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Stability and degradation profiles of Spantide II in aqueous solutions
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
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Stability and degradation profiles of Spantide II in aqueous solutions Loice Kikwai, R. Jayachandra Babu, Narayanasamy Kanikkannan, Mandip Singh ∗ Division of Pharmaceutics, College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL 32307, USA
a r t i c l e
i n f o
a b s t r a c t
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 chromatographymass 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 degradation 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 identified as hydrolysis, racemization, cleavage and formation of diketopiperazine. © 2005 Elsevier B.V. All rights reserved.
1.
Introduction
Spantide II is a synthetic peptide consisting of 11 amino acid residues (Fig. 1). It has a structure similar to substance P, a neuropeptide released from the C-fiber of the sensory nerves. It has been shown to antagonize the neurokinin-1 (NK1) receptors and inhibits the inflammatory response associated with substance P (Brown et al., 1990; Scholzen et al., 1998). We are investigating the potential use of Spantide II for the treat∗
Corresponding author. Tel.: +1 850 561 2790; fax: +1 850 599 3347. E-mail address: [email protected] (M. Singh).
0928-0987/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2005.09.005
ment of inflammatory skin disorders such as psoriasis and contact dermatitis. Currently, these disorders are primarily treated with corticosteroids, which have side effects such as stinging, itching, irritation, dryness, scaling, atrophy and hypo-pigmentation when applied to the skin (Epstein et al., 1963; Takeda et al., 1988). Targeting the neurogenic pathway of inflammation by utilizing Spantide II may reduce systemic side effects while improving patient compliance, thus leading to promising anti-inflammatory therapies. In earlier studies,
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Fig. 1 – Structure of Spantide II.
we demonstrated the anti-inflammatory effect of Spantide II in an allergic contact dermatitis mouse model (Jaiani et al., 2002; Babu et al., 2004). Due to the increased interest in proteins and peptides as pharmaceutical therapies, it is necessary to have a broad understanding of the chemical and physical stability of these agents in order to design suitable formulations. Generally, the amino acid residues on a peptide sequence are sensitive to various degradation pathways such as hydrolysis, deamidation and metal catalyzed oxidation (Manning et al., 1989; Reubsaet et al., 1998). In a previous study, we reported the preformulation stability of Spantide II as a function of pH, temperature, salt concentration and various dermatological vehicles (Kikwai et al., 2004). However, the mechanism of degradation of Spantide II and the resulting degradation products under various accelerated conditions have not been fully characterized. The characterization of peptide degradation is of importance, since an unstable peptide product may worsen product purity, potency and appearance. The purpose of this study was to further characterize the stability and degradation mechanisms of Spantide II in aqueous solutions. The HPLC assay method used in the present study was stability indicating, with a clear distinction between active peptide and the degradation products and further analysis was done by LC-MS to characterize the degradation products. The stability of Spantide II as a function of pH and temperature as well as the degradation profile of Spantide II under various forced degradation conditions were investigated.
2.
Material and methods
2.1.
Materials
Spantide II was custom synthesized by Bio Peptide Co. LLC (San Diego, CA) and used without further purification. Potassium chloride, mono and dibasic potassium phosphate, hydrochloric acid, potassium biphthalate, sodium hydroxide, hydrogen peroxide, trifluoroacetic acid (TFA) and boric acid were procured from Sigma Chemical Co. (St. Louis, MO, USA). Ethanol USP (200 proof) was obtained from Florida Distillers Co. (Lake Alfred, FL, USA). Water, acetonitrile and methanol (HPLC
grade) were purchased from Fisher Scientific (Atlanta, GA, USA). All other chemicals were standard reagent grade.
2.2. assay
High-performance liquid chromatography (HPLC)
A HPLC system (Waters Corporation) along with a Vydac ˚ pore size silica) analytical column reverse phase C18 (300 A (5 m, 4.6 mm × 250 mm) were used for the analysis of Spantide II. The HPLC system consisted of an autosampler (model 717 plus), two pumps (model 515), and an UV detector (model 996 PDA), all interfaced with EmpowerTM software. The mobile phases used were 0.1% TFA in water (solvent A) and 0.1% TFA in acetonitrile (solvent B). The mobile phase was filtered, degassed by sonication prior to use and analysis was run at a gradient of 68%:32% (solvent A:B, respectively), which was reversed to 32%:68% (solvent A:B, respectively) over 30 min, with a flow rate of 1 ml/min. Spantide II content in the samples was determined using a PDA-UV detector set at 230 nm. All analyses were performed at room temperature, and the retention time of Spantide II was 20.8 min.
2.3. Liquid chromatography-mass spectrometry (LC-MS) The LC system consisted of Beckman Gold 125S Solvent Module equipped with a Beckman Gold 166 UV detector. The 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˚ pore size silica, 5 m particles, 1.0 mm × 250 mm) umn (300 A attached to a 2 mm guard cartridge of same column chemistry was used for the analysis of Spantide II. The LC system was operated by the Beckman Gold V712 software. Mass spectra were acquired using an AccuTOFTM Timeof-Flight Mass Spectrometer (Model JMS-T100LC, JEOL Inc., Peabody, MA, USA). The instrument was interfaced with a positive ion mode electro spray inlet probe used to ionize the molecules. The spectra were obtained in the low-resolution mode with a voltage of 10 kV. Mass spectra were collected in the positive ion mode, scanning from 200–2000 mass units every 1 s.
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2.4.
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Standard stability conditions
Spantide II was dissolved in 50% ethanol:water to prepare a stock solution with a concentration of 1 mg/ml. A 50% ethanol:water solution was used due to the poor water solubility of Spantide II. The final concentration for all stability samples was 0.5 mg/ml, which was obtained by mixing a 1:1 ratio of the stock solution with the appropriate experimental solution. Stability samples were prepared in glass vials capped with silicone lined polypropylene caps and were kept at predefined temperature conditions. Samples were collected at intervals of 0, 2, 4, 8, 12, 24, 48 96, 168, 240, 336, 720, and 1440 h for analysis. The physical appearance of the sample was noted. The concentration of Spantide II in the samples was determined by HPLC, and the study was performed in triplicate. Samples containing degradation peaks as identified by HPLC were further analyzed by LC-MS in order to elucidate the degradation pathway of Spantide II.
2.4.1.
Effect of pH
Buffer solutions (0.025 M) of pH 1–10 were prepared as per USP 28 procedures (The United States Pharmacopoeia, 2004). Buffers used were hydrochloric acid buffer for pH 1.0 and 2.0; acid phthalate buffer for pH 3.0 and 4.0, neutralized phthalate buffer for pH 5.0; phosphate buffer for pH 6.0 and 7.0; and alkaline borate buffer for pH 8.0–10.0. Test samples were prepared by mixing a 1:1 ratio of each buffer with Spantide II stock solution and stability studies were conducted at 60 ◦ C.
2.4.3.
Accelerated degradation by change in pH
Acidic and alkaline media were prepared by mixing Spantide II stock with 0.1 M HCL or 0.1 M NaOH at 1:1 ratio, while for the neutral condition Spantide II stock was mixed with a 50% ethanol:water solution at 1:1 ratio. All the samples were subjected to forced degradation at 60 ◦ C.
2.4.4.
Accelerated degradation by oxidation
Hydrogen peroxide (H2 O2 ) was used as an oxidizing agent at a concentration of 0.3% (v/v) and was mixed with Spantide II stock in a 1:1 ratio to a final concentration of 0.15% (v/v). The samples were stored at room temperature and 40 ◦ C.
2.5.
ln[C] = ln[C0 ] − ko t where C0 is the initial concentration of the sample.
3.
Results and discussion
3.1.
