
ucts. The stability of Spantide II as a function of pH and tem- The LC system consisted of Beckman Gold 125S Solvent Mod-
perature as well as the degradation profile of Spantide II under ule equipped with a Beckman Gold 166 UV detector. The
various forced degradation conditions were investigated. mobile phase and the gradient system used were identical
to that described in the above section of HPLC assay, but
with a flow rate of 0.2 ml/min. A Vydac C18 reverse phase col-
2. Material and methods umn (300 Å pore size silica, 5 m particles, 1.0 mm × 250 mm)
attached to a 2 mm guard cartridge of same column chemistry
2.1. Materials was used for the analysis of Spantide II. The LC system was
operated by the Beckman Gold V712 software.
Spantide II was custom synthesized by Bio Peptide Co. LLC (San Mass spectra were acquired using an AccuTOFTM Time-
Diego, CA) and used without further purification. Potassium of-Flight Mass Spectrometer (Model JMS-T100LC, JEOL Inc.,
chloride, mono and dibasic potassium phosphate, hydrochlo- Peabody, MA, USA). The instrument was interfaced with a pos-
ric acid, potassium biphthalate, sodium hydroxide, hydrogen itive ion mode electro spray inlet probe used to ionize the
peroxide, trifluoroacetic acid (TFA) and boric acid were pro- molecules. The spectra were obtained in the low-resolution
cured from Sigma Chemical Co. (St. Louis, MO, USA). Ethanol mode with a voltage of 10 kV. Mass spectra were collected in
USP (200 proof) was obtained from Florida Distillers Co. (Lake the positive ion mode, scanning from 200–2000 mass units
Alfred, FL, USA). Water, acetonitrile and methanol (HPLC every 1 s.
160 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 7 ( 2 0 0 6 ) 158–166
Table 1 – Degradation of Spantide II at 60 ◦ C in aqueous ethanol solution: m/z ratios of the degradation products
Cleavage site Fragment A Fragment B Fragment A M + H+ Fragment B M + H+
theoretical mass theoretical mass (m/z): (peak) (m/z): (peak)
Table 2 – Degradation products of Spantide II in acidic medium at 60 ◦ C, showing theoretical masses, peak occurrence,
and m/z ratios of major hydrolytic products
Cleavage site Fragment A Fragment B Fragment A M + H+ Fragment B M + H+
theoretical mass theoretical mass (m/z): (peak) (m/z): (peak)
The degradation products that occurred at the HPLC peak acids of Spantide II are not required for binding of Spantide
P3 were relatively polar in nature compared to Spantide II II to neurokinin-1 receptors while the terminal amino acids
as they eluted prior to the parent peak. MS spectral anal- are required for binding and receptor recognition. Therefore,
ysis of peak P3 identified products that resulted from the hydrolytic cleavage of the terminal amino acid residues can
hydrolytic cleavage of Spantide II at (nicotinoyl) Lys1 -Pro2 , lead to the deactivation of Spantide II antagonist activity and
Pro2 -(pyridyl)Ala3 and Phe8 -Trp9 peptide bonds. Hydrolysis thus decreasing its anti-inflammatory activity (Ljungqvist et
at (nicotinoyl)Lys1 -Pro2 produced a fragment-A with an m/z al., 1989).
