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 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.

1. Introduction ment of inflammatory skin disorders such as psoriasis and

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) recep-
tors 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-
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,

∗
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
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 var-
ious 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 preformu-
lation stability of Spantide II as a function of pH, tempera-
ture, 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 character-
ized. The characterization of peptide degradation is of impor-
tance, 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 aque-
ous 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 anal-
ysis was done by LC-MS to characterize the degradation prod-
ucts. The stability of Spantide II as a function of pH and tem-
perature 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, hydrochlo-
ric acid, potassium biphthalate, sodium hydroxide, hydrogen
peroxide, trifluoroacetic acid (TFA) and boric acid were pro-
cured 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
159
grade) were purchased from Fisher Scientific (Atlanta, GA,
USA). All other chemicals were standard reagent grade.
2.2. High-performance liquid chromatography (HPLC)
assay
A HPLC system (Waters Corporation) along with a Vydac
reverse phase C18 (300 Å pore size silica) analytical column
(5 ␮m, 4.6 mm × 250 mm) were used for the analysis of Span-
tide 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 sam-
ples 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 Mod-
ule 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-
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
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 Time-
of-Flight Mass Spectrometer (Model JMS-T100LC, JEOL Inc.,
Peabody, MA, USA). The instrument was interfaced with a pos-
itive 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 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
2.4. 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 sol-
ubility 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 experimen-
tal 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 trip-
licate. 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 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 deter-
mined 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. 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 phtha-
late 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 Span-
tide 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 sub-
jected 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 Span-
tide 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. Statistical analysis
The logarithmic concentration of Spantide II in the stability
samples was plotted as a function of time. The observed reac-
tion rate constant (ko ) was obtained from the slope of the natu-
ral logarithmic (ln) plot of Spantide II concentration (C) versus
time (t) profiles by linear regression analysis using Graph-
pad Prizm® software (Graphpad Software Inc., San Diego, CA),
according to equation:
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 degrada-
tion 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.
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 con-
centration of Spantide II decreased to 30% of the initial con-
centration, which represented a decline of two half lives. The
influence of temperature on the observed reaction rate con-
stant (kobs ) is given by the natural logarithmic form (ln) of the
Arrhenius equation:
Ea
ln kobs = ln A −
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 good-
ness 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 decom-
pose at a given temperature. To determine the shelf life of
Spantide II at 25 ◦ C, the following equation was used:
0.105
T90% =
k25
Fig. 2 – Arrhenius plot of Spantide II.
 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 deter-
mined 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 pharmaceuti-
cal 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 prod-
ucts were identified by analysis of the MS spectra correspond-
ing 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
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
theoretical mass theoretical mass
Pro2 -(pyridyl)Ala3 330.4 1338.7
Pro4 -PheCl2 5 593.7 1093.1
Trp7 -Phe8 576.3 1110.1
161
associated with peak P1 confirms that it corresponds to degra-
dation products of Spantide II with m/z ratios of 1339.8, 331.5
and 577.3. While MS spectrum associated with peak P2 corre-
sponds 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 diketopiper-
azine degradation that was elucidated in an earlier study
(Kikwai et al., 2004). Diketopiperazine (DKP) occurs at the N-
terminal of the peptide and forms a cyclic product that has
two N-terminal amino acids. The mechanism of DKP forma-
tion 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). Simi-
larly, 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 degrada-
tion. 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 frag-
ment 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. Csapá 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 for-
mation, 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 solu-
tions over a range of pH values (1–10) in various buffers was
Fragment A M + H+ Fragment B M + H+
(m/z): (peak) (m/z): (peak)
331.5 (P1 ) 1339.8 (P1 )
– 1094.7 (P1 , P2 )
577.3 (P1 ) 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 degrada-
tion 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 degrada-
tion was rapid at pH <2.0 indicating specific hydrogen-ion-
catalysis 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, indicat-
ing 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).
Fig. 5 – HPLC chromatograms of Spantide II degradation in acidic and alkaline medium at 60 ◦ C.
Reubsaet et al. (1995) also reported a similar acid catalyzed
degradation profile for antagonist G, which has a similar struc-
ture 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 chro-
matograms 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 alka-
line media, respectively. This degradation was instantaneous
and rapid as observed by the appearance of degradation prod-
ucts 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 sig-
nificantly lower compared to 0 h. There were barely any traces
of Spantide II seen in the chromatogram after 12 h of expo-
sure 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 Span-
tide 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 reten-
tion 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).
 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 ) 158–166 163