Analytical techniques
A previously developed and validated reverse phase HPLC method was used for analysis of Spantide II samples (Kikwai et al., 2004). This method was checked for its stability-indicating properties to differentiate the active drug from the degradation products. The Spantide II peak was well resolved from other degradation products, which eluted before or after the parent peak. The peaks were further analyzed by LC-MS in order to verify the identity of the degradation products.
Effect of temperature
The influence of temperature on the observed degradation rate constant (kobs ) of Spantide II in ethanol–water mixture (1:1) was determined. The reaction rate constant was determined at 25, 40, 50, 60, and 70 ◦ C. The resulting Arrhenius plot was used to determine the activation energy (Ea ) and shelf life (T90% ) of Spantide II.
2.4.2.
according to equation:
3.2.
Effect of temperature
The degradation of Spantide II at 25, 40, 50, 60 and 70 ◦ C in aqueous ethanol (50%) was monitored by HPLC. There was no significant degradation of Spantide II at 25 and 40 ◦ C over a period of 1 month. On the other hand, at 70 ◦ C, the concentration of Spantide II decreased to 30% of the initial concentration, which represented a decline of two half lives. The influence of temperature on the observed reaction rate constant (kobs ) is given by the natural logarithmic form (ln) of the Arrhenius equation: ln kobs = ln A −
Ea RT
where A is the frequency factor and is a constant, Ea the energy of activation, R the universal gas constant, and T is the absolute temperature. The kobs values were fitted using the equation as shown in Fig. 2. The relationship between ln kobs and 1/T was determined by linear regression and the goodness of fit for the line was R2 = 0.9783. The calculated Ea was 85 kJ mol−1 and A was 4.4 × 106 s−1 . Shelf life (T90% ) indicates the period of storage of a product without loss of potency. It is described as the time taken for 10% of the product to decompose at a given temperature. To determine the shelf life of Spantide II at 25 ◦ C, the following equation was used: 0.105 T90% = k25
Statistical analysis
The logarithmic concentration of Spantide II in the stability samples was plotted as a function of time. The observed reaction rate constant (ko ) was obtained from the slope of the natural logarithmic (ln) plot of Spantide II concentration (C) versus time (t) profiles by linear regression analysis using Graphpad Prizm® software (Graphpad Software Inc., San Diego, CA),
Fig. 2 – Arrhenius plot of Spantide II.
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Fig. 3 – HPLC chromatogram of Spantide II degradation in aqueous ethanol at 60 ◦ C.
The ln kobs at 25 ◦ C for Spantide II was calculated and determined to be −19.06. From this result, shelf life of Spantide II at 25 ◦ C was determined to be 230 days. This indicates that Spantide II is stable at room temperature and pharmaceutical products of this compound can be stored at 4 ◦ C for an extended shelf life of the product. To determine the degradation mechanism of Spantide II, the degradation profiles were monitored by HPLC and LC-MS after exposure of the samples to 60 ◦ C. The degradation products were identified by analysis of the MS spectra corresponding to peaks observed in the LC chromatograms produced by LC-MS. The degradation of Spantide II in aqueous ethanol (50%), at 60 ◦ C yielded two degradation peaks P1 and P2 as depicted in Fig. 3. The degradation products occurring at these peaks are non-polar compared to Spantide II as they elute after the parent compound. Further examination of the MS spectra
associated with peak P1 confirms that it corresponds to degradation products of Spantide II with m/z ratios of 1339.8, 331.5 and 577.3. While MS spectrum associated with peak P2 corresponds to degradation product of Spantide II with m/z ratio of 1111.7. Table 1 shows the m/z ratios of the product occurring in peak P1 and P2 as determined by LC-MS. The fragments with m/z ratio of 331.5 and 1339.8 are associated with Spantide II (nicotinoyl)Lys1 -Pro2 diketopiperazine degradation that was elucidated in an earlier study (Kikwai et al., 2004). Diketopiperazine (DKP) occurs at the Nterminal of the peptide and forms a cyclic product that has two N-terminal amino acids. The mechanism of DKP formation involves nucleophilic attack of the N-terminal nitrogen on the amide carbonyl between the second and third amino acids. This intramolecular aminolysis reaction occurs readily in aqueous solution (Goolcharran and Borchardt, 1998). The degradation pathway of a model peptide l-Ala-l-Pro-l-Met (APM) under the influence of pH and temperature was studied by Goolcharran et al. (2000). This tripeptide APM underwent diketopiperazine formation (Ala-Pro-diketopiperazine). Similarly, the results of our study demonstrated the formation of DKP after cleavage at (nicotinoyl)Lys1 -Pro2 of Spantide II. The hydrolytic cleavage of Spantide II at the peptide bond between Pro4 -PheCl2 5 results in two products; fragment-A, of MW 593.7 after addition of a hydroxyl group (m/z = +17) and fragment-B, of 1093.1 after addition of a hydrogen. The m/z ratio corresponding to fragment-A was not observed in the MS spectrum therefore, it might have undergone further degradation. Further hydrolytic degradation products of fragment-A were not observed in the MS spectrum. Fragment-B with a MW of 1093.1 and an m/z ratio of 1094.7 was observed in both P1 and P2 . Resolution of this fragment at different retention times on the HPLC chromatogram indicates that this fragment underwent further racemization. Additionally Spantide II underwent hydrolytic cleavage at the peptide bond between Trp7 -Phe8 . The resulting products of MW 1110.1 after addition of a hydroxyl group (m/z = +17) and 576.3 after addition of hydrogen (m/z = +1) were observed in MS spectrum with m/z ratios of 1111.7 and 577.7 and occurred in P2 and P1 in the HPLC chromatogram, respectively. Csapa´ et al. (1997) demonstrated that protein and peptide molecules hydrolyzed at elevated temperatures and racemized if the exposure is extended over longer periods. The results indicate that in addition to DKP formation, hydrolytic and racemized degradation products were formed when Spantide II is exposed to elevated temperature conditions.
3.3.
Effect of pH
The degradation of Spantide II at 60 ◦ C in aqueous solutions over a range of pH values (1–10) in various buffers was
Table 1 – Degradation of Spantide II at 60 ◦ C in aqueous ethanol solution: m/z ratios of the degradation products Cleavage site Pro2 -(pyridyl)Ala3 Pro4 -PheCl2 5 Trp7 -Phe8
Fragment A theoretical mass
Fragment B theoretical mass
Fragment A M + H+ (m/z): (peak)
330.4 593.7 576.3
1338.7 1093.1 1110.1
331.5 (P1 ) – 577.3 (P1 )
Fragment B M + H+ (m/z): (peak) 1339.8 (P1 ) 1094.7 (P1 , P2 ) 1111.7 (P2 )
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Fig. 4 – pH–log kobs profile of Spantide II at 60 ◦ C.
monitored by HPLC. The Spantide II plots of ln concentration versus time were linear at all pH’s indicating pseudo-first order degradation rate kinetics. The pseudo-first order rate constant (ko ) values were calculated from the slopes of linear plots. The observed reaction rate constant (kobs ) can be expressed as: kobs = ko + kOH [OH− ] + kH [H+ ] + kbuffer [buffer] where ko is the pseudo-first order rate constant for degradation in water only, while kOH , kH , and kbuffer are second order rate constants for the degradation catalyzed by hydrogen ions, hydroxide ions and buffer components respectively. The influence of pH on the stability of Spantide II is shown in Fig. 4. The data indicate that Spantide II is susceptible to degradation at pH <2.0 and pH >5. The rate of degradation was rapid at pH <2.0 indicating specific hydrogen-ioncatalysis of the positively charged Spantide II species. In the pH region (pH 2–5), the degradation rate plateaus and can be attributed to specific buffer-species-catalysis of the neutral species. The rate of degradation is rapid at pH >5, indicating specific hydroxide-ion-catalysis of the negatively charged species. Similar results were obtained for Aplidine, which showed optimal stability of the peptide in the pH region 2–6 under accelerated condition at 60 ◦ C and at pH below 2 and above 6 a clear proton-catalyzed and hydroxyl-catalyzed hydrolyses, respectively, were observed (Waterval et al., 2001).
Reubsaet et al. (1995) also reported a similar acid catalyzed degradation profile for antagonist G, which has a similar structure to Spantide II and identical amino acids at positions 7, 9, and 10. The degradation mechanism of Spantide II in acidic and alkaline media at 60 ◦ C was monitored by HPLC and LC-MS. The degradation products were identified by analysis of the MS spectra corresponding to peaks observed in the LC chromatograms produced by LC-MS. Degradation of the peptide backbone occurs by hydrolysis, in peptides exposed to both acidic and alkaline media (Manning et al., 1989; Oliyai et al., 1992). Fig. 5A and B shows chromatograms of Spantide II and its degradation products after exposure to acidic and alkaline media, respectively. This degradation was instantaneous and rapid as observed by the appearance of degradation products on the 0 h chromatograms in both acidic and alkaline conditions. In the acidic medium, Spantide II peak was still detectable at 24 h, although its height and intensity was significantly lower compared to 0 h. There were barely any traces of Spantide II seen in the chromatogram after 12 h of exposure to the alkaline medium. The degradation rate in acidic medium was slower than in alkaline medium. Consequently, there were more degradation peaks formed in the alkaline medium. These data were consistent with that published by Reubsaet et al. (1995) who observed high degradation rates and more degradation peaks in alkaline medium compared to acidic medium. In acidic medium, degradation products that occurred in peaks P4 –P6 were relatively non-polar as they eluted after the parent peak. MS spectral analysis of peaks P4 –P6 determine that Spantide II had undergone hydrolytic cleavage at different peptide bonds. The major hydrolytic cleavage sites on Spantide II in acidic medium, and their theoretical masses, peak occurrence and m/z ratios are shown in Table 2. These products seemed to racemize since they occurred at multiple retention times on the HPLC chromatograms but had identical m/z ratios. These results agree with data obtained for antagonist G where its hydrolytic degradation products racemized and were detected at different retention times on the HPLC (Reubsaet et al., 1999).
Fig. 5 – HPLC chromatograms of Spantide II degradation in acidic and alkaline medium at 60 ◦ C.
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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 (nicotinoyl)Lys1 -Pro2 Pro2 -(pyridyl)Ala3 Trp7 -Phe8 Phe8 -Trp9 Nle11 -NH2
Fragment A theoretical mass 251.1 348.1 1110.1 1257.2 1669.7
Fragment B theoretical mass 1436.5 1338.5 576.7 429.5 17.0
The degradation products that occurred at the HPLC peak P3 were relatively polar in nature compared to Spantide II as they eluted prior to the parent peak. MS spectral analysis of peak P3 identified products that resulted from the hydrolytic cleavage of Spantide II at (nicotinoyl) Lys1 -Pro2 , Pro2 -(pyridyl)Ala3 and Phe8 -Trp9 peptide bonds. Hydrolysis at (nicotinoyl)Lys1 -Pro2 produced a fragment-A with an m/z ratio of 251.1 (m/z = +17), and fragment-B with an m/z ratio of 1436.5 (m/z = +1) after addition of hydrogen. Fragment-A racemized and was detected in P5 and P6 in the HPLC chromatogram. The m/z ratio corresponding to fragment-B was not observed in the MS spectrum therefore, it might have undergone further degradation. Further hydrolytic degradation products of fragment-A were not observed in the MS spectrum. Hydrolytic cleavage at Pro2 -(pyridyl)Ala3 produced two degradation fragments. The fragment-A with m/z ratios of 349.2 (m/z = +17) occurred in the MS spectrum corresponding to HPLC peak P3 , while the fragment-B with m/z ratio of 1339.4, racemized and was identified in the MS spectrum corresponding to HPLC peaks P5 and P6 . Hydrolytic cleavage at Phe8 -Trp9 produced two degradation products, fragmentA with an m/z ratio of 1258.3 (m/z = +17) and fragment-B, with an m/z ratio of 430.1, which occurred in the HPLC peak P4 and P3 , respectively. The hydrolytic fragment-A at Trp7 Phe8 with an m/z ratio 1111.1 (m/z = +17) was detected in the MS spectrum corresponding to peak P4 . The corresponding fragment-B may have been further degraded, as it was not detected on the MS spectrum. The hydrolytic cleavage of the amidated Nle11 amino acid residue of Spantide II was detected in the MS spectrum corresponding to HPLC peak P4 . This fragment had an m/z ratio of 1670.5 (m/z = +17). Under highly acidic conditions (pH 1.0–2.0), Spantide II decomposed by hydrolysis at several cleavage sites and the resulting products were further racemized and detected at various retention times. Oliyai and Borchardt (1993) reported the degradation of Asp-hexapeptide under highly acidic condition was predominantly via intermolecular cleavage of the Asp-Gly amide bond forming a tetrapeptide and a dipeptide. The first four amino
Fragment A M + H+ (m/z): (peak) 252.3 (P5 , P6 ) 349.2 (P3 ) 1111.1 (P4 ) 1258.3 (P4 ) 1670.5 (P4 )
Fragment B M + H+ (m/z): (peak) – 1339.4 (P5 , P6 ) 577.6 (P5 ) 430.1 (P3 ) –
acids of Spantide II are not required for binding of Spantide II to neurokinin-1 receptors while the terminal amino acids are required for binding and receptor recognition. Therefore, hydrolytic cleavage of the terminal amino acid residues can lead to the deactivation of Spantide II antagonist activity and thus decreasing its anti-inflammatory activity (Ljungqvist et al., 1989). In alkaline medium, the degradation products are both polar and non-polar relative to Spantide II, because the peaks elute before and after the parent peak. Table 3 shows major hydrolytic degradation products of Spantide II in alkaline media. Analysis of the degradation products in alkaline medium revealed m/z ratios that were similar to those that occurred in acidic medium. The degradation products racemized and occurred at different retention times on the chromatograms, however, m/z ratios (m/z = +17) were identical as determined by MS. There is a correlation between hydrolytic degradation in acidic and alkaline medium as observed by degradation products resulting from cleavage at Pro2 -(pyridyl)Ala3 , Trp7 -Phe8 , and Nle11 -NH2 were observed in both MS spectra of acidic and alkaline conditions. It should be noted that from Table 3, three of the degradants, of Pro2 -(pyridyl)Ala3 , and Pro4 -Phe5 , Nle11 -NH2 in the alkaline medium were eluted along with the parent peak, making LCMS analysis more complex in alkaline medium. These results are consistent with the data published by Reubsaet et al. (1999), who examined tripeptide fragments of antagonist G a peptide with similar amino acid residues as Spantide II. The authors observed that the hydrolytic degradation product: Phe-Trp resulting from the carboxylic tripeptide, Phe-Trp-Arg-COOH was detected on MS of both acidic and alkaline media.
3.4.
Effect of oxidizing agent
Peptide oxidative products are relatively hydrophilic/polar and are expected to elute prior to the parent molecule (Reubsaet 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 Pro2 -(pyridyl)Ala3 (pyridyl)Ala3 -Pro4 Pro4 -Phe5 Trp7 -Phe8 Nle11 -NH2
Fragment A theoretical mass 348.1 496.6 593.7 1110.1 1669.7
Fragment B theoretical mass
Fragment A M + H+ (m/z): (peak)
Fragment B M + H+ (m/z): (peak)
1338.5 1190.2 1093.1 576.7 17.03
349.2 (P2 ) – – 1111.1 (P3 ) 1670.5 (all peaks)
1339.4 (P4 , P5 ) 1191.3 (Parent, P3 , P4 , P5 ) 1094.2 (P1 , P2 , Parent) 577.6 (P3 , P4 , P5 ) –
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Fig. 6 – LC-MS chromatogram of Spantide II oxidation in hydrogen peroxide.
H2 O2 at 40 ◦ C. Spantide II degradation yielded polar oxidative products, which eluted before the parent peak. Products corresponding to oxidative peaks (OP1 to OP3 ) were examined for product identity by LC-MS. The schemes of oxidative degradation of Spantide II are shown in Fig. 7. In Fig. 7A, Pro2,4 undergoes possible ring opening and addition of an oxygen and hydrogen molecule thus increasing the overall mass of Spantide II. If one Pro ring opens, the mass will increase by +16 to 1684.8 while opening at both Pro residues the mass will increase by +32 to 1700.8. This increase in mass was observed in LC-MS studies that were conducted to further identify the oxidative degradation products of Spantide II. Table 4 shows the m/z ratios observed by LC-MS for Spantide II oxidation in H2 O2 . Pro2,4 ring opening was observed in LC peaks OP1 , OP2 , and OP2 . The scheme of oxidative degradation of Pro2,4 by peptide bond cleavage is shown in Fig. 7B. Pro2,4 can undergo cleavage of the peptide bonds at Pro2 -Ala3 and/or at Pro4 -PheCl2 5 , resulting in the addition of oxygen and expulsion of carbon dioxide. Based on LC-MS evaluation we found that cleavage of Pro at Pro2 -Ala3 did not occur because there was no evidence
of m/z ratio of matching mass 1340.8 or 349.3 in the MS spectra of all degradation peaks. However, cleavage at Pro4 -PheCl2 5 was identified by the m/z ratio of a fragment with a mass of 1095.6, which corresponds to (PheCl2 5 -Nle11 )-Spantide II. The corresponding m/z ratios for Pro4 cleavage observed in the LC peak OP3 are seen in Table 4. Oxidation of the Phe5,8 residues on Spantide II is a possible mechanism of degradation in the presence of H2 O2 leading to 2-hydroxyphenylalanine (Wang, 1999). The scheme of oxidative degradation as can be seen in Fig. 7C, indicates the addition of OH group at position 2 of the aromatic rings of PheCl2 5 or Phe8 , resulting in a mass increase of 16 (m/z = +16) (Table 4). Addition of the hydroxyl group at both PheCl2 5 and Phe8 results in a mass increase of 32 (m/z = +32). The oxidative product 2-hydroxyphenylalanine is more hydrophilic/polar than Spantide II and eluted prior to the parent peak. In this study, we focused on identification of the major peaks from the LC chromatogram. Several small peaks in Fig. 7 were probably the oxidative products from Spantide II, but the corresponding m/z values in LC-MS were not detectible due to very low intensity of these products (due to noise interference of MS spectrum with the m/z values). Oxidation could dominate other degradation pathways in a protein or peptide (Wang, 1999). There are several potential oxidative degradation reactions in hIGF-I (Fransson et al., 1996). Similarly, oxidation is the major degradation pathway for KGF-2 (Kaushal et al., 1998). The results of the present study indicate no signs of oxidative degradation of Spantide II in aqueous solution under various pH and temperature conditions, but under forced degradation conditions (H2 O2 ) we found several oxidative products indicating possible amino acid residues that could undergo oxidative degradation under extreme conditions. In conclusion, the study of the degradation profiles of Spantide II in aqueous solution was successfully accomplished using HPLC and LC-MS techniques. Spantide II is susceptible to degradation at pH <2.0 and pH >5 while maximal stability was observed between pH 2–5. Spantide II degrades by Lys-Pro diketopiperazine, hydrolysis and racemization in both acidic and alkaline media, and by oxidation of Pro and Phe amino acid residues as assessed by LC-MS analysis. This study further demonstrates that Spantide II is stable in aqueous ethanol (50%, v/v) with a shelf life (T90% ) of 230 days at 25 ◦ C. The longterm objective of the study is to develop a topical formulation of Spantide II for the treatment of inflammatory skin disorders. We demonstrated the topical anti-inflammatory activity of this compound in allergic and irritant contact dermatitis animal models. We are currently investigating the effect
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 Spantide II Pro2 ring opening Pro2,4 ring opening Pro4 bond cleavage Phe5 or 8 oxidation Phe5 and 8 oxidation
Theoretical mass
M + H+ (m/z)
M + 2H+ (m/z)
1668.8 1684.8 1700.8 1094.8 1684.8 1700.8
1669.0 1685.0 1701.0 1095.6 1685.1 1701.0
835.6 843.6 851.6 548.8 843.6 851.6
Peak Parent OP1 , OP3 OP2 , OP3 OP3 OP1 , OP3 OP2 , OP3
european journal of pharmaceutical sciences
27
( 2 0 0 6 ) 158–166
165
Fig. 7 – Oxidative pathways of Spantide II.
of formulation variables on the release and skin permeation kinetics of gel formulation of Spantide II as a topical antiinflammatory agent.
Acknowledgments The authors acknowledge the financial assistance provided by NIAMS (NIH) grant number AR47455-02 and RCMI (NIH) grant number G12RR03020.
references
Babu, R.J., Kikwai, L., Jaiani, L.T., Kanikkannan, N., Armstrong, C.A., Ansel, J.C., Singh, M., 2004. Percutaneous absorption and anti-inflammatory effect of a substance P receptor
antagonist: Spantide II. Pharm. Res. 21, 108– 113. 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, pp. 451–456. ´ J., Csapo-Kiss, ´ ´ ´ Csapa, Z., Wagner, L., Talos, T., Martin, T.G., ´ Folestad, S., Tivesten, A., Nemethy, S., 1997. Hydrolysis of proteins performed at high temperatures and for short times with reduced racemization, in order to determine the enantiomers of d- and l-amino acids. Anal. Chim. Acta 339, 99–107. Epstein, N.N., Epstein, W.L., Epstein, J.H., 1963. Atrophic striae in patients with inguinal intertrigo. Arch. Dermatol. 87, 450–457. Fransson, J., Florin-Robertsson, E., Axelsson, K., Nyhle’n, C., 1996. Oxidation of human insulin-like growth factor I in formulation studies: kinetics of methionine oxidation in
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aqueous solution and in solid state. Pharm. Res. 13, 1252–1257. Goolcharran, C., Borchardt, R.T., 1998. Kinetics of diketopiperazine formation using model peptides. J. Pharm. Sci. 87, 283–288. Goolcharran, C., Cleland, J.L., Keck, R., Jones, A.J., Borchardt, R.T., 2000. Comparison of the rates of deamidation, diketopiperazine formation and oxidation in recombinant human vascular endothelial growth factor and model peptides. AAPS PharmSci. 2, E5. Jaiani, L.T., Sachdeva, M., Jaychandra, B.R., Kanikkannan, N., Legat, F.J., Neparidze, N.G., Kikwai, L., Armstrong, C.A., Ansel, J.C., 2002. Inhibition of allergic contact dermatitis by a topically applied SP receptor antagonist. Invest. Dermatol. 119, Abstract 655. Kaushal, P., Burducea, I., Hunt, S., Geblaoui, A., Gentz, R., Khan, F., 1998. Identification of chemical modifications in proteins, myeloid progenitor inhibitory factor-1 (MPIF-1) and keratinocyte growth factor-2 (KGF-2) by matrix-assisted laser desorption mass spectrometry. Pharm. Sci. 1, S537. Kikwai, L., Babu, R.J., Kanikkannan, N., Singh, M., 2004. Preformulation stability of Spantide II, a promising topical anti-inflammatory agent for the treatment of psoriasis and contact dermatitis. J. Pharm. Pharmacol. 56, 19–25. Ljungqvist, A., Chu, J.Y., Tang, P.F., Bender, W., Hakanson, R., Leander, S., Rosell, S., Folkers, K., 1989. Increased potency of antagonists of substance P having asparagine in position 6. Regul. Pept. 24, 283–291. Manning, M.C., Patel, K., Borchardt, R.T., 1989. Stabil. Protein Pharmaceut. Pharmacol. Res. 6, 903–918. Oliyai, C., Borchardt, R.T., 1993. Chemical pathways of peptide degradation. IV. Pathways, kinetics, and mechanism of degradation of an aspartyl residue in a model hexapeptide. Pharm. Res. 10, 95–102.
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Oliyai, C., Schoneich, C., Wilson, G.S., Borchardt, R.T., 1992. Topics in pharmaceutical sciences. In: Crommelin, D.J.A., Mihda, K.K. (Eds.), Medpharm. Scientific, Stuttgart, pp. 23–46. Reubsaet, J.L.E., Beijnen, J.H., Belshof, E.H.M., Bouyakhrichan, M., Bult, A., Hop, E., Kellekule, Y., van Maanen, R.J., Teeuwsen, J., Underberg, W.J.M., 1999. Qualitative and quantitative aspects of the degradation of several tripeptides derived from the anti-tumour peptide antagonist [Arg6 , d-Trp7,9 , MePhe8 ] substance P{6–11}. J. Pharm. Biomed. Anal. 19, 277–284. Reubsaet, J.L.E., Beijnen, J.H., Bult, A., van der Houwen, O.A.G.J., Teeuwsen, J., Koster, E.H.M., Underberg, W.J.M., 1995. Degradation kinetics of antagonist [Arg6 , d-Trp7 ,9 MePhe8 ]-substance P{6–11} in aqueous solution. Anal. Biochem. 227, 334–341. Reubsaet, J.L.E., Beijnen, J.H., Bult, A., van Maanen, R.J., Marchal, J.A.D., Underberg, W.J.M.J., 1998. Analytical techniques used to study the degradation of proteins and peptides: chemical instability. Pharm. Biomed. Anal. 17, 955–978. Scholzen, T., Armstrong, C.A., Bunnett, N.W., Luger, T.A., Olerud, J.E., Ansel, J.C., 1998. Neuropeptides in the skin: interactions between the neuroendocrine and the skin immune systems. Exp. Derm. 7, 81–96. Takeda, K., Arase, S., Takahashi, S., 1988. Side effects of topical corticosteroids and their prevention. Drugs 36, 15–23. The United States Pharmacopoeia 28, The National Formulary 23, United States Pharmacopoeial Convention, Rockville, MD, 2004. Wang, W., 1999. Instability, stabilization, and formulation of liquid protein pharmaceuticals. Int. J. Pharm. 185, 129–188. Waterval, J.C., Bloks, J.C., Sparidans, R.W., Beijnen, J.H., Rodriguez-Campos, I.M., Bult, A., Lingeman, H., Underberg, W.J., 2001. Degradation kinetics of aplidine, a new marine anti-tumoural cyclic peptide, in aqueous solution. J. Chromatogr. B Biomed. Sci. 754, 161–168.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ejps
Stability and degradation profiles of Spantide II in aqueous solutions Loice Kikwai, R. Jayachandra Babu, Narayanasamy Kanikkannan, Mandip Singh ∗ Division of Pharmaceutics, College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL 32307, USA
a r t i c l e
i n f o
a b s t r a c t
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 chromatographymass 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 degradation 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 identified as hydrolysis, racemization, cleavage and formation of diketopiperazine. © 2005 Elsevier B.V. All rights reserved.
1.
Introduction
Spantide II is a synthetic peptide consisting of 11 amino acid residues (Fig. 1). It has a structure similar to substance P, a neuropeptide released from the C-fiber of the sensory nerves. It has been shown to antagonize the neurokinin-1 (NK1) receptors and inhibits the inflammatory response associated with substance P (Brown et al., 1990; Scholzen et al., 1998). We are investigating the potential use of Spantide II for the treat∗
Corresponding author. Tel.: +1 850 561 2790; fax: +1 850 599 3347. E-mail address: [email protected] (M. Singh).
0928-0987/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2005.09.005
ment of inflammatory skin disorders such as psoriasis and contact dermatitis. Currently, these disorders are primarily treated with corticosteroids, which have side effects such as stinging, itching, irritation, dryness, scaling, atrophy and hypo-pigmentation when applied to the skin (Epstein et al., 1963; Takeda et al., 1988). Targeting the neurogenic pathway of inflammation by utilizing Spantide II may reduce systemic side effects while improving patient compliance, thus leading to promising anti-inflammatory therapies. In earlier studies,
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
Fig. 1 – Structure of Spantide II.
we demonstrated the anti-inflammatory effect of Spantide II in an allergic contact dermatitis mouse model (Jaiani et al., 2002; Babu et al., 2004). Due to the increased interest in proteins and peptides as pharmaceutical therapies, it is necessary to have a broad understanding of the chemical and physical stability of these agents in order to design suitable formulations. Generally, the amino acid residues on a peptide sequence are sensitive to various degradation pathways such as hydrolysis, deamidation and metal catalyzed oxidation (Manning et al., 1989; Reubsaet et al., 1998). In a previous study, we reported the preformulation stability of Spantide II as a function of pH, temperature, salt concentration and various dermatological vehicles (Kikwai et al., 2004). However, the mechanism of degradation of Spantide II and the resulting degradation products under various accelerated conditions have not been fully characterized. The characterization of peptide degradation is of importance, since an unstable peptide product may worsen product purity, potency and appearance. The purpose of this study was to further characterize the stability and degradation mechanisms of Spantide II in aqueous solutions. The HPLC assay method used in the present study was stability indicating, with a clear distinction between active peptide and the degradation products and further analysis was done by LC-MS to characterize the degradation products. The stability of Spantide II as a function of pH and temperature as well as the degradation profile of Spantide II under various forced degradation conditions were investigated.
2.
Material and methods
2.1.
Materials
Spantide II was custom synthesized by Bio Peptide Co. LLC (San Diego, CA) and used without further purification. Potassium chloride, mono and dibasic potassium phosphate, hydrochloric acid, potassium biphthalate, sodium hydroxide, hydrogen peroxide, trifluoroacetic acid (TFA) and boric acid were procured from Sigma Chemical Co. (St. Louis, MO, USA). Ethanol USP (200 proof) was obtained from Florida Distillers Co. (Lake Alfred, FL, USA). Water, acetonitrile and methanol (HPLC
grade) were purchased from Fisher Scientific (Atlanta, GA, USA). All other chemicals were standard reagent grade.
2.2. assay
High-performance liquid chromatography (HPLC)
A HPLC system (Waters Corporation) along with a Vydac ˚ pore size silica) analytical column reverse phase C18 (300 A (5 m, 4.6 mm × 250 mm) were used for the analysis of Spantide II. The HPLC system consisted of an autosampler (model 717 plus), two pumps (model 515), and an UV detector (model 996 PDA), all interfaced with EmpowerTM software. The mobile phases used were 0.1% TFA in water (solvent A) and 0.1% TFA in acetonitrile (solvent B). The mobile phase was filtered, degassed by sonication prior to use and analysis was run at a gradient of 68%:32% (solvent A:B, respectively), which was reversed to 32%:68% (solvent A:B, respectively) over 30 min, with a flow rate of 1 ml/min. Spantide II content in the samples was determined using a PDA-UV detector set at 230 nm. All analyses were performed at room temperature, and the retention time of Spantide II was 20.8 min.
2.3. Liquid chromatography-mass spectrometry (LC-MS) The LC system consisted of Beckman Gold 125S Solvent Module equipped with a Beckman Gold 166 UV detector. The 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˚ pore size silica, 5 m particles, 1.0 mm × 250 mm) umn (300 A attached to a 2 mm guard cartridge of same column chemistry was used for the analysis of Spantide II. The LC system was operated by the Beckman Gold V712 software. Mass spectra were acquired using an AccuTOFTM Timeof-Flight Mass Spectrometer (Model JMS-T100LC, JEOL Inc., Peabody, MA, USA). The instrument was interfaced with a positive ion mode electro spray inlet probe used to ionize the molecules. The spectra were obtained in the low-resolution mode with a voltage of 10 kV. Mass spectra were collected in the positive ion mode, scanning from 200–2000 mass units every 1 s.
160
2.4.
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Standard stability conditions
Spantide II was dissolved in 50% ethanol:water to prepare a stock solution with a concentration of 1 mg/ml. A 50% ethanol:water solution was used due to the poor water solubility of Spantide II. The final concentration for all stability samples was 0.5 mg/ml, which was obtained by mixing a 1:1 ratio of the stock solution with the appropriate experimental solution. Stability samples were prepared in glass vials capped with silicone lined polypropylene caps and were kept at predefined temperature conditions. Samples were collected at intervals of 0, 2, 4, 8, 12, 24, 48 96, 168, 240, 336, 720, and 1440 h for analysis. The physical appearance of the sample was noted. The concentration of Spantide II in the samples was determined by HPLC, and the study was performed in triplicate. Samples containing degradation peaks as identified by HPLC were further analyzed by LC-MS in order to elucidate the degradation pathway of Spantide II.
2.4.1.
Effect of pH
Buffer solutions (0.025 M) of pH 1–10 were prepared as per USP 28 procedures (The United States Pharmacopoeia, 2004). Buffers used were hydrochloric acid buffer for pH 1.0 and 2.0; acid phthalate buffer for pH 3.0 and 4.0, neutralized phthalate buffer for pH 5.0; phosphate buffer for pH 6.0 and 7.0; and alkaline borate buffer for pH 8.0–10.0. Test samples were prepared by mixing a 1:1 ratio of each buffer with Spantide II stock solution and stability studies were conducted at 60 ◦ C.
2.4.3.
Accelerated degradation by change in pH
Acidic and alkaline media were prepared by mixing Spantide II stock with 0.1 M HCL or 0.1 M NaOH at 1:1 ratio, while for the neutral condition Spantide II stock was mixed with a 50% ethanol:water solution at 1:1 ratio. All the samples were subjected to forced degradation at 60 ◦ C.
2.4.4.
Accelerated degradation by oxidation
Hydrogen peroxide (H2 O2 ) was used as an oxidizing agent at a concentration of 0.3% (v/v) and was mixed with Spantide II stock in a 1:1 ratio to a final concentration of 0.15% (v/v). The samples were stored at room temperature and 40 ◦ C.
2.5.
ln[C] = ln[C0 ] − ko t where C0 is the initial concentration of the sample.
3.
Results and discussion
3.1.
Analytical techniques
A previously developed and validated reverse phase HPLC method was used for analysis of Spantide II samples (Kikwai et al., 2004). This method was checked for its stability-indicating properties to differentiate the active drug from the degradation products. The Spantide II peak was well resolved from other degradation products, which eluted before or after the parent peak. The peaks were further analyzed by LC-MS in order to verify the identity of the degradation products.
Effect of temperature
The influence of temperature on the observed degradation rate constant (kobs ) of Spantide II in ethanol–water mixture (1:1) was determined. The reaction rate constant was determined at 25, 40, 50, 60, and 70 ◦ C. The resulting Arrhenius plot was used to determine the activation energy (Ea ) and shelf life (T90% ) of Spantide II.
2.4.2.
according to equation:
3.2.
Effect of temperature
The degradation of Spantide II at 25, 40, 50, 60 and 70 ◦ C in aqueous ethanol (50%) was monitored by HPLC. There was no significant degradation of Spantide II at 25 and 40 ◦ C over a period of 1 month. On the other hand, at 70 ◦ C, the concentration of Spantide II decreased to 30% of the initial concentration, which represented a decline of two half lives. The influence of temperature on the observed reaction rate constant (kobs ) is given by the natural logarithmic form (ln) of the Arrhenius equation: ln kobs = ln A −
Ea RT
where A is the frequency factor and is a constant, Ea the energy of activation, R the universal gas constant, and T is the absolute temperature. The kobs values were fitted using the equation as shown in Fig. 2. The relationship between ln kobs and 1/T was determined by linear regression and the goodness of fit for the line was R2 = 0.9783. The calculated Ea was 85 kJ mol−1 and A was 4.4 × 106 s−1 . Shelf life (T90% ) indicates the period of storage of a product without loss of potency. It is described as the time taken for 10% of the product to decompose at a given temperature. To determine the shelf life of Spantide II at 25 ◦ C, the following equation was used: 0.105 T90% = k25
Statistical analysis
The logarithmic concentration of Spantide II in the stability samples was plotted as a function of time. The observed reaction rate constant (ko ) was obtained from the slope of the natural logarithmic (ln) plot of Spantide II concentration (C) versus time (t) profiles by linear regression analysis using Graphpad Prizm® software (Graphpad Software Inc., San Diego, CA),
Fig. 2 – Arrhenius plot of Spantide II.
161
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
Fig. 3 – HPLC chromatogram of Spantide II degradation in aqueous ethanol at 60 ◦ C.
The ln kobs at 25 ◦ C for Spantide II was calculated and determined to be −19.06. From this result, shelf life of Spantide II at 25 ◦ C was determined to be 230 days. This indicates that Spantide II is stable at room temperature and pharmaceutical products of this compound can be stored at 4 ◦ C for an extended shelf life of the product. To determine the degradation mechanism of Spantide II, the degradation profiles were monitored by HPLC and LC-MS after exposure of the samples to 60 ◦ C. The degradation products were identified by analysis of the MS spectra corresponding to peaks observed in the LC chromatograms produced by LC-MS. The degradation of Spantide II in aqueous ethanol (50%), at 60 ◦ C yielded two degradation peaks P1 and P2 as depicted in Fig. 3. The degradation products occurring at these peaks are non-polar compared to Spantide II as they elute after the parent compound. Further examination of the MS spectra
associated with peak P1 confirms that it corresponds to degradation products of Spantide II with m/z ratios of 1339.8, 331.5 and 577.3. While MS spectrum associated with peak P2 corresponds to degradation product of Spantide II with m/z ratio of 1111.7. Table 1 shows the m/z ratios of the product occurring in peak P1 and P2 as determined by LC-MS. The fragments with m/z ratio of 331.5 and 1339.8 are associated with Spantide II (nicotinoyl)Lys1 -Pro2 diketopiperazine degradation that was elucidated in an earlier study (Kikwai et al., 2004). Diketopiperazine (DKP) occurs at the Nterminal of the peptide and forms a cyclic product that has two N-terminal amino acids. The mechanism of DKP formation involves nucleophilic attack of the N-terminal nitrogen on the amide carbonyl between the second and third amino acids. This intramolecular aminolysis reaction occurs readily in aqueous solution (Goolcharran and Borchardt, 1998). The degradation pathway of a model peptide l-Ala-l-Pro-l-Met (APM) under the influence of pH and temperature was studied by Goolcharran et al. (2000). This tripeptide APM underwent diketopiperazine formation (Ala-Pro-diketopiperazine). Similarly, the results of our study demonstrated the formation of DKP after cleavage at (nicotinoyl)Lys1 -Pro2 of Spantide II. The hydrolytic cleavage of Spantide II at the peptide bond between Pro4 -PheCl2 5 results in two products; fragment-A, of MW 593.7 after addition of a hydroxyl group (m/z = +17) and fragment-B, of 1093.1 after addition of a hydrogen. The m/z ratio corresponding to fragment-A was not observed in the MS spectrum therefore, it might have undergone further degradation. Further hydrolytic degradation products of fragment-A were not observed in the MS spectrum. Fragment-B with a MW of 1093.1 and an m/z ratio of 1094.7 was observed in both P1 and P2 . Resolution of this fragment at different retention times on the HPLC chromatogram indicates that this fragment underwent further racemization. Additionally Spantide II underwent hydrolytic cleavage at the peptide bond between Trp7 -Phe8 . The resulting products of MW 1110.1 after addition of a hydroxyl group (m/z = +17) and 576.3 after addition of hydrogen (m/z = +1) were observed in MS spectrum with m/z ratios of 1111.7 and 577.7 and occurred in P2 and P1 in the HPLC chromatogram, respectively. Csapa´ et al. (1997) demonstrated that protein and peptide molecules hydrolyzed at elevated temperatures and racemized if the exposure is extended over longer periods. The results indicate that in addition to DKP formation, hydrolytic and racemized degradation products were formed when Spantide II is exposed to elevated temperature conditions.
3.3.
Effect of pH
The degradation of Spantide II at 60 ◦ C in aqueous solutions over a range of pH values (1–10) in various buffers was
Table 1 – Degradation of Spantide II at 60 ◦ C in aqueous ethanol solution: m/z ratios of the degradation products Cleavage site Pro2 -(pyridyl)Ala3 Pro4 -PheCl2 5 Trp7 -Phe8
Fragment A theoretical mass
Fragment B theoretical mass
Fragment A M + H+ (m/z): (peak)
330.4 593.7 576.3
1338.7 1093.1 1110.1
331.5 (P1 ) – 577.3 (P1 )
Fragment B M + H+ (m/z): (peak) 1339.8 (P1 ) 1094.7 (P1 , P2 ) 1111.7 (P2 )
162
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
Fig. 4 – pH–log kobs profile of Spantide II at 60 ◦ C.
monitored by HPLC. The Spantide II plots of ln concentration versus time were linear at all pH’s indicating pseudo-first order degradation rate kinetics. The pseudo-first order rate constant (ko ) values were calculated from the slopes of linear plots. The observed reaction rate constant (kobs ) can be expressed as: kobs = ko + kOH [OH− ] + kH [H+ ] + kbuffer [buffer] where ko is the pseudo-first order rate constant for degradation in water only, while kOH , kH , and kbuffer are second order rate constants for the degradation catalyzed by hydrogen ions, hydroxide ions and buffer components respectively. The influence of pH on the stability of Spantide II is shown in Fig. 4. The data indicate that Spantide II is susceptible to degradation at pH <2.0 and pH >5. The rate of degradation was rapid at pH <2.0 indicating specific hydrogen-ioncatalysis of the positively charged Spantide II species. In the pH region (pH 2–5), the degradation rate plateaus and can be attributed to specific buffer-species-catalysis of the neutral species. The rate of degradation is rapid at pH >5, indicating specific hydroxide-ion-catalysis of the negatively charged species. Similar results were obtained for Aplidine, which showed optimal stability of the peptide in the pH region 2–6 under accelerated condition at 60 ◦ C and at pH below 2 and above 6 a clear proton-catalyzed and hydroxyl-catalyzed hydrolyses, respectively, were observed (Waterval et al., 2001).
Reubsaet et al. (1995) also reported a similar acid catalyzed degradation profile for antagonist G, which has a similar structure to Spantide II and identical amino acids at positions 7, 9, and 10. The degradation mechanism of Spantide II in acidic and alkaline media at 60 ◦ C was monitored by HPLC and LC-MS. The degradation products were identified by analysis of the MS spectra corresponding to peaks observed in the LC chromatograms produced by LC-MS. Degradation of the peptide backbone occurs by hydrolysis, in peptides exposed to both acidic and alkaline media (Manning et al., 1989; Oliyai et al., 1992). Fig. 5A and B shows chromatograms of Spantide II and its degradation products after exposure to acidic and alkaline media, respectively. This degradation was instantaneous and rapid as observed by the appearance of degradation products on the 0 h chromatograms in both acidic and alkaline conditions. In the acidic medium, Spantide II peak was still detectable at 24 h, although its height and intensity was significantly lower compared to 0 h. There were barely any traces of Spantide II seen in the chromatogram after 12 h of exposure to the alkaline medium. The degradation rate in acidic medium was slower than in alkaline medium. Consequently, there were more degradation peaks formed in the alkaline medium. These data were consistent with that published by Reubsaet et al. (1995) who observed high degradation rates and more degradation peaks in alkaline medium compared to acidic medium. In acidic medium, degradation products that occurred in peaks P4 –P6 were relatively non-polar as they eluted after the parent peak. MS spectral analysis of peaks P4 –P6 determine that Spantide II had undergone hydrolytic cleavage at different peptide bonds. The major hydrolytic cleavage sites on Spantide II in acidic medium, and their theoretical masses, peak occurrence and m/z ratios are shown in Table 2. These products seemed to racemize since they occurred at multiple retention times on the HPLC chromatograms but had identical m/z ratios. These results agree with data obtained for antagonist G where its hydrolytic degradation products racemized and were detected at different retention times on the HPLC (Reubsaet et al., 1999).
Fig. 5 – HPLC chromatograms of Spantide II degradation in acidic and alkaline medium at 60 ◦ C.
163
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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 (nicotinoyl)Lys1 -Pro2 Pro2 -(pyridyl)Ala3 Trp7 -Phe8 Phe8 -Trp9 Nle11 -NH2
Fragment A theoretical mass 251.1 348.1 1110.1 1257.2 1669.7
Fragment B theoretical mass 1436.5 1338.5 576.7 429.5 17.0
The degradation products that occurred at the HPLC peak P3 were relatively polar in nature compared to Spantide II as they eluted prior to the parent peak. MS spectral analysis of peak P3 identified products that resulted from the hydrolytic cleavage of Spantide II at (nicotinoyl) Lys1 -Pro2 , Pro2 -(pyridyl)Ala3 and Phe8 -Trp9 peptide bonds. Hydrolysis at (nicotinoyl)Lys1 -Pro2 produced a fragment-A with an m/z ratio of 251.1 (m/z = +17), and fragment-B with an m/z ratio of 1436.5 (m/z = +1) after addition of hydrogen. Fragment-A racemized and was detected in P5 and P6 in the HPLC chromatogram. The m/z ratio corresponding to fragment-B was not observed in the MS spectrum therefore, it might have undergone further degradation. Further hydrolytic degradation products of fragment-A were not observed in the MS spectrum. Hydrolytic cleavage at Pro2 -(pyridyl)Ala3 produced two degradation fragments. The fragment-A with m/z ratios of 349.2 (m/z = +17) occurred in the MS spectrum corresponding to HPLC peak P3 , while the fragment-B with m/z ratio of 1339.4, racemized and was identified in the MS spectrum corresponding to HPLC peaks P5 and P6 . Hydrolytic cleavage at Phe8 -Trp9 produced two degradation products, fragmentA with an m/z ratio of 1258.3 (m/z = +17) and fragment-B, with an m/z ratio of 430.1, which occurred in the HPLC peak P4 and P3 , respectively. The hydrolytic fragment-A at Trp7 Phe8 with an m/z ratio 1111.1 (m/z = +17) was detected in the MS spectrum corresponding to peak P4 . The corresponding fragment-B may have been further degraded, as it was not detected on the MS spectrum. The hydrolytic cleavage of the amidated Nle11 amino acid residue of Spantide II was detected in the MS spectrum corresponding to HPLC peak P4 . This fragment had an m/z ratio of 1670.5 (m/z = +17). Under highly acidic conditions (pH 1.0–2.0), Spantide II decomposed by hydrolysis at several cleavage sites and the resulting products were further racemized and detected at various retention times. Oliyai and Borchardt (1993) reported the degradation of Asp-hexapeptide under highly acidic condition was predominantly via intermolecular cleavage of the Asp-Gly amide bond forming a tetrapeptide and a dipeptide. The first four amino
Fragment A M + H+ (m/z): (peak) 252.3 (P5 , P6 ) 349.2 (P3 ) 1111.1 (P4 ) 1258.3 (P4 ) 1670.5 (P4 )
Fragment B M + H+ (m/z): (peak) – 1339.4 (P5 , P6 ) 577.6 (P5 ) 430.1 (P3 ) –
acids of Spantide II are not required for binding of Spantide II to neurokinin-1 receptors while the terminal amino acids are required for binding and receptor recognition. Therefore, hydrolytic cleavage of the terminal amino acid residues can lead to the deactivation of Spantide II antagonist activity and thus decreasing its anti-inflammatory activity (Ljungqvist et al., 1989). In alkaline medium, the degradation products are both polar and non-polar relative to Spantide II, because the peaks elute before and after the parent peak. Table 3 shows major hydrolytic degradation products of Spantide II in alkaline media. Analysis of the degradation products in alkaline medium revealed m/z ratios that were similar to those that occurred in acidic medium. The degradation products racemized and occurred at different retention times on the chromatograms, however, m/z ratios (m/z = +17) were identical as determined by MS. There is a correlation between hydrolytic degradation in acidic and alkaline medium as observed by degradation products resulting from cleavage at Pro2 -(pyridyl)Ala3 , Trp7 -Phe8 , and Nle11 -NH2 were observed in both MS spectra of acidic and alkaline conditions. It should be noted that from Table 3, three of the degradants, of Pro2 -(pyridyl)Ala3 , and Pro4 -Phe5 , Nle11 -NH2 in the alkaline medium were eluted along with the parent peak, making LCMS analysis more complex in alkaline medium. These results are consistent with the data published by Reubsaet et al. (1999), who examined tripeptide fragments of antagonist G a peptide with similar amino acid residues as Spantide II. The authors observed that the hydrolytic degradation product: Phe-Trp resulting from the carboxylic tripeptide, Phe-Trp-Arg-COOH was detected on MS of both acidic and alkaline media.
3.4.
Effect of oxidizing agent
Peptide oxidative products are relatively hydrophilic/polar and are expected to elute prior to the parent molecule (Reubsaet 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 Pro2 -(pyridyl)Ala3 (pyridyl)Ala3 -Pro4 Pro4 -Phe5 Trp7 -Phe8 Nle11 -NH2
Fragment A theoretical mass 348.1 496.6 593.7 1110.1 1669.7
Fragment B theoretical mass
Fragment A M + H+ (m/z): (peak)
Fragment B M + H+ (m/z): (peak)
1338.5 1190.2 1093.1 576.7 17.03
349.2 (P2 ) – – 1111.1 (P3 ) 1670.5 (all peaks)
1339.4 (P4 , P5 ) 1191.3 (Parent, P3 , P4 , P5 ) 1094.2 (P1 , P2 , Parent) 577.6 (P3 , P4 , P5 ) –
164
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
Fig. 6 – LC-MS chromatogram of Spantide II oxidation in hydrogen peroxide.
H2 O2 at 40 ◦ C. Spantide II degradation yielded polar oxidative products, which eluted before the parent peak. Products corresponding to oxidative peaks (OP1 to OP3 ) were examined for product identity by LC-MS. The schemes of oxidative degradation of Spantide II are shown in Fig. 7. In Fig. 7A, Pro2,4 undergoes possible ring opening and addition of an oxygen and hydrogen molecule thus increasing the overall mass of Spantide II. If one Pro ring opens, the mass will increase by +16 to 1684.8 while opening at both Pro residues the mass will increase by +32 to 1700.8. This increase in mass was observed in LC-MS studies that were conducted to further identify the oxidative degradation products of Spantide II. Table 4 shows the m/z ratios observed by LC-MS for Spantide II oxidation in H2 O2 . Pro2,4 ring opening was observed in LC peaks OP1 , OP2 , and OP2 . The scheme of oxidative degradation of Pro2,4 by peptide bond cleavage is shown in Fig. 7B. Pro2,4 can undergo cleavage of the peptide bonds at Pro2 -Ala3 and/or at Pro4 -PheCl2 5 , resulting in the addition of oxygen and expulsion of carbon dioxide. Based on LC-MS evaluation we found that cleavage of Pro at Pro2 -Ala3 did not occur because there was no evidence
of m/z ratio of matching mass 1340.8 or 349.3 in the MS spectra of all degradation peaks. However, cleavage at Pro4 -PheCl2 5 was identified by the m/z ratio of a fragment with a mass of 1095.6, which corresponds to (PheCl2 5 -Nle11 )-Spantide II. The corresponding m/z ratios for Pro4 cleavage observed in the LC peak OP3 are seen in Table 4. Oxidation of the Phe5,8 residues on Spantide II is a possible mechanism of degradation in the presence of H2 O2 leading to 2-hydroxyphenylalanine (Wang, 1999). The scheme of oxidative degradation as can be seen in Fig. 7C, indicates the addition of OH group at position 2 of the aromatic rings of PheCl2 5 or Phe8 , resulting in a mass increase of 16 (m/z = +16) (Table 4). Addition of the hydroxyl group at both PheCl2 5 and Phe8 results in a mass increase of 32 (m/z = +32). The oxidative product 2-hydroxyphenylalanine is more hydrophilic/polar than Spantide II and eluted prior to the parent peak. In this study, we focused on identification of the major peaks from the LC chromatogram. Several small peaks in Fig. 7 were probably the oxidative products from Spantide II, but the corresponding m/z values in LC-MS were not detectible due to very low intensity of these products (due to noise interference of MS spectrum with the m/z values). Oxidation could dominate other degradation pathways in a protein or peptide (Wang, 1999). There are several potential oxidative degradation reactions in hIGF-I (Fransson et al., 1996). Similarly, oxidation is the major degradation pathway for KGF-2 (Kaushal et al., 1998). The results of the present study indicate no signs of oxidative degradation of Spantide II in aqueous solution under various pH and temperature conditions, but under forced degradation conditions (H2 O2 ) we found several oxidative products indicating possible amino acid residues that could undergo oxidative degradation under extreme conditions. In conclusion, the study of the degradation profiles of Spantide II in aqueous solution was successfully accomplished using HPLC and LC-MS techniques. Spantide II is susceptible to degradation at pH <2.0 and pH >5 while maximal stability was observed between pH 2–5. Spantide II degrades by Lys-Pro diketopiperazine, hydrolysis and racemization in both acidic and alkaline media, and by oxidation of Pro and Phe amino acid residues as assessed by LC-MS analysis. This study further demonstrates that Spantide II is stable in aqueous ethanol (50%, v/v) with a shelf life (T90% ) of 230 days at 25 ◦ C. The longterm objective of the study is to develop a topical formulation of Spantide II for the treatment of inflammatory skin disorders. We demonstrated the topical anti-inflammatory activity of this compound in allergic and irritant contact dermatitis animal models. We are currently investigating the effect
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 Spantide II Pro2 ring opening Pro2,4 ring opening Pro4 bond cleavage Phe5 or 8 oxidation Phe5 and 8 oxidation
Theoretical mass
M + H+ (m/z)
M + 2H+ (m/z)
1668.8 1684.8 1700.8 1094.8 1684.8 1700.8
1669.0 1685.0 1701.0 1095.6 1685.1 1701.0
835.6 843.6 851.6 548.8 843.6 851.6
Peak Parent OP1 , OP3 OP2 , OP3 OP3 OP1 , OP3 OP2 , OP3
european journal of pharmaceutical sciences
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Fig. 7 – Oxidative pathways of Spantide II.
of formulation variables on the release and skin permeation kinetics of gel formulation of Spantide II as a topical antiinflammatory agent.
Acknowledgments The authors acknowledge the financial assistance provided by NIAMS (NIH) grant number AR47455-02 and RCMI (NIH) grant number G12RR03020.
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