ratio of 251.1 (m/z = +17), and fragment-B with an m/z ratio In alkaline medium, the degradation products are both
of 1436.5 (m/z = +1) after addition of hydrogen. Fragment-A polar and non-polar relative to Spantide II, because the
racemized and was detected in P5 and P6 in the HPLC chro- peaks elute before and after the parent peak. Table 3 shows
matogram. The m/z ratio corresponding to fragment-B was major hydrolytic degradation products of Spantide II in alka-
not observed in the MS spectrum therefore, it might have line media. Analysis of the degradation products in alka-
undergone further degradation. Further hydrolytic degrada- line medium revealed m/z ratios that were similar to those
tion products of fragment-A were not observed in the MS that occurred in acidic medium. The degradation products
spectrum. Hydrolytic cleavage at Pro2 -(pyridyl)Ala3 produced racemized and occurred at different retention times on the
two degradation fragments. The fragment-A with m/z ratios of chromatograms, however, m/z ratios (m/z = +17) were iden-
349.2 (m/z = +17) occurred in the MS spectrum correspond- tical as determined by MS. There is a correlation between
ing to HPLC peak P3 , while the fragment-B with m/z ratio hydrolytic degradation in acidic and alkaline medium as
of 1339.4, racemized and was identified in the MS spectrum observed by degradation products resulting from cleavage at
corresponding to HPLC peaks P5 and P6 . Hydrolytic cleavage Pro2 -(pyridyl)Ala3 , Trp7 -Phe8 , and Nle11 -NH2 were observed in
at Phe8 -Trp9 produced two degradation products, fragment- both MS spectra of acidic and alkaline conditions. It should
A with an m/z ratio of 1258.3 (m/z = +17) and fragment-B, be noted that from Table 3, three of the degradants, of
with an m/z ratio of 430.1, which occurred in the HPLC peak Pro2 -(pyridyl)Ala3 , and Pro4 -Phe5 , Nle11 -NH2 in the alkaline
P4 and P3 , respectively. The hydrolytic fragment-A at Trp7 - medium were eluted along with the parent peak, making LC-
Phe8 with an m/z ratio 1111.1 (m/z = +17) was detected in MS analysis more complex in alkaline medium.
the MS spectrum corresponding to peak P4 . The correspond- These results are consistent with the data published by
ing fragment-B may have been further degraded, as it was Reubsaet et al. (1999), who examined tripeptide fragments of
not detected on the MS spectrum. The hydrolytic cleavage antagonist G a peptide with similar amino acid residues as
of the amidated Nle11 amino acid residue of Spantide II was Spantide II. The authors observed that the hydrolytic degrada-
detected in the MS spectrum corresponding to HPLC peak P4 . tion product: Phe-Trp resulting from the carboxylic tripeptide,
This fragment had an m/z ratio of 1670.5 (m/z = +17). Under Phe-Trp-Arg-COOH was detected on MS of both acidic and
highly acidic conditions (pH 1.0–2.0), Spantide II decomposed alkaline media.
by hydrolysis at several cleavage sites and the resulting prod-
ucts were further racemized and detected at various retention 3.4. Effect of oxidizing agent
times. Oliyai and Borchardt (1993) reported the degradation of
Asp-hexapeptide under highly acidic condition was predomi- Peptide oxidative products are relatively hydrophilic/polar and
nantly via intermolecular cleavage of the Asp-Gly amide bond are expected to elute prior to the parent molecule (Reubsaet
forming a tetrapeptide and a dipeptide. The first four amino et al., 1998). Fig. 6 shows an LC chromatogram of Spantide II in
Table 3 – Degradation products of Spantide II in alkaline medium at 60 ◦ C, showing theoretical masses, peak occurrence,
and m/z ratios of major hydrolytic products
Cleavage site Fragment A Fragment B Fragment A M + H+ Fragment B M + H+
theoretical mass theoretical mass (m/z): (peak) (m/z): (peak)
Table 4 – Degradation products of Spantide II in H2 O2 at 40 ◦ C, showing theoretical masses, peak occurrence, and m/z
ratios of major oxidation products
Product Theoretical mass M + H+ (m/z) M + 2H+ (m/z) Peak
of formulation variables on the release and skin permeation antagonist: Spantide II. Pharm. Res. 21, 108–
kinetics of gel formulation of Spantide II as a topical anti- 113.
inflammatory agent. Brown, J.R., Perry, P., Hefeneider, S., Ansel, A.C., 1990.
Neuropeptide modulation of keratinocyte cytokine
production. In: Oppenheim, P., Kluger, D. (Eds.), Molecular
and Cellular Biology of Cytokines. Wiley-Liss Inc., New York,
Acknowledgments pp. 451–456.
Csapá, J., Csapó-Kiss, Z., Wágner, L., Tálos, T., Martin, T.G.,
The authors acknowledge the financial assistance provided by Folestad, S., Tivesten, A., Némethy, S., 1997. Hydrolysis of
NIAMS (NIH) grant number AR47455-02 and RCMI (NIH) grant proteins performed at high temperatures and for short
number G12RR03020. times with reduced racemization, in order to determine the
enantiomers of d- and l-amino acids. Anal. Chim. Acta 339,
99–107.
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