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)

(nicotinoyl)Lys1 -Pro2 251.1 1436.5 252.3 (P5 , P6 ) –

Pro2 -(pyridyl)Ala3 348.1 1338.5 349.2 (P3 ) 1339.4 (P5 , P6 )
Trp7 -Phe8 1110.1 576.7 1111.1 (P4 ) 577.6 (P5 )
Phe8 -Trp9 1257.2 429.5 1258.3 (P4 ) 430.1 (P3 )
Nle11 -NH2 1669.7 17.0 1670.5 (P4 ) –

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 anal-
ysis 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 chro-
matogram. The m/z ratio corresponding to fragment-B was
not observed in the MS spectrum therefore, it might have
undergone further degradation. Further hydrolytic degrada-
tion 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 correspond-
ing 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, fragment-
A 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 correspond-
ing 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 prod-
ucts were further racemized and detected at various retention
times. Oliyai and Borchardt (1993) reported the degradation of
Asp-hexapeptide under highly acidic condition was predomi-
nantly via intermolecular cleavage of the Asp-Gly amide bond
forming a tetrapeptide and a dipeptide. The first four amino
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 alka-
line media. Analysis of the degradation products in alka-
line 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 iden-
tical 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 LC-
MS 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 degrada-
tion 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 Fragment A Fragment B Fragment A M + H+ Fragment B M + H+
theoretical mass theoretical mass (m/z): (peak) (m/z): (peak)

Pro2 -(pyridyl)Ala3 348.1 1338.5 349.2 (P2 ) 1339.4 (P4 , P5 )

(pyridyl)Ala3 -Pro4 496.6 1190.2 – 1191.3 (Parent, P3 , P4 , P5 )
Pro4 -Phe5 593.7 1093.1 – 1094.2 (P1 , P2 , Parent)
Trp7 -Phe8 1110.1 576.7 1111.1 (P3 ) 577.6 (P3 , P4 , P5 )
Nle11 -NH2 1669.7 17.03 1670.5 (all peaks) –
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 cor-
responding to oxidative peaks (OP1 to OP3 ) were examined for
product identity by LC-MS. The schemes of oxidative degra-
dation 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 cleav-
age 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
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
Spantide II 1668.8
Pro2 ring opening 1684.8
Pro2,4 ring opening 1700.8
Pro4 bond cleavage 1094.8
Phe5 or 8 oxidation 1684.8
Phe5 and 8 oxidation 1700.8
of m/z ratio of matching mass 1340.8 or 349.3 in the MS spec-
tra 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 oxida-
tive degradation as can be seen in Fig. 7C, indicates the addi-
tion 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 correspond-
ing 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 poten-
tial 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 con-
ditions, 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 Span-
tide 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 fur-
ther demonstrates that Spantide II is stable in aqueous ethanol
(50%, v/v) with a shelf life (T90% ) of 230 days at 25 ◦ C. The long-
term objective of the study is to develop a topical formulation
of Spantide II for the treatment of inflammatory skin disor-
ders. We demonstrated the topical anti-inflammatory activity
of this compound in allergic and irritant contact dermati-
tis animal models. We are currently investigating the effect
M + H+ (m/z) M + 2H+ (m/z) Peak
1669.0 835.6 Parent
1685.0 843.6 OP1 , OP3
1701.0 851.6 OP2 , OP3
1095.6 548.8 OP3
1685.1 843.6 OP1 , OP3
1701.0 851.6 OP2 , OP3
 european journal of pharmaceutical sciences
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 anti-
inflammatory 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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