Proteins, peptides and polymers -...
Transcript of Proteins, peptides and polymers -...
er Proteins, peptides and polymers
Chapter2
2.1 PROTEIN AND PEPTIDE DRUGS
2.1.1 Bovine Serum albumin
Bovine serum albumin (BSA) is the most abundant protein in the circulatory system
and contributes 80% to colloid osmotic blood pressure (Carter eta!, 1994). Serum
albumin is mainly responsible for maintenance of blood pH. It is a highly water
soluble (>50% w/v in water at pH 7) non-glyoprotein and has been used as a model
protein in numerous studies. It is characterized by a single polypeptide chain and
consists of 5 83 amino acids with a molecular weight of 66kDa. It has 17 disulphide
bridges and 1 free cysteine in position 34. The high solubility of BSA is mainly due to
its high total charge. BSA also contains 48 aromatic amino acid residues (2 Trp, 19
Tyr and 27 Phe). The fluorescence of BSA is mainly from its Trp residues. Serum
albumin undergoes reversible conformational isomerization with changes in pH
(Peters eta!, 1996).
It possess following properties:
1. Solubility in aqueous phase
2. Biocompatible and biodegradable
3. Stable at working condition
4. Molecular weight, amino acid and structure is fully defined
5. Simple in nature
6. Non-hygroscopic
2.1.1.1 Physicochemical properties
BSA is commonly used as a model protein surrogate for costlier protein due to its
characteristic properties (Table 2T-1 ).
Table 2T-1. Physicochemical properties ofBSA
Description White to light tan colour powder that contains 96% pure protein and 3%w/w ofwater(BP' 1988)
Molecular wei2ht 69,000 I so-electric point 4.7 Structure Structure ofBSA consist of polypeptide chain with non-unifonn four globular
segments Stabilitv Disulphide linkage (S-S) at 17"' position Solubility 5%w/v is easily soluble at room temperature within 10 minutes
Denaturation of BSA Bv heat or at higher ammonium sulphate concentration Storage Protection from light and moisture at temperature between 2 and 25°C
• Development end Cherac:terlxatlon of Novel Delivery Systems lor Proteins end Peptldes • 68
Chapter2
2.1.1.2ldentification test
BSA is identified by various colour reactions which could be used as identification
tests for BSA.
a) Modified Millons test
BSA solution is boiled with I 0% w/v mercuric sulphate in 10% w/v sulphuric acid
and gives yellow precipitate.
b) Biurette test
Reaction with alkaline CuS04 solution (1 0% CuS04 in O.SN NaOH), protein gives
violet colour.
c) Ninhydrin test
When protein is treated with ninhydrin reagent it gives violet or purple colours that
show presence of protein.
2.1.1.3 Analytical techniques
Various methods have been reported for estimation ofBSA.
2.1.1.3.1 Chromatographic methods
Various methods have been reported in literature for the estimation of BSA. These
methods are based on various principle i.e. UV spectroscopy, colorimetry,
chromatography, chromatography electrophoresis and volumetric analysis.
Liquid chromatographic method was utilized initially (Peters, 1996) for the
determination of Albumin in normal saline solution using column of spherule TSK
2000 with a mobile phase sodium phosphate disodium EDT A salt and
mercaptoethanol and finally spectroscopic determination was done at 300 nm.
The HPLC method for the estimation of BSA was reported using N-methyl
pyridinium polymer crosslinked with ethylene glycol dimethylacrylate column
(25cm X 4 em). The mobile phase used was 0.5 M NaiCo in 0.05 M tris HCL buffer
(pH7) and absorbance was measured at 280 nm (Nishimura eta!., 1990) .
• Development and Charactertzallon of Novel Delivery Systems for Proteins and Peptldes • 69
Chapter2
The reverse phase chromatography method was utilized for the estimation of serum
albumin. The used column (5crnX4.6cm) of spherisorb RPC6 (J.lm) with 0.067 M
potassium phosphate buffer of pH 7.4 Using acetonitrile as mobile phase and
absorbance was measured at 280 run (Nishimura et al., 1990).
2.1.1.3.2 Spectrophotometric Method
A colorimetric method described by Lowry et al. (1951) for the estimation of protein
based on developed blue colour by the reaction of Folin-ciocalteau reagent and
alkaline copper sulphate reagent.
A simple rapid and inexpensive colorimetric method given by Bradford et al. (1976)
that is based on the ability of protein to bind with coomassie blue G-250 dye and
forms a complex that have extinction coefficient much greater than that of free dye.
A simple UV spectrophotometric method described by Lowry (1951) is based on
scanning the BSA solution in PBS (pH 7.4). Protein estimation is done by measuring
absorbance at 280 nm and PBS (pH 7.4) is taken as blank.
Another very simple and accurate method was presented by Kang et al. (1996) using
bromocresol purple reagent or bromocresol green reagent and by measuring
absorbance of mixture at 603 run and 628 run respectively. Protein follows Beer
Lambert law in the range of I 0-100 J.lg/ml.
A very sensitive and accurate method presented by Smith et al. (1985) for protein
estimation was BCA protein assay. This assay uses (Bicinchoninic acid) BCA so
detect the cuprous ions generated from cupric ions by reaction with protein under
alkaline conditions. The BCA-cuprous ion complex is a relatively stable chromophore
absorbing at 562 nm. The analysis has a working range of 1-2000 J.lg/ml.
2.1.1.3.3 Miscellaneous Method
Microstimation of albumin was reported by Motonaka et al. (1988) which was based
on reaction on reaction with iodine. BSA sample solution was rreated with methanolic
iodine and IN H2S04 and titrated potentiometrically with 0.5-20 mM silver nitrate.
A very sensitive method used for the estimation of protein developed using colloidal
gold solution. Colloidal gold solution, acidified to pH 3.0 with 60% acetic acid and
• Development and Characterization of Novel Delivery Systems tor Proteins and Peptldes • 70
Chapter 2
stabilized with polysorbate 20, was added to protein aqueous solution and incubated
for I 0 mint.~tes at room temperature. The absorbance was measured at 615 nm against
water.
An electrophoresis method developed by Wang et a!. (2007) used for estimation of
proteins using SDS-PAGE (Sodium dodecyl sulphate-polyacrylamide gel
electrophoresis). After electrophoresis the separated fraction was visualized by
staining with Zncl3 or CaCI3 solution.
Another electrophoresis method developed by Lee eta!. (1991) was used for specific
protein visualization in SDS-PAGE with iodine staining.
2.1.2 Serratiopeptidase
Serratiopeptidase (STP) is an endopeptidase. It is a stronger caseinolytic agent than
any other known alkaline or neutral protease. This powerful proteolytic enzyme is
obtained from silkworms. It is also known as serrapeptidase, Serratia peptidase, or
serrapeptase. The enzyme is obtained from microorganism Serratia E 15 and HY-6,
which live in the gut wall of the silkworm (Yamazaki et a!, 1967). It plays a crucial
role in morphological transformation of silkworms. It helps in the proteolysis of
cocoons and emergence of the moth. It has anti-inflammatory, analgesic, and
proteolytic activity with molecular weight of about 60 K Dalton (Sweetman et al,
2002a; 2002b).
2.1.2.1 Proprietary Names
Aniflazime, Aniflazym, Bidanzen, Brasan, Cipzen, Dailat, Danzen, Danzen (FM),
Danzyme, Dasen, Dazen, Denzo, Enziflur, Eze, Flanzen (FM), Infladase, Kineto,
Korzen, Lergan, Medizyme (FM), Podase, Rodase (FM), Septirose (FM), Seraim,
Seramed, Sera to-M, Serradase, Serrano, Serrao, Serra pep, Serrason, Serrazyme,
Serrin, Sinsia, Sumidin, Unizen, Unizen (FM), Verolin
2.1.2.2 Prod11ction
Serratiopeptidase is naturally processed commercially through fermentation from the
culture of the Serratia marcescens. The controlled fermentation of Serratia sp.
secretes this enzyme in the highly selective medium. The recovery process involves
various types of filtration. concentration and steps to make enzyme useful for
• Development and Characterization of Novel Dellvery Systems for Proteins and Peptldes • 71
· Chapter2
pharmaceutical applications and finally dried to fine free flowing powder form
(Tanimoto eta!, 1983; Scull, 1997; Aiyappaet a!, 1976).
2.1.2.3 Mechanism of action
It binds to alpha-2-macroglobulin in the blood in ratio of I: I which helps to mask its
antigenicity but retain its enzymatic activity. Levels of serratiopeptidase are slowly
transferred to the exudates at the site of inflammation and gradually the blood level
declines (Marly, 1985; Odagiri, 1979).
It reduces inflammation in three ways (Vicari et al, 2005; Esch et al, 1989):
I. It breaks down the insoluble protein by-products of blood coagulation
known as fibrin.
2. It thins the fluids formed from inflammation and injury as well as
facilitating their drainage that speeds the tissue repair process.
3. It alleviates pain by inhibiting the release of specific pain-inducing amines
called bradykinin.
Analgesic effect of proteolytic enzymes is due to their cleavage of bradykinin, a
messenger molecule involved in pain signaling (Klein eta!, 2000).
2.1.2.4 Pharmacokinetics
Serratiopeptidase is an acid labile enzyme, so when consumed in unprotected form is
destroyed by acid in the stomach. However, enterically coated tablets enable the
enzyme to pass through the stomach unchanged, and are absorbed in the intestine. It is
found in negligible amounts in the urine, suggesting that it is transported directly from
the intestine into the bloodstream (Miyata, 1980; Moriya et a!, 1994). Its optimum pH
is 8.5-9.5 and optimum temperature is 40°C (Stable at 40°C but rapidly losses activity
at 60°C in 10 minutes).
2.1.2.5 Clinical uses
Clinical studies have shown that serrapeptase induces fibrinolytic, anti-inflammatory
and anti-edemic (prevents swelling and fluid retention) activity in a number of tissues.
It is used for the treatment of arthritis, synovitis, and several other inflammatory
conditions of muscle and bones (Selan et al, 1993). Its anti-inflammatory effects have
been found to be superior to other proteolytic enzymes (Mazzone ct al, 1990).
·Development and Characterization ol Novel Delivery Systems tor Proteins and Peptldes • 72
Chapter2
Japanese patents even suggest that oral serrapeptase may help treat or prevent viral
diseases such as AIDS and hepatitis B and C (Mazzone et a!, 1990; Marly, 1985). But
perhaps it's most spectacular application is in reversing cardiovascular disease
(Marly, 1985).
In fact, serrapeptase appears so effective in unblocking carotid arteries that one
researcher-Dr. Hans Nieper, the late, eminent internist from Hannover, Germany
called it a "miracle" enzyme(Table 4T-2) (Odagiri, 1979).
2.1.2.6 Dosage
I Omg three times per day on an empty stomach
2.1.2. 7 Interactions
SIP is inhibited by Ni++, Mg++, Cd++, Cu++ and EDT A. However, activities are
regained by addition of Zn++, Mn++ and Co++. Concomitant use of drug with an
anticoagulant may intensify the anticoagulant effect (Miyata, 1980; Selan eta!, 1993).
2.1.2.8 Contraindications
It is contraindicated in patients with blood coagulation disorder, severe hepatic/renal
disorders and hypersensitivity (Miyata, 1980).
Table 2T-2. Clinical significance of serratiopeptidase
s. Clinical use Symptoms Remarks Symptoms treated Effects Ref No I. Cystic breast Breast More than 88% Reduction in breast No adverse Kee et al,
disease engorgement persons pain, swelling and reactions 1989 reported marked induration reported improvement
2. Sinusitis/bronc Hypersecretion More than 97% Reduction in the Effective in Kee et al, hi tis of thick mucus persons viscosity of the laryngitis, 1989
reported marked mucus improving catarrhal improvement the elimination of rhinophary-
bronchopulmon-ary ngitis and secretions sinusitis
3. Microbial Biofilm- More than 87% significant Effective in Perna, infections embedded treated group improvement in perennial 1985
bacteria reported marked rhinorrhea, nasal rhinitis, improvement stuffiness, coryza chronic
and paranasal sinus rhinitis with shadows sinusitis or
chronic relapsing bronchitis
• Development and Characterization o1 Novel Delivery Systems tor Proteins and Peptldes • 73
Chapter2
4. Carpal Tunnel Musculoligam Sixty five Improvement in No adverse Panagariy syndrome entous strain of percent of the pain and reactions a et al,
the hand and patients showed inflammation reported 1999 wrist clinical
improvement 5. Arteriosclerosis Partial or Significantly Improvement in Due to Panagariy
complete effective blood flow through protein· a et al, blockage of the an artery dissolving 1999 blood flow action of through an serrapeptase artery
6. Periodontal Periodontitis better relief than Serratiopeptida-se Effective in Maheshwa disorders antibiotic alone improves scaling in ri et al,
microcirculatio-n root planning 2006 and reduces pain
7. Obstetrics Post-partum Significantly Reduction in pain Resolution Maheshwa haematomas, effective and swelling due to anti· ri et al, breast inflammatory 2006 engorgements activity and pregnancy related thrombophlebit is
2.1.2.9 Adverse drug reactions
Hypersensitivity: infrequently hypersensitivity reaction such as rash and redness may
occur.
Digestive: diarrohea, anorexia, gastric discomfort, nausea or vomiting.
Hemolysis: rarely bleeding tendency such as epistasis and blood sputum may occur.
A case of pneumonitis and subepidermal bullous dermatosis due to serrapeptase was
also reported (Nihon, 1989; Shimizu, 1999).
2.1.2.1 0 Analytical profile
STP is not listed in any pharmacopoeia till date. The literature survey reveals a
crescent number of publications related to STP determination. Tomoda et a! (I 972)
developed a highly specific and sensitive radioimmunossay (RIA) for the
determination of STP (Tomoda et a!, 1972). RIA was based upon competition of
protease with 125-I labelled protease for anti-protease, followed by antibody to
separate bound enzyme from free enzyme. For tablet analysis, there was a
chromatographic method reported (Garcia eta!, 2004). Also, capillary electrophoresis
was applied for tablets in aqueous media (Garcia et al, 2005) and for bulk substance in
nonaqueous media (Tivcsten et a!, 1999). HPLC (Choulis et a!, 1989) and steric
~Development and Choractertzatlon of Novel Dellvery Systems for Proteins and Peptldes • 74
Chapter2
exclusion chromatography (ljitsu et a!, 1986) have also been reported for the
estimation of STP. It can be estimated in biological fluids by HPLC. The column
required is Lichrosorb-CN-10-and the mobile phase is CH30H/ CH3C00Na (4:6)
buffer. Flow rate is I ml/min. at 800 psi and the system sensitivity was 0.02 with
A.max at 278nm (Choulis et a!, 1989). STP can also be estimated in terms of
proteolytic activity by flurometric method. Fluorescence is developed by using
fluorescein isothiocyanate (FITC)-labeled casein measured at an excitation
wavelength of 490nm and emission wavelength of 529 nm. The enzyme can be
measured in nanogram and sub nanogram range using the assay (Twining, 1984). It
can be estimated as protein by using Bicinchonic acid (BCA) protein assay method.
BCA, sodium salt is stable, water soluble compound capable of forming an intense
purple color complex with cuprous ion in an alkaline environment. The color
produced from this reaction is stable and increases in proportional fashion over a
broad range of increasing protein concentration (Smith et al, 1985).
2.1.3 Enalapril Maleate
Enalapril Maleate is an antihypertensive drug with ACE inhibitor activity available as
white to nearly white, hygroscopic crystalline powder. Chemically it is (S)-l-[N-[1-
(Ethoxycarbonyl)-3-phenylpropyl]-L-alanyl]-L-proline. Enalapril Maleate contains
not less than 98.0 percent and not more than I 02.0 percent of C20H28N20 5·C4H404,
calculated on the dried basis (Galichet, 2003).
Fig 2F-1. Structure ofEnalapril Maleate
O HOOC, •
~~() 0
2.1.3.1 Proprietary Names
Ampracc; Bitensil; Cardiovet; Enacard; Enaloc; Enapren; Glioten; Hipoartel;
lnnovace; Lotrial; Olivin; Pres; Renitcc; Renitcn; Rcnivace; Yasotec; Xanef.
-Development and Characterization ol Novel Delivery Systems for Proteins and PepHdes-75
Chapter2
2.1.3.2 Physicochemical Properties
Its melting point range is 148° to !51 °.with dissociation constant value ofpKa2.97
and 5.35 (25°) (maleate). Partition Coefficient is Log P(octanol!buffer pH 7.4)- 2.45
(Florey, 2005).
2.1.3.3 Pharmacodynamics
Competitively inhibits angiotensin !-converting enzyme, preventing conversion of
angiotensin I to angiotensin II, a potent vasoconstrictor. Clinical consequences
include decreased sodium and fluid retention, decreased BP, and increased dieresis
(Fig 2F-2) (Colson eta!, 1999).
Fig 2F -2. Mechanism of blood pressure regulation
Angiotensinogen
(Plasmaa2 Renin
~· (J.G. cells)
Angiotensin- I
( decapeptide)
... -........ ·-
l Converting enzyme/ Kininase II
Angiotensin- II
Aminopeptidase
Angiotensin- Ill
-. •.
Low macula densa Na + Na+ loss
Low glomerular afferent pressure
~-~·c·;;:;--;;:;.c~~ Fall in blood
volume
- • _ Inhibiilol\ -·-. Increased blood volume
·- -. -.
Rise in BP
Vasoconstriction
-Na & water retention
(Kidney)
i !heotaoeotide) f---------- Aldosterone secretion
Angiotensinases (Adrenal cortex)
Inactive fragments
• Development ond Choroctertzatlon of Novel Oellvery Systems for Proteins and Peptldes • 76
Chapler2
2.1.3.4 Pharmacokinetics
After oral administration, 60% of a dose is absorbed, and rapidly and extensively
hydrolysed in the liver to enalaprilat. Peak plasma concentrations of enalaprilat are
achieved 3 to 4 h after oral dose. Enalapril is excreted in urine as the metabolite,
enalaprilat, and the rest in faeces as the unchanged drug. After a single oral dose of
20 mg enalapril, enalapril and enalaprilat can be detected in breast milk with a
concentration of 1 to 2.3 )lg/L for the latter. Enalaprilat is removed by haemodialysis
and peritoneal dialysis. Oral bioavailability, 53 to 74% (enalapril).
2.1.3.5 Therapeutic concentration.
The serum therapeutic concentration range is 0.01 to 0.05 mg/L for the metabolite,
desethylenalapril (Galichet, 2003).
2.1.3. 6 Toxicity
Treatment with enalapril can result in renal failure, with the possibility of death.
Severe hypotension is the main toxic effect and loss of hearing has also been reported
by some.
2.1.3.7 Half-life
Elimination half-life is approx. 2 h for enalapril.
2.1.3.8 Indications
Treatment of hypertension, symptomatic CHF and asymptomatic left ventricular
dysfunction after myocardial infarction. Treatment of diabetic nephropathy, childhood
hypertension, and hypertension related to scleroderma renal crisis.
2.1.3.9 Therapeutic Dosage
Dose varies according to the age and condition of the patient. In case of hypertension
initial dose is 2.5 to 5 mg/day. In renal function impairment an initial dosage of 5
mg/day; in normal renal function and mild impairment 2.5 mg/day; in moderate-to
severe renal impairment 2.5 mg on the day of dialysis in dialysis patients (adjust
dosage on non-dialysis days based on blood pressure response). In heart failure, 2.5
mg bid is given. Hypertensive patients at risk (eg, those with heart failure,
• Development and Choroctertzatlon of Novel Dellvery Sysfems for Proteins and Peptldes • 77
Chapter2
hyponatremia, high-dose diuretic therapy, recent intensive diureses or increase in
diuretic dose, renal dialysis, or severe volume or salt depletion of any etiology) have
potential for extremely hypotensive response. The starting dose should be 0.625 mg
administered IV over a period of 5 minutes and preferably longer (up to 1 hour) and in
Left Ventricular Dysfunction dose is 2.5 mg bid (Alfonso et al, 2000).
2.1.3.10 Contraindications
Metronidazole is not used in persons with history of hypersensitivity to nitroimidazole
derivatives or metronidazole preparation. It is contraindicated in trichonosomiasis in
the first trimester of pregnancy. Avoid use during breast-feeding because
metronidazole is excreted in breast milk. Nursing should be discontinued during
therapy and for two day following metronidazole therapy. It should be used with
caution in cases with CNS diseases and should be discontinued immediately if
abnormal neurological signs develop during treatment (USP'2003; Maryadele, 2001).
It should not be used in pregnancy especially during Category D (second, third
trimester); Category C (first trimester). It is also excreted in breast milk. It should be
used with caution in patients with history of angioedema. Neutropenia and
agranulocytosis might occur and risk appears greater with renal dysfunction, heart
failure or immunosuppression.
2.1.3.11 Drug Interactions and/or Related Problems
Greater risk of hypersensitivity and toxicity possible with co-administration with
allopurinol, capsaicin, digoxin, lithium, Phenothiazine, Potassium preparations and
potassium-sparing diuretics. Enalapril bioavailability may be decreased in presence of
antacids. Hypotensive effects may be reduced in presence of Indomethacin and
rifampin.
2.1.3.12 Side/Adverse Effects
CV: Chest pain; myocardial infarction; hypotension; angina; orthostatic hypotension;
tachycardia; syncope; vasculitis. CNS: Headache; vertigo; dizziness; fatigue; asthenia.
DERM: Rash; photosensitivity. GI: Nausea; abdominal pain; vomiting; diarrhea.
Urinary tract infection. HEMA: Decreased hemoglobin and hematocrit; neutropenia;
agranulocytosis; thrombocytopenia; pancytopenia; eosinophilia. META:
-Development and Charactertzcrtlon ot Novel Dellvery Systems for Proteins and Peptldes-78
Chapter 2
Hyperkalemia. RESP: Bronchitis; continuing cough; dyspnea. OTHER: Fever;
myalgia; arthralgia; arthritis.
2.1.3.13 Analytical Methods
a) Colour Test.
Bromothymol blue (acidic aqueous, pH 2: CHC!3)-yellow.
b) Thin-layer Chromatography.
Plate: silica gel (Analtech GF, Whatman KLF orE Merck G60). Mobile phase:
chloroform: methanol: acetic acid (90: 10:1 ). Rf 0.6;
Plate: silica gel (Analtech GF, Whatman KLF orE Merck G60). Mobile phase: n
butanol: water: acetic acid (3:1:1). Rf0.7
c) Gas Chromatography
Column: silica (DB-1, 10m x 0.25 mrn i.d., 0.25 !JlU). Column temperature: 150°,
held I min, and increased to 280° at 30°/min. Injector temperature: 280°. Carrier gas:
helium. MS detection (NICI, SIM (selected-ion monitoring) at m/z 302, Reference
compound: RS-5139. Retention time: enalapril4.58 min (Wang eta!, 2007).
d) High Performance Liquid Chromatography (Schmitt et al, 1997).
I) Column: Hypersil ODS (250 x 4.5 mrn i.d., 5 J.Lm). Mobile phase: sodium
heptanesulfonate (20 mM, pH 2.5): acetonitrile (5% THF) (63:37), 1.0 mL!min flow
rate. UV detection (A.=215 nm). Retention time: 10 min.
2) Column: Hypersil CIS (250 x 4.6 mrn i.d., 12 J.Lm). Mobile phase:
acetonitrile:water (20:80), 1.0 mL/min flow rate. UV detection (A.=215 nm). Retention
time: 1.9 min (Hejazi eta!, 2003).
e) Ultraviolet Spectrum.
Aqueous acid (0.2 M H2S04)--257, 268 nm; (0.1 M phosphate buffer, pH 4.5:
methanol (80:20)--229 nm (enalapril maleate).
f) Infra-red Spectrum .
• Development and Choroctertzotlon of Novel OeDvery Systems lor Proteins and Peptldes-79
Chapter2
Principal peaks at wavenumbers 1750, 1645, 1425, 1390, 1187 cm-1 (maleate).
g) Mass Spectrum.
Principal ions at m/z 91, 70,208,254, 117, 56, 160, 54 (maleate) (Galichet, 2003;
Florey, 2005).
2.2 POLYMERS
2.2.1 Chitosan
Chitosan is a linear polysaccharide composed of randomly distributed ~-(1-4)-1inked
D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit).
Chitosan is structurally similar to glycosaminoglycans with a chemical formula of
(C6Hll04N)n (Hejazi eta!, 2003), schematically represented in Figure 2F-3.
Fig 2F -3. Chemical structure of chitosan
' OH NH2 ~OH NH2 ~OH NH2 H~0 o H~0 o H~o-
H 0 0 HO 0 0 HO 0 0 ~ ~ ~ ~ ~ 00
2.2.1.1 Source and synthesis
Chitosan is produced commercially by deacetylation of chitin , which is the structural
element in the exoskeleton of crustaceans (crabs, shrimp, etc) Fig 2F-4.
Fig 2F-4. Synthesis of chitosan
Crab/ D1mineraliz Deproteinati Decolouriza B Slmmshells C, ..,,•,lion=-:::>! C.- v...,,on=::.!L~ ..,.=ti=-.on=~(. . C ·- dil HC! - dil NaOH - KMn04 .
Deacetylatio EJ n ' Chitosan Cone _,
NaOH
2.2.1.2 Biophysicochemical properties of clzitosan
Chitosan is a weak base with a pKa of about 6.2-7.0, and it requires a certain amount
of acid to become soluble (Hamman eta!, 2000). The word 'chitosan' refers to a large
number of polymers, which differ in their degree of N-deacetylation (40-98%) and
molecular weight (50,000-2,000,0000Daltons). These two characteristics are very
• Development and Characterization ol Novel Delivery Systems lor Proteins and Peptldes • 80
Chapter2
important to its physicochemical properties and may have a major effect on the
biological properties. Pharmaceutical grade chitosan is deacetylated between 90 and
95% and food grade between 75 and 80% (Paul eta!, 2000). Chitosan salts are soluble
in water; the solubility depends on the degree of deacetylation and the pH of the
solution. The pharmaceutical requirements of chitosan are: particle size <30 !liD,
density between 1.35 and 1.40 g/cm3, pH 6.5-7.5, insoluble in water, and partially
soluble in acids (Hejazi et a!, 2003). The chemical and biological properties of
chitosan are summarized in table 2T-3.
Table 2T -3. Chemical and biological properties of chitosan
Chemical properties of chitosan Biological pro]l_erties of chitosan Cationic polyamine Biocompatible High charge density at ]l_H <6.5 Natural polymer Adheres to negatively charged surfaces Biodegradable to normal body constituents Forms _gels with polyanions Safe and non-toxic High molecular weight, linear polyelectrolyte Hemostatic, bacteriostatic and fungistatic Viscosity, high to low Spermicidal Chelates certain transitional metals Anti-cancerogen Amiable to chemical modification Anti-cholesteremic Reactive amino!hy_droxyl groups Versatile
2.2.1.3 Applications
The intriguing properties of chitosan have been known for many years and this
polycationic polymer (in acidic environments) has been used in the fields of
agriculture, industry and medicine (Hamman et a!, 2000). It is widely used in the
management of wounds and burns (Muzzarelli, 1997). Chitosan oligosaccharide
stimulates fibroblasts production by means of affecting fibroblasts growth factor
(FGF). Thus, collagen production is stimulated as well as other components of
connective tissue. The preparation promotes acceleration of the wound-healing
process, and connective tissue gets an ordered structure. Chitosan oligosaccharide
application prevents rough scar formation (Dodane et a!, 1998). Due to its unique
polymeric cationic character, gel and film forming properties, non-toxicity,
biocompatibility and biodegradability, chitosan has been extensively examined in the
pharmaceutical industry for its potential in the development of drug delivery systems
(Shu et al, 2002). It is presently considered as a novel carrier material in drug delivery
systems (Shu ct a!, 2002). Medical and pharmaceutical applications of chitosan
include drug delivery, wound healing ointments and dressings, artificial skin,
- Development and Characterization of Novel Oellvery Systems lor Proteins and Peplldes • 81
Chapter2
homeostatic agents, enzyme immobilization, dialysis membranes, contact lenses or
eye bandages, orthopaedics, surgical sutures and dentistry (Paul et al, 2000). The
ability of chitosan to enhance the paracellular transport of several peptide drugs, both
in vivo and in vitro, is considered to be one of the most important pharmaceutical
application of chitosan (Hamman et al, 2000; Paul et al, 2000).
2.2.2 Eudragit SIOO
Polymethacrylates are synthetic cationic and anionic polymers of dimethylaminoethyl
methacrylates, methacrylic acid, and methacrylic acid esters in varying ratios.
Eudragit S 100 is one such polymer belonging to this class. It is a copolymer of
methacrylic acid and methyl methacrylate (30:70). The ratio of the free carboxyl
groups to the ester groups is approx. 1:2 in EUDRAGIT® S 100. The average
molecular weight is approx. 135,000.
2.2.2.1 Synthesis
IH3 IH3 -CH2-<f-CH2-1-
J=o c=o OH bH3
Fig 2F -5. Structure of Eudragit SIOO
Eudragit S I 00 is prepared by the polymerization of methacrylic acids or their esters,
e.g. methyl ester or dimethylaminoethyl ester.
2.2.2.2 Physicochemical Properties
Polymer is water-insoluble and films prepared from Eudragit S I 00 are only slightly
permeable to water. White powders with a faint characteristic odour. Dry powder
polymer forms are stable at temperatures less than 30°C. Above this temperature,
powders tend to form clumps, although this does not affect the quality of the
substance and the clumps can readily be broken up. Dry powders are stable for at least
3 years if stored in a tightly closed container at Jess than 30°C (Rohm Pharma, 2005).
A daily intake of 2 mglkg body-weight of Eudragit (equivalent to approximately
• Development and Characterization of Novel Delivery Systems for Proteins and Peptldes • 82
Chapler2
150 mg for an average adult) may be regarded as essentially safe in humans (Gumy et
a!, 1977; Dew et a!, 1982).
It is insoluble in acid medium and dissolves above neutral pH. Dissolution occurs as a
result of structural change of the polymer associated with ionization of the carboxylic
functional group. At acid pH, ES I 00 possess low permeability due to hydrogen
bonding between the hydroxyl groups of the carboxylic moiety and the carbonyl
oxygen of ester groups in the polymer molecules. This binding increases the degree of
compactness of the polymer and decreases its porosity and permeability (El-Kamel et
al, 2001). When the pH of the aqueous medium is increased, ESIOO microparticle
starts to dissolve as the carboxylic functional group ionizes. The reported theoretical
dissolution threshold is pH 7.0 and the pKa of polymer molecules is believed to be
approximately 6 (Nguyen et a!, 2005). Eudragit S I 00 swells at pH above 6.5
(Bhagwat et a!, 2005). Therefore, the release of the active substances may occur due
to a combination of swelling and dissolution.
2.2.2.3 Applications
Polymethacrylate copolymers are widely used as film-coating materials in oral
pharmaceutical formulations (Okor et a!, 1990; Umejima et a!, 1993). They are also
used in topical formulations and are generally regarded as nontoxic and nonirritant
materials. Included in the FDA Inactive Ingredients Guide (oral capsules and tablets),
nonparenteral medicines licensed in the UK, Canadian List of Acceptable Non
medicinal Ingredients (Smolinske, 1992).
Table 2T -4. General properties of Eudragit 8100
Viscosity Loss on Residue Heavy Limit of methyl Polymer Solubility/ drying on metals methacrylate drywt permeability
ignition content
50-200 S5.0% SO. I% S20ppm :::00.005% 95% Low mPas
2.2.3 Cetyl alcohol
Cetyl alcohol, used in pharmaceutical preparations, is a mixture of solid aliphatic
alcohols comprising mainly 1-hexadecanol (C 16H340). The USPNF 23 specifies not
less than 90.0% of cetyl alcohol, the remainder consisting chiefly of related alcohols .
• Development and Characterization ol Novel Delivery Systems for Proteins and Peptldes • 83
Chapter2
Fig 2F-6. Structure of Cetyl alcohol
H H
I I H-~-(CH;,) 14-~-0H
H H
2.2.3.1 Source and Synthesis
Cetyl alcohol may be manufactured by a number of methods such as esterification and
hydrogenolysis of fatty acids or by catalytic hydrogenation of the triglycerides
obtained from coconut oil or tallow. Cetyl alcohol may be purified by crystallization
and distillation (Eccleston, 1984 ).
2.2.3.2 Biophysicochemical Properties
Cetyl alcohol occurs as waxy, white flakes, granules, cubes, or castings. It has a faint
characteristic odor and bland taste. It is freely soluble in ethanol (95%) and ether,
solubility increases with increasing temperature; practically insoluble in water. It is
miscible when melted with fats, liquid and solid paraffins, and isopropyl myristate
(Table 2T-5).
Table 2T-5. General properties of cetyl alcohol
Viscosity M.pt B.pt Refractive index Density Flash point 7 mPas at SO'C 45-52'C 316-344 n79D = 1.4283 0.908 glcm' 165°C
'C
2.2.3.3 Applications
Cetyl alcohol is widely used in cosmetics and pharmaceutical formulations such as
suppositories, modified-release solid dosage forms, emulsions, lotions, creams, and
ointments. In suppositories, cetyl alcohol is used to raise the melting point ofthe base,
and in modified-release dosage forms it may be used to form a permeable barrier
coating. In lotions, creams, and ointments cetyl alcohol is used because of its
emollient, water-absorptive, and emulsifying properties. It enhances stability,
improves texture, and increases consistency_ The emollient properties are due to
absorption and retention of cetyl alcohol in the epidem1is, where it lubricates and
softens the skin while imparting a characteristic 'velvety' texture. Cetyl alcohol is
also used for its water absorption properties in water-in-oil emulsions. Cetyl alcohol
acts as a weak emulsifier of the water-in-oil type, thus allowing a reduction of the
• O~elopmenl and Choraclertzollon of Novel Oellvery Syslems lor Proteins and Peptldes-84
Chapter2
quantity of other emulsifying agents used in a formulation (Eccleston, 1984). Cetyl
alcohol has also been reported to increase the consistency of water-in-oil emulsions.
In semisolid emulsions, excess cetyl alcohol combines with the aqueous emulsifier
solution to form a viscoelastic continuous phase that imparts semisolid properties to
the emulsion and also prevents droplet coalescence (Mapstone, 1974). Therefore,
cetyl alcohol is sometimes referred to as a 'consistency improver' or a 'bodying
agent', although it may be necessary to mix cetyl alcohol with a hydrophilic
emulsifier to impart this property. It is included in the FDA Inactive Ingredients
Guide (ophthalmic preparations, oral capsules and tablets, otic and rectal preparations,
topical aerosols, creams, emulsions, ointments and solutions, and vaginal
preparations). It is also included in non-parenteral medicines licensed in the UK and
the Canadian List of Acceptable Non-medicinal Ingredients.
2.3 PREFORMULATION STUDIES
Preformulation is an integral part of entire development process. It encompasses the
phase of formulation development in which the pharmaceutical scientists take an
initial look at the protein molecule to identifY the conditions that are likely to be best
suited for development of a formulation possessing long term stability.
Preformulation studies relate to the pharmaceutical and analytical investigations that
serve as proceedings and supporting formulation development effort. So before
starting the development of delivery systems, preformulation studies of proteins
selected were carried out.
The drug was tested for identification; solubility in various solvents, UV absorption
maxima, HPLC chromatograms, preparation of calibration curve of the drug and IR
investigations. Activity of enzyme was accessed by in vitro proteolytic activity.
Calibration curve of proteins were prepared by reported or by developed methods.
2.3.1 Bovine serum albumin (BSA)
2.3.1.1 UV-spectropfwtometry
A 20 J.lg/rnl solution of BSA in PBS (pH 7.4) was taken in I em standard cuvettes and
scanned in range of 200-400nm using Shimadzu 1700 Pharmaspec UV -visible
spectrophotometer .
• Development and Characterization of Novel Delivery Systems for Proteins and Peptldes -85
Chapter2
2.3.1.2 Preparation of calibration curve
The measurement of protein was done by Lowry protein assay using copper and the
Folin reagent having many advantages (Lowry eta\, 1951). It is sensitive as Nessler's
reagent, yet requires no digestion. It is 10 to 20 times more sensitive than
measurement of the ultraviolet absorption at f.. =280nm with more specificity and less
liability of disturbance by turbidities. It is several folds more sensitive than the
ninhydrin reaction. Free amino acids give much more color than proteins with the
ninhydrin reaction, whereas the reverse is true with the Folin reagent. It is 100 times
more sensitive than the Biuret reaction.
2.2.1.2.1 Reagents
Reagent A 2% NazC03 in 0.10 N NaOH
I% CuS04.5HzO in water
2% NaK tartrate.
Folin & Ciocalteu's phenol reagent, 2N (Sigma F-9252).
ReagentBl
Reagent B2
Reagent C
ReagentD Carbonated copper solution is the same reagent except for omission
ofNaOH.
2. 2.1. 2. 2 Preparation of Bovine Serum Albumin (BSA) standard curve
Stock solution of BSA of 1000 f.lg/ml in 1 OOml volumetric flask was prepared with
50mg (0.05%w/v) of sodium K tartarate. Volume was made up to the 100 ml mark
with phosphate buffer saline (pH 7.4). Aliquots of 0.1, 0.2, 0.3, 0.4, .... .lml was
withdrawn from stock BSA solution (1 OOOf.lglml) in !Om! volumetric flask to give a
concentration range of 10-IOOf.lg/ml.
Prepared a 100:1:1 mixture of solutions of A, Bl, B2 reagents. Solution Bl was added
to A first, then solution B2 was added. 1 ml of this reagent mixture was added to each
tube.
Folin & Ciocalteu's phenol reagent was diluted with the distilled H20 (1:1). 0.1 ml of
this reagent was added to each tube. Solution was mixed well and allowed to stand for
1Om in at room temperature. 0.1 Om! of reagent D was added very rapidly and was
mixed thoroughly. After 30min the sample was analyzed in spectrophotometer.
Volume was made up to lOml mark with PBS (pH 7.4) in each tube and absorbance
was measured at 750nm against blank in UV-Vis Spectrophotometer .
• Development and Characterization o1 Novel Dellvery Systems tor Proteins and Peptldes • 86
Chapter2
2.3.2 Serratiopeptidase
2.3.2.1 UV-spectroplwtometry
UV absorption maxima of STP in PBS (pH 7.4) were determined by scanning the
solution of protein (I OOJ.!g/ml) in Shimadzu 1700 Pharmaspec UV -visible
spectrophotometer between 200nm to 400nm and the absorption maxima was
observed.
2.3.2.2 IR spectroscopy
Weighed 1 mg ofSTP and triturated with 100 mg of dried KBr in mortor. Pellets were
prepared using pellet press. Pellet was scanned between the range of 400 to 4000 cm-1
The spectrum was recorded and major peaks were determined (FTIR Fourier
transform infrared-8400S Shimadzu (Japan).
2.3.2.3 Preparation of calibration curve
Simplified first derivative spectrophotometric based estimation procedure was
developed. The first-order derivative spectra were obtained over the 200-400 run
range and N=1, ~A.=l.O nm. A solution of STP was prepared by dissolving 100 mg
(accurately weighed) of the standard STP in 100 ml of phosphate buffer PH 7.4. This
stock solution was further diluted to get a working standard solution of 100 llgfml.
Aliquots of working standard solution were suitably diluted with buffer to give final
concentrations. The peak amplitude of the obtained first-derivative spectra was
measured at 229.5 run against reagent blank and the calibration curve plotted.
2.3.2.4 In vitro proteolytic activity
The enzyme STP was assayed by carrying out the in vitro proteolytic activity
specified by Food and chemical codex (2003). STP solution in different concentration
ranging from 10-100!-lg/ml were placed in phosphate buffer (pH 7.4) 5ml maintained
at 37±0.5°C and stirred constantly at IOOrpm. After 2 hrs, protein was recovered by
centrifugation at 9000g for I 0 min and the supernatant was used for protein analysis
by measunng absorbance at 229.5 nm by first derivative method
spectrophotometerically (Shimadzu UV -1700, Pharmaspec, Tokyo, Japan). Samples
were then assayed for proteolytic activity (n=3). The assay was based on a 30 min
proteolytic hydrolysis of casein at 37°C and pH 7.0. Unhydrolyzed casein was
-Development and Characterization of Novel Delivery Sytlems for Profelns and Peptldes-87
Chapter 2
removed by filtration and the solubilized casem was determined
spectrophotometrically at wavelength of 275 nm. In this method, the protease activity
is expressed as protease units (PC) of preparation derived from Bacillus subtilis var.
One bacterial PC is defined as quantity of enzyme that produces l.5f!g/ml equivalent
ofL-tyrosine per minute under the condition of the assay.
2.3.3 Enalapril maleate
2.3.3.1 UV-spectrophotometry
UV absorption maxima of EM in PBS (pH 7.4) were determined by scarming the
solution of protein (I OO).lg/ml) in Shimadzu 1700 Pharmaspec UV -visible
spectrophotometer between 200nm to 400nm and the absorption maxima observed.
2.3.3.2 JR spectroscopy
Weighed I mg of protein and triturated with I 00 mg of dried KBr in mortar. Pellets
were prepared using pellet press. Pellet was scarmed between the range of 400 to 4000
em-!. The spectrum was recorded and major peaks were determined (FTIR Fourier
transfonn infrared-8400S Shimadzu (Japan).
2.3.3.3 Preparation of calibration curve
A solution of EM was prepared by dissolving 100 mg (accurately weighed) of the
standard EM in I OOml of phosphate buffer pH 7.4. This stock solution was further
diluted to get a working standard solution of I 00 f!g/ mi. The resulting solution was
filtered using Whattman filter (0.22).lm pore size) and analyzed for EM content. The
EM concentration in the supernatant solution was analyzed by HPLC system
(Shimadzu LC-1 OAT vp, binary gradient) equipped with detector (Shimadzu UV
visible SPD-10A vp), software (Spinchrom CFR V.2.2, Spincotech Pvt. Ltd.,
Chennai) and Column (Phenomenex Luna, C-18, 5 ).lm, 25x 4.6 mm i. d.) using
acetonitrile: water (20: 80) as mobile phase with the flow rate of 1.0 mL!min and
wavelength of215 nm (Walily, 1995).
2.3.4 Drug-polymer interaction studies
Investigation of physico-chemical properties of the proteins (BSA, STP and EM) and
the polymers (Chi to san, Eudragit S I 00, and Cetyl alcohol) were the important criteria
-Development and Charactertzotlon ol Novel Delivery Systems lor Proteins and Peplldes-88
Chapter 2
which have to be considered before using polymers to prepare a microsphere system.
FTIR spectrophotometric analyses were carried out to investigate polymer and drug
interactions. Drugs were mixed with the polymers in the ratio of I :6. The pellets were
prepared on KBr press. FTIR spectra were obtained on FTIR-84008 Shimadzu
(Japan). The spectra were recorded over the wave number 4 700 to 400 cm-1•
2.3.5 Results and discussion
The primary aim of preformulation studies is to determine the inherent stability of the
molecule and to identity the key problems that are likely to be encountered during
development of a stable formulation. In the present study UV, HPLC and IR
spectrophotometry were used for the identification of chemical and physical
properties of drugs.
2.3.5.1 Bovine Serum albumin (BSA)
BSA was used as a model protein. UV spectroscopic method helps in identification of
BSA as protein. The BSA was freely soluble in water, ethanol and chloroform;
sparingly soluble in dilute acids and slightly soluble in ether. UV absorption maxima
of BSA in PBS (pH 7.4) were determined by scanning the solution of drug
(IOOJ.!g/ml) in Shimadzu 1700 Pharmaspec UV-visible spectrophotometer and the
absorption maxima was observed at 280nm (Table 2T-6, Fig 2F-7).
Table 2T -6. Preparation of BSA calibration curve
Concentration (!lgimL) Absorbance Regressed
10 0.063 0.0637
20 0.083 0.0847
30 0.107 O.I057
40 O.I24 0. I267
50 O.I47 O.I477
60 O.I62 O.I687
70 O.I88 0. I 897
80 0.217 0.2107
90 0.225 0.23 I 7
IOO 0.247 0.2527
• Development and Charoctertzatlon of Novel Delivery Systems for Proteins and Peptldes • 89
Chapter2
0.3
0.25
0.2
0.15
0.1
0.05
0 20
Fig 2F-7. Standard curve of BSA
40
y = 0.0021x + 0.0427 R2 = 0.9963
60 80 100 120
f Series1 -Linear (Series1)
2.3.5.2 Serratiopeptidase
The STP was freely soluble in water, ethanol and chloroform; sparingly soluble in
dilute acids and slightly soluble in ether. The identification of STP was carried out by
UV, IR spectroscopy and in vitro proteolytic activity.
UV absorption maxima of STP in PBS (pH 7.4) were determined by scanning the
solution of drug (I OOf!g/ml) in Shimadzu 1700 Pharmaspec UV -visible
spectrophotometer and the absorption maxima was observed at 220nm (Merck Index,
2001b).
IR analysis of STP was done by weighing 1 mg of STP and triturated with 100 mg of
dried KBr in mortor. Pellets were prepared using pellet press. The pellets were
scarmed between the range of 400 to 4000 cm-1. The spectrum was recorded and
determined the major peaks (FTlR-84008 Shimadzu (Japan). TheIR spectra of STP
indicated the presence of bands at about 3320 cm·1 (-NH2 group), 1690 cm·1 (-COOH,
carboxylic stretching) and 1550 cm·1 (-CONH, peptide linkage) characteristic of
peptide STP (Fig 2F -8) .
• Developmenl and Characlerlzal!on of Novel Dellvert Syslemslor Prolelns and Peplldes • 90
Chapter2
Fig 2F-8. IR spectra of serratiopeptidase
67.5
%T
60
52.5
45
37.5
30
22.5
15
4500 4000 3500 3000 2500 2000 1750 1500 1250 1000 750 500 Serretopeptidase 1/cm
Simplified first derivative spectrophotometric based estimation procedure was
developed for preparation of calibration curve of STP. Shimadzu 1700 Pharmaspec
UV -visible spectrophotometer with a matched pair of 10 mm quartz cells was used in
the present study. The first-order derivative spectra were obtained over the 200-400
nm range and N=1, .1.1.=1.0 nm. A solution ofSTP was prepared by dissolving 100
mg (accurately weighed) of the standard STP in 100 ml of phosphate buffer PH 7.4.
This stock solution was further diluted to get a working standard solution of 100
f!g/ml. Aliquots of working standard solution were suitably diluted with buffer to give
final concentrations. The peak amplitude of the obtained first-derivative spectra was
measured at 229.5 run against reagent blank and the calibration curve plotted (Table
2T-7; Fig 2F-9, 10). The powdered amount equivalent to 1 OOmg (accurately weighed)
of drug was dissolved in 50 ml of distilled water and the insoluble excipients were
separated by centrifugation at 3000rpm for l 0 min. The supernatant liquid was
transferred to lOOm! volumetric flask quantitatively with distilled water and volume
made up and drug content was determined from the calibration curve.
• Development and Choraclerlzotlon of Novel Delivery Systems tor Proteins and Peptldes • 91
Chapter2
Table 2T-7. UV study ofserratiopcptidase by first derivative spectroscopy
S.no Concentration (ltg/ml) Absorbance I. 10 -0.004 2. 20 -0.007 3. 30 -0.011 4. 40 -0.013 5. 50 -0.016 6. 60 -0.019 7. 70 -0.023 8. 80 -0.026 9. 90 -0.029 10. 100 -0.032
Regression equation y=oU.Ull x+u.uuu, L.:orre1at1on coernc1ent (K2J u.'J'J':J t, une arrange (~g mi-l) 10-100
Figure 2F -9. UV spectra of STP by first derivative spectroscopy
e.a3A
(8.028 /div)
--·----··:r--"""'<.:i'------+
-a.B8A 2~88~.a~n-.~~<-=sa-./d~iv~)-+~48~B.~8n~m
Fig 2F-10. Standard curve ofSerratiopeptidase by first derivative spectroscopy
0
120 .0.005
.{).Ql
~ u
.Q.QIS c ~
.Q
" -t-STP 0 .0.02 ~
"' ~ .0.025
.0.03
.O.D35 concentration
• Development and Characterization o1 Novel Delivery Systems lor Proteins and Peplldes-92
Chapler2
The enzyme STP was assayed by carrying out the in vitro proteolytic activity
specified by Food and chemical codex (2003) (Table 2T-8, Fig 2F-9, 10).
Table 2T -8. Calibration curve of serratiopeptidase in terms of activity
S.No Concentration Activity (units/mg) (uelml)
I. 10 21.34 2. 20 42.76 3. 30 60.98 4. 40 88.75 5. 50 107.09 6. 60 125.43 7. 70 150.28 8. 80 172.05 9. 90 187.45 10. 100 230.67
Fig 2F-11. Standard curve of serratiopeptidase in terms of activity
0 20 40 60 80 100 120
concentration
y: 2.2!Sx· 3.156 R'=0.993
-+-STP
- Unear (STP)
In order to study the interaction between SIP and polymers (Chi to san, Eudragit S 100
and cetyl alcohol) IR studies were performed. Drugs were mixed with polymers
(Chitosan, Eudragit S I 00 and cetyl alcohol) in the ratio of I :6. The IR spectra of
serratiopeptidase (STP) indicated the presence of bands at about 3320 em·' (-NH2
group), 1690 cm-1 (-COOH, carboxylic stretching) and 1550 cm-1 (-CONH, peptide
linkage) characteristic of peptide SIP.
-Development and Chorocterlzollon ol Novel Delivery Systems lor Proteins and Peptldes • 93
Chapler2
Fig 2F -12. IR spectra of serratiopeptidase with chitosan
4<;00 .. ooo 3!>00 :woo 2500 2000 1750 1600 1250 1000 750
Fig 2F-13. IR spectra of serratiopeptidase with Eudragit S 100
.,.
"
Fig 2F-14. IR spectra of serratiopeptidase with cetyl alcohol
%T .,.
•
• Development ond Charactertzotlon of Novel Dellvery Systems for Proteins and Peptldes • 94
Chapter2
IR spectra of STP with polymers studied also showed the peaks at the same bands.
This indicated that there is no interaction between STP and polymers employed (Fig
2F-12-14).
2.2.5.3 Enalapril maleate
Enalapril maleate was freely soluble in water, methanol and dimethyl formamide,
slightly soluble in isopropyl alcohol; very slightly soluble in acetone, alcohol and
hexane; practically insoluble in chloroform (IP' 1996; Gennaro, 2000).
UV absorption maxima of EM in PBS (pH 7.4) were determined by scanning the
solution of drug (IOOf.tg/ml) in Shimadzu 1700 Pharmaspec UV-visible
spectrophotometer and the only end absorption was observed with no max1ma
(Florey, 200lb). As only end absorption was present, HPLC method was used for
estimation of EM (O'Niel J M, 2001).
Fig 2F-15. UV spectra ofEnalapril maleate
0 ·'j(l A ..) + ~ -::h'i
(8. 500 /d i v)
EM was characterized by FTIR analysis. Weighed I mg of EM and triturated with
I 00 mg of dried KBr in mortor. Pellets were prepared using pellet press. The pellets
were scanned between the range of 400 to 4000 cm-1. The spectrum was recorded and
determined the major peaks (FTJR-8400S Shimadzu (Japan). The IR spectra of EM
indicated the presence of bands at about 3315 cm-1 ( -NH2 group), 1685 cm-1 (-
• Otvelopment and Characterization of Novel Oeltvery Systems for Proteins and Peptldes • 95
Chapter2
COOH, carboxylic stretching) and 1560 cm-1 (-CONH, peptide linkage)
characteristic of peptide EM (Galichet, 2005) (Fig 2F-16).
120
%T
100
80
80
40
20
0
-20
-40
Fig 2F-16. IR spectra of EM
--?'-'M .... --r:--r- ---:--~--r-~: ---r-~--
- -:------.:.------:--- --:------ .;.. ----- .:... ----- -:...- ---- -~-- --- -:... ---: . : j ~ l ; ~
---:-------: ------------ !-------~------r------~ -----~------r---. ' ' ' . . . ' ' --- -·---- --------- ------~- ----- ~ ---- ---------·------~- --. ' ' ' '
' ' ' ; i [ ~ i j
--~~~~,!~~~~~~ -r----~-:- -----~--------------.----- ~------r------r --- -----:----r------r------------r-- --r- -- ·--
-- --- -·---- -- ------r--- ---:----------- ---r ------:------ ------:------. ' ' ' ' ' ' ------.------ ----·-r·-----r··---- ------r------:·----- ------ .. ------ ------
------ ---- ---- :----+--- -------[- ---:----- ---- -: ----- ---4500 4000 3500 3000 2500 2000 1750 1500 1250 1000 750 500 250
E1 1~m
The calibration curve of EM was analyzed and developed by HPLC system
(Shimadzu LC-IOAT vp, binary gradient)equipped with detector (Shimadzu UV
visible SPD-IOA vp), software (Spinchrom CFR V.2.2, Spincotech Pvt. Ltd.,
Chennai) and Column (Phenomenex Luna, C-18, 5 J-Uil, 25x 4.6 mm i. d.) using
acetonitrile: water (20: 80) as mobile phase with the flow rate of 1.0 mL/min and
wavelength of215 nm (Walily, 1995). Further, peptide content was determined from
the calibration curve (Table 2T-9).
Table 2T-9. HPLC study ofEnalapril maleate
Concentration fu!!/m)) Area 10 356.645 20 414.056
30 656.764
40 753.776 50 976.864
60 1130.117
70 1350.175 80 1387.518 90 1547.058 100 1752.239
• Development and Chorocterlzollon of Novel Delivery Systems for Proteins and Peptldes • 96
. ~ .. 0 ,.
Chapter2
Fig 2F-17. HPLC spectra ofEnalapril maleate
[mVJ ,-----------------------------, 150
100
50
- e :'.man ju rn· at1.m an JU eul~p riJ'.m an j11 e na/.1pr1IG
+-------_,..J ..... \ .....
2000 ~ 1800 VJ
:> 1600 .5. 1400 (I)
1200 > ... :I 1000 0 1.. <D 800 "C r: 600 :I ~ 400 <D ... <( 200
0
' ' J I
Time
Fig 2F-18. Calibration curve ofEnalapril maleate
0 50 100
Cone (mlcrog/ml)
y = 15.81x + 162.9 R2 = 0.991
--EM
-Linear (EM)
150
' l [mull
In order to study the interaction between EM and polymers (Chitosan, Eudragit S 100
nd cetyl alcohol) IR studies were performed. Drugs were mixed with polymers
(Chitosan, Eudragit S 100 and cetyl alcohol) in the ratio of I :6. The IR spectra of
Enalapril maleate (EM) indicated the presence of bands at about 3315 em-! ( -NH2
group), !685 em-! (-COOH, carboxylic stretching) and 1560 cm-1 (-CONH, peptide
linkage) characteristic of peptide EM (Fig 2F-19-21 ).
• Development end Charactertzotlon of Novel Delivery Systems lor Prole Ins and Peplldes • 97
Chapler2
Fig 2F-19. IR spectra of Enalapril maleate with chitosan
. ·- --- ~- ---- ~- -- ---,-- --- ..--- -- -~----,---
00
"'
_,
4'llO 4nnn 1:';00 'Vlll'l ?~nn ?fllll no;n 1~m 1?'11 1ni'Wl ?'>O <;M ?."1'1
Fig 2F -20. IR spectra of Enalapril maleate with Eudragit S 100
"" rc---:::""...---,---,---,--,---,----,---,--,-----,----,-----,nn %<
'"" 00
0
_,
Fig 2F -21. IR spectra of Enalapril maleate with cetyl alcohol
.,
.,
-20 --·-. ___ , __ -
IR spectra of EM with polymers studied also showed the peaks at the same bands.
This indicated that there is no interaction between EM and polymers employed.
• Development and Charoclerlzallon of Novel Delivery Systems for Proteins and Peptldes • 98
Chapler2
References I. Aiyappa PS, Harris JO. The extracellular metalloprotease of Serratia
marcescens. I. Purification and characterization. Mol. Cell Biochem. 1976; 13:
95-100.
2. Alfonso OR, Remington: The science and practice of Pharmacy, vol II, 201h
edition, Lipincott Williams and Wilkins; 2000, pp 1280-1281.
3. Bhagwat D, Diehl D and Baichwal AR. Novel once a day controlled release
sulphonylurea formulation. In PCT ntemational application. US: Edward
Mendell company, Inc.
4. Bradford MM. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal.
Biochem. 1976, 72: 248-254.
5. Carter DC, Ho JX. Structure of serum albumin. Adv. Protein Chern. 1994; 45:
153-203.
6. Choulis NH, Rekkas D, Dallas P. HPLC determination of serratiopeptidase in
biological fluids. Pharmazie. 1989; 44: I I I -1 I 6.
7. Colson P, Ryckwaert F, and Coriat Pierre. Renin Angiotensin System
Antagonists and Anesthesia. Anesth. Analg. 1999; 89: ll43-II55.
8. Committee on Institute of Medicine. Food and Chemical Codex. Washington,
DC: National Academic Press; 2003.
9. Dew MJ, Hughes PJ, Lee MG. An oral preparation to release drugs in the
human colon. Br. J. Clin. Pharmacal. 1982; 14: 405-08.
10. Dodane V, Vilivalam VD. Pharmaceutical applications of chitosan. Pharm Sc
Techno! Today 1998; I: 246-53.
I I. Eccleston OM. Properties of fatty alcohol mixed emulsifiers and emulsifYing
waxes. In: Florence AT, ed. Materials Used in Pharmaceutical Formulation:
Critical Reports on Applied Chemistry, volume 6. Oxford: Blackwell
Scientific, 1984: 124-156.
12. El-Kamel AH, Sokar MS, AI Gamal SS, Naggar VF. Preparation and
evaluation of ketoprofen floating oral delivery system. Int. J. Pham1. 2001;
- Development and Characterization ol Novel Delivery Systems for Proteins and Peplldes • 99
Chapter2
220: 13-21.
13. Esch PM, Gerngross H, Fabian A. Reduction of postoperative swelling.
Objective measurement of swelling of the upper ankle joint in treatment with
serrapeptase-a prospective study. Fortschr Med 1989; 107: 67-72.
14. Florey K. Analytical profiles of drug substance, 16, Academic press, 2005,
207-243.
15. Galichet L. "Clark's analysis of drug and Poisons", 3rd edition, Pharmaceutical
Press, pp 971-933.
16. Garcia C, Paim C, Steppe M. New liquid chromatographic method for
determination of serratiopeptidasein coated tablets. J. AOAC Int. 2004; 87:
842-46.
17. Garcia C, Sippel J, Sfair L, Garcia S, Jablonski A, Steppe M, Schapoval E.
Validation of capillary electrophoresis method for analysis of
serratiopeptidasein pharmaceutical dosage form. J. AOAC Int. 2005; 88: 1081-
85.
18. Gennaro R. Alfonso, Remington: the science and practice of Pharmacy, vol II,
20 ed. Lipincott Williams & Wilkins, 2000; 1280-1281.
19. Gurny R, Guitard P, Buri P, Sucker H. Realization and theoretical
development of controlled-release drug forms using methacrylate films 3:
preparation and characterization of controlled-release drug forms. Pharm. Acta
Helv. 1977; 52: 182-87.
20. Hamman JH, Stander M, Junginger HE, Kotze AF. Enhancement of
paracellular drug transport across mucosal epithelia by N-trimethyl chitosan
chloride. STP Pharma Sciences 2000; 10: 35-38.
21. Hejazi R, Arniji M. Chitosan-based gastrointestinal delivery systems. J.
Control Rei. 2003; 89: 151-65.
22. Kang R, Saito H, Ihara Y, Miyoshi E, Koyama N, Sheng Y, Taniguchi N.
Transcriptional Regulation of the N-Acetylglucosaminyltransferase V Gene in
Human Bile Duct Carcinoma Cells (HuCC-Tl) Is Mediated by Ets-1. J. Bioi
Chern. 1996; 271:26706-26712.
23. Ijitsu T, Yonezawa K, Ueno M. Improved analysis of serrapeptase by high
performance steric exclusion chromatography. Yakugaku Zasshi 1986; I 06:95-
98.
• Development and Charactertzotlon ol Novel Dellvery Systems tor Proteins and Peptldes • 100
Chapter2
24. Indian Pharmacopoeia, Govt. of India, Ministry of Health and family welfare,
the controller of publication, Delhi, 1996; I: 281-282.
25. Kee WH, Tan SL and Lee V. The treatment of breast engorgement with
Serrapeptase (Danzen): a randomized double-blind controlled trial. Singapore
Med. J. 1989; 30: 48-54.
26. Klein G, Kullich W. Short-term treatment of painful osteoarthritis of the knee
witb oral enzymes. A randomized, double-blind study versus diclofenac. Clin.
Drug Invest. 2000; 19: 15-23.
27. Lee, K.-K., Ellis, A. E. A novel method for specific visualisation of serum
albumin in polyacrylamide gels by iodine staining. Electrophoresis. 1991; 12:
382-383.
28. Lowry OH, Rosenberg NJ, Farr AJ, Randall RJ, 1951, Protein measurement
with the Folin phenol reagent. J. Bioi. Chern. 193, 265-232.
29. Maheshwari M, Miglani G, Mali A, Paradkar A, Yamamura S, Kadam S.
Development of Tetracycline-Serratiopeptidase-Containing Periodontal Gel:
Formulation and Preliminary Clinical Study. AAPSPharmSciTech 2006; 7(3):
article 76.
30. Mapstone GE. Crystallization of cetyl alcohol from cosmetic emulsions.
Cosmet Perfum 1974; 89: 31-33.
31. Marly M. Enzymotherapie anti-inflammatoire a !'aide de Ia Serrapeptase:
resultats cliniques en traumatologie et en ORL. C R Therapeut 1985; 3: 9-19.
32. Maryadele ONJ, Merck Index, 30th edition, Merck research laboratories, NJ,
200 I, 630-631.
33. Mazzone A, Catalan M, Costanzo M, Drusian A, Mandol A, Russo S, Guarini
E, Vesperini G. Evaluation of Serratia peptidase in acute or chronic
inflammation of otorhinolaryngology pathology: a multicentre, double-blind,
randomized trial versus placebo. J. Int. Med. Res. 1990; 18:379-88.
34. Miyata K. Intestinal absorption of Serratia Peptidase. J. Appl. Biochem. 1980;
2:111-16.
35. Moriya N, Nakata M, Nakamura M, Takaoka M, Iwasa S, Kato K, Kakinuma
A. Intestinal absorption of serrapeptase (TSP) in rats. Biotechnol. Appl.
Biochem. 1994; 20: 101-8.
~ Development and Choracfertzaflon of Novel Oelfvery Systems for Proteins and Peptldes • 101
Chapter2
36. Motonaka J, Takabuyashi Hand Ekeda S. Nippon Kagaku Kaishi, 1988; 7:
1021.
37. Muzzarelli RAA. Human enzymatic activities related to the therapeutic
administration of chitin derivatives. Cell Mol. Life Sci. 1997; 53: 131-40.
38. Nguyen DA, Fogler HS. Facilitated diffusion in the dissolution of carboxylic
polymers. Am. Instt. Chern. Engg. J. 2005; 51:415-425.
39. Nihon KS. A case of pneumonitis due to serrapeptase. Gakkai Zasshi 1989;
27: 1231-36.
40. Nishimura K, Harada K, Masuda S, et.al. J. Chrom. Biomed. App. 1990; 91:
176.
41. O'Niel JM. Merck Index, 30 ed. Merck Research Laboratories, Div of Merck
and Company Inc. NJ, 2001; 630-631.
42. Odagiri J. Clinical applications of Serrapeptase in sinusitis. Med Consult New
Remedy. 1979; 6: 201-09.
43. Okor RS, Obi CE. Drug release through aqueous-based film coatings of
acrylate-methacrylate, a water-insoluble copolymer. Int. J. Pharm. 1990; 58:
89-91.
44. Panagariya A, Sharma AK. A preliminary trial of serratiopeptidase in patients
with carpal tunnel syndrome. J. Assoc. Physicians India. 1999; 4 7: 1170-72.
45. Paul W, Sharma CP. Chitosan, a drug carrier for the 21st century: a review.
S.T.P. Pharma Sciences 2000; 10: 5-22.
46. Perna L. Osservazionl Clniche sui traitarnento in osppio cleco con Serratio
peptidasl nella neifre perenna naila ninite cronica nacutizzata con sinusopattia,
nella bronchia cronica nacutizzata. Rlv Pat Clin Tuberc Penumol 1985; 56:
509-516.
47. Peters T, Jr. All about albumin: biochemistry, genetics and medical
applications, Academic press, San Diego, CA. 1996.
48. Rohm Pharma GmbH. Eudragit. http://www.roehm.com/enlpharmapolymers
(accessed 20 May 2005).
49. Schmitt N, Lunn C, HPLC method for pharmaceutical analysis, Wiley
Intersciencc, 1997, pp. 543-545 .
• Development and Characterization of Novel Oellvery Sys!ems lor Proteins and Peptldes • 102
Chapter2
50. Scull T. In compliance with Serratia, cultivations the research regarding the
induction and a suppression of Serrapeptase creations. Korean microbe life
science gangrene 1997; 25: 4232-39.
51. Selan L, Berlutti F, Passariello C, Comodi-Ballanti MR, Thaller MC.
Proteolytic enzymes: a new treatment strategy for prosthetic infections?
Antimicrob. Agents Chemother. 1993; 37: 2618-621.
52. Shimizu H. A case of serratiopeptidase-induced subepidermal bullous
dermatosis. Br. J. Dermatol. 1999; 141: 1139-40.
53. Shu XZ, Zhu KJ. Controlled drug release properties of ionically cross-linked
chi to san beads: the influence of anion structure. Int. J. Pharm. 2002; 23 3: 217-
25.
54. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano
MD, Fujimoto EN, Goeke NM, Klenk DC, Olson BJ. Measurement of protein
using bicinchoninic acid. Anal. Biochem. 1985; 150: 76-85.
55. Smolinske SC. Handbook of Food, Drug and Cosmetic Excipients. Boca
Raton, FL: CRC Press, 1992: 75-77.
56. Sweetman CS. Martindale- the complete drug reference. 33ed. Pharmaceutical
Press: London. 2002a; 210-13.
57. Sweetman CS. Martindale- the complete drug reference. 33ed. Pharmaceutical
Press: London. 2002b; 594-97.
58. Tanimoto T, Fukuda H, Kawamura J. On the quality of enzyme preparation
(V): Serratiopeptidase preparation. Eisei Shikenjo Hokoku. 1983; 101: 92-5.
59. The United States Pharmacopoeia (USP26/NF21), Asian edition, 2003, 700-
704.
60. Tivesten A, Folestad S; Schanbacher V, Svensson K. Nonaqueous capillary
electrophoresis for the analysis of labile pharmaceutical compounds.
Chromatographia. 1999; 49: 7-11.
61. Tomoda K, Miyata K. Some information on the composition of trachael
secretions before and after the administration of Danzen. Exper. Ther. 1972;
477: 9-16.
62. Twining SS. Fluorescein isothiocyanate-labeled casein assay for proteolytic
enzymes. Anal. Biochem. 1984; 143: 30-34 .
• Development and Choroctertzatlon of Novel Delivery Systems for Proteins and Peptldes • 103
Chapler2
63. Umejima H, Kim NS, Ito T. Preparation and evaluation ofEudragit gels VI: in
vivo evaluation of Eudispert rectal hydrogel and Xerogel containing
salicylamide. J. Pharm. Sci. 1993; 82: 195-99.
64. Vicari E, La Vignera S, Battiato C et.al. Treatment with non-steroidal anti
inflammatory drugs in patients with amicrobial chronic prostato-vesiculitis:
transrectal ultrasound and seminal findings. Minerva Urol. Nefrol. 2005; 57:
53-9.
65. Walily AFME, Belala SF, Heabab EA, Kershb AE. Simultaneous
determination of enalapril maleate and hydrochlorothiazide by first-derivative
ultraviolet spectrophotometry and high-performance liquid chromatography. J.
Pharm. Biomed. Anal. 1995; 13: 851-856.
66. Yamazaki H, Tsuji H, Saeki K. Anti-inflammatory action of a protease, TSP,
produced by Serratia. Nippon Yakurigaku Zasshi 1967; 63: 302-14 .
• Development ond Charactertzatlon of Novel Oellvery Systems for Proteins and Peptldes • 104