Optimization of spray drying process for formulation of solid dispersion containing polypeptide-k powder through quality by design approach

    Optimization of spray drying process for formulation of solid dispersion containing polypeptide-k powder through quality by design approach Puneet Kaur, Sachin Kumar Singh, Varun Garg, Monica Gulati, Yogyata Vaidya PII: DOI: Reference:

S0032-5910(15)00492-1 doi: 10.1016/j.powtec.2015.06.034 PTEC 11077

To appear in:

Powder Technology

Received date: Revised date: Accepted date:

22 January 2015 10 June 2015 13 June 2015

Please cite this article as: Puneet Kaur, Sachin Kumar Singh, Varun Garg, Monica Gulati, Yogyata Vaidya, Optimization of spray drying process for formulation of solid dispersion containing polypeptide-k powder through quality by design approach, Powder Technology (2015), doi: 10.1016/j.powtec.2015.06.034

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Optimization of spray drying process for formulation of solid dispersion containing polypeptide-k powder through quality by design approach

T

Puneet Kaur, Sachin Kumar Singh*, Varun Garg, Monica Gulati, Yogyata Vaidya

IP

School of Pharmaceutical Sciences, Lovely Professional University, Phagwara-144411, Punjab, India.

SC R

* Corresponding Author: School of Pharmaceutical Sciences, Lovely Professional University, Phagwara - 144411, Punjab, India. Tel.:

AC

CE P

TE

D

MA

NU

+919888720835; Fax: +91 1824501900; E-mail address: [email protected];[email protected]

1

ACCEPTED MANUSCRIPT ABSTRACT Polypeptide-k is an antidiabetic phytochemical isolated from dried and ripened seeds of

T

Momordica charantia. The peptide has not been able to find much clinical use despite its

IP

established therapeutic effect. This is mainly attributed to its poor aqueous solubility. Present

SC R

study presents the formulation of solid dispersion containing polypeptide-k to enhance its aqueous solubility. The study was carried out by employing the optimization approach using

NU

spray drying technique. Variables were evaluated using Box Behnken Design. These include, trehalose to drug ratio, tween-80 to drug ratio, inlet air temperature and feed flow rate.

MA

Responses measured were moisture content, solubility, product yield and angle of repose. Data analysis by ANOVA test indicated that ratio of trehalose to PPK significantly affected

D

product flow properties and yield, whereas ratio of tween-80 to PPK was found to

TE

significantly affect moisture content, solubility and flow properties of the resultant formulation. Inlet air temperature significantly influenced moisture content while feed flow

CE P

significantly affected moisture content and product yield. The optimized batch of formulation exhibited higher solubility in water as well as various aqueous buffers as compared to pure

AC

polypeptide-k. Through PXRD and SEM, it was inferred that enhanced solubility was due to reduced particle size and increased surface area of the formulation. Keywords: Polypeptide-k, Poor solubility, Spray drying, Solid dispersion, Box Behnken Design, ANOVA

2

ACCEPTED MANUSCRIPT 1. Introduction Polypeptide-k (PPK) is obtained from the dried seeds and ripened fruits of Momordica

T

charantia, family Cucurbitaceae [1]. This phytochemical is known for its anti-diabetic

IP

potential and has gained importance in therapy of diabetes in last few decades. It is available

SC R

in Asian markets as sublingual tablets [1, 2]. Past decade has witnessed extensive research in order to fully explore its antidiabetic potential. PPK shows high homology to human insulin and has been reported to activate the inactive insulin [1]. In addition, it is also known to

NU

rejuvenate pancreas [1]. Till date, the goal of oral delivery of PPK has not been achieved.

MA

Major impediment in this direction has been poor aqueous solubility of PPK which results in its reduced bioavailability. Several approaches have been explored to enhance solubility and

D

dissolution rate of poorly soluble drugs. These include, particle size reduction to micron and

TE

submicron levels [3, 4]; complexation with cyclodextrins [5]; preparation of metastable polymorphs [6]; nanoemulsion; conversion of crystalline drug candidates to amorphous one

CE P

[7-9]; liquisolid compacts [10]; solid dispersion [11] etc. However, all techniques suffer from one or more limitations. Micro or nanoparticles tend to undergo Ostwald’s ripening during

AC

storage, which leads to increase in particle size. Thus in order to improve the stability of micro or nanoparticles concentration of steric (e.g. hydroxypropylmethyl cellulose (HPMC), hydroxypropyl cellulose (HPC), povidone (PVP K-30), pluronics (F68 and F127)) and electrostatic stabilizers like surfactants (polysorbate (Tween-80), sodium lauryl sulfate (SLS)) are required to be added to the formulation [12]. These additives adversely affect the safety profiles of these formulations [4, 13]. Amorphous systems are thermodynamically unstable and tend to recrystallize during manufacturing or storage [14, 15]. Cyclodextrins have generated interest in the fields of pharmaceuticals due to their ability to modify physical, chemical and biological properties of hydrophobic drugs. Cyclodextrins form inclusion complexes with these drugs [16, 17]. However, due to their high molecular

3

ACCEPTED MANUSCRIPT weight, relatively low water solubility and possible parenteral toxicity, the amount of cyclodextrins that can be used in most pharmaceutical formulations is limited [18].

T

Solid-dispersion technique is a simple and economical method and has, till date, proven to be

IP

the most successful in improving the dissolution and bioavailability of poorly soluble, active

SC R

pharmaceutical ingredients. Preparation of solid dispersion involves deposition of drug on the surface of an inert carrier resulting in larger surface area of the drug. This leads to a faster dissolution rate. A variety of hydrophilic inert substances with high surface area have been

NU

employed for the preparation of solid dispersions [11, 19]. Commonly reported excipients

MA

used in preparation of solid dispersion include propylene glycol (PG), polyethylene glycol (PEG) 4000, 6000 and 8000, Tween – 80 and Povidone (PVPK-30) [11]. For converting solid

D

dispersed powders into a free flowing powder and protecting them from heat generated

TE

during drying process, suitable carriers like sugars are added into the formulation. Lactose, mannitol, sorbitol and trehalose etc. [20], have been commonly used for this purpose. They

CE P

have been reported not only to improve flow properties of the powders but also to improve their stability (as cryoprotectant) as well as solubility [21, 22].The technique has proven to be

AC

very useful for protein and peptide drugs. The techniques that are routinely used for the preparation of solid dispersions include solvent evaporation method, melt extrusion, kneading, co-grinding as well as co-precipitation method [11, 23]. Solvent evaporation technique is the most preferred choice because thermal decomposition of drugs or carriers can be prevented due to the relatively low temperatures required for the evaporation of solvents [24]. Choice of drying technique employed to evaporate solvent plays a major role in the product performance. On the other hand, a welldesigned drying protocol also helps in improving the interaction of poorly soluble drug with the inert carriers, which ultimately results in increased solubility and better flow property of the drug. The drying processes routinely used are: rotary evaporation, freeze drying and spray

4

ACCEPTED MANUSCRIPT drying. Amongst these, spray and freeze drying processes have shown better performance in terms of product yield and ease of scale up. These are, in particular, more suitable for drying

T

of proteins and peptides.

IP

The technique of solid dispersion has never been explored to enhance the aqueous solubility

SC R

of PPK [25]. In the present study, an attempt has been made to develop and optimize a solid dispersion system of poorly soluble polypeptide-k using different drug to carrier ratios and altering the conditions for spray drying process. Final product has low water activity and

NU

lesser weight which results in easy storage and transportation. Spray drying technique

MA

involves atomization of a liquid product in hot gas current to obtain instantaneous powder [26, 27, 28, 29]. Air is the most preferred choice as a drying gas, however, in some of the

D

cases nitrogen gas has also been used [30]. Physicochemical properties of the final product

TE

mainly depend on inlet temperature, air flow rate, feed flow rate and type of carrier used. The formulation and processing parameters were optimized using response surface methodology

CE P

(RSM). RSM uses a group of mathematical and statistical techniques to investigate the relationships between response and the independent variables [31-33]. Box Behnken design

AC

was used to investigate ratio of drug to carriers, feed flow and inlet temperature in order to optimize the best formulation. Best formulation was said to be the one which had better solubility, good powder flow, better product yield and least moisture content. 2. Materials and methods 2.1. Materials Polypeptide-k (PPK) was isolated from dried seeds of ripened fruits of Momordica charantia by the procedure reported by Khanna (2004). Poly ethylene glycol 200, 400, 600, 4000 and 6000, propylene glycol (PG), tween 20, 60 and 80, span 20, 40, 60 and 80 and Pluronic F-68 were purchased from Central Drug House (CDH), New Delhi, India. Formic acid, trehalose, mannitol and sorbitol were purchased from Lobachemie, Mumbai, India. Spray dryer,

5

ACCEPTED MANUSCRIPT SprayMate, Jay Instruments and systems, Navi Mumbai, India was used for drying the solid dispersion of PPK. UV-Visible double beam spectrophotometer, UV-1800, Shimadzu, Japan

T

was used for quantitative estimation of the drug. Magnetic Stirrer, REMI, India was used for

SC R

2.2. Isolation of Polypeptide-K from Momordica Charantia

IP

mixing of solutions.

Dried seeds of Momordica charantia were collected from the ripened fruits. As polypeptide-k is a storage protein, it is found in more quantity in the dried seeds. Seeds were ground to a

NU

fine powder. The pulverised seeds were treated for de-oiling with a mixture of water and

MA

acetone in the ratio of 3:1. The powder was dried and suspended in a mixture of water and acetone. The resultant powder was dried and dispersed in quantity sufficient water and pH

D

was adjusted to 9.5 by using ammonium hydroxide. The supernatant was collected by

TE

centrifugation and its pH was adjusted to 3 by using diluted sulphuric acid. The flocculent precipitate so formed was collected and dried. The dried mass was powdered and grounded

CE P

powder was washed with a mixture of water and acetone (3:1) to remove oil, salts and undesirable material till this gave a single spot on HPTLC and TLC [1].

AC

2.3. Preliminary studies to select the suitable solubilizers PEG 200, 400, 600, 4000 and 6000, propylene glycol (PG), tween 20, 40, 60 and 80, span 20, 40, 60 and 80 and Pluronic F-68 were evaluated for their solubility enhancing effect on polypeptide-k (PPK). Liquid solubilizers were used as such, whereas, solid agents (1g each) were dissolved in 10 ml water. In each case, 50 mg of PPK samples were added to 2 ml of solubilizer and the mixtures were vortexed for 2 min at regular time intervals for 48 h. Solutions were centrifuged at 3500 rpm for 15 min and the supernatant was collected. Suitable dilutions were made and solution was passed through 0.45 µm membrane filter. Filtered solutions were analyzed through UV-visible double beam spectrophotometer at 272.23 nm.

6

ACCEPTED MANUSCRIPT 2.4. Preliminary studies to select the suitable solid carrier A suitable carrier is required to adsorb the liquid solution of drug properly and provide free

T

flow without aggregation of the powder [20-22]. Trehalose, mannitol and sorbitol were

IP

evaluated for their flow improving properties. It was found from the solubility studies carried

SC R

out in different aqueous and organic solvents that 10 % v/v formic acid (in water) was the only solvent in which PPK was found to exhibit significant solubility. Though the percentage of formic acid used in the present study was just 10 % v/v in water, which is a very dilute

NU

solution, for safety purpose mask and gloves were used to cover our body parts.

MA

PPK (10g) was added in 10% v/v formic acid solution containing 0.2% of tween-80. Solution was divided into three equal parts and trehalose, mannitol and sorbitol were added separately

D

to one of the parts. Solutions were spray dried at an inlet temperature of 125°C, feed rate 18

TE

ml/min and aspiration air flow rate of 1200 rpm. Dried powder was collected and evaluated for angle of repose, moisture content and particle agglomeration through scanning electron

CE P

microscopy (SEM).

2.5. Design of experiments

AC

A set of experiments with Box–Behnken Design (BBD) was adopted to develop the PPK solid dispersion system by spray drying technique. Initial screening trials were carried out for evaluating the formation and processing aspects of solid dispersion. Results from the initial screening trials suggested that carrier to drug ratio, surfactant to drug ratio, feed flow and inlet air temperature were the main factors which affected the powder flow, moisture content, product yield and solubility. Based on the number of factors and their level, a Box–Behnken design was used to evaluate the effect of formulation and processing parameters affecting the physical properties of solid dispersion system. The four independent factors identified for this study were carrier to drug ratio, surfactant to drug ratio, feed flow and inlet air temperature. All these factors were operated at three levels (+1, 0, −1). The concentration of drug, type of

7

ACCEPTED MANUSCRIPT carrier and type of surfactant were kept same for all the experiments. Design-Expert 9.0.3 software was used to conduct the study. A total of 25 experiments were designed by the

T

software with 2 center points. Experiments were run in random order to increase the

IP

predictability of the model. Table 1 shows the independent factors and their design level used

SC R

in this study. 2.6. Procedure for preparation of solid dispersion system

Results of solubility studies of PPK using various solubilizing agents and flow properties of

NU

powders using three different carriers indicated that PPK was most soluble in tween-80 while

MA

trehalose provided the best flow with least moisture content (see results and discussion section). Hence, tween-80 was selected as solubilizer and trehalose as carrier and

D

cryoprotectant. The use of trehalose as cryoprotectant to retain the structure and thereby

TE

stability of protein has been well proven in previous research [34-36]. It is pertinent to mention here that since the drug is a poorly soluble peptide, combination of tween-80 and

CE P

trehalose would not only enhance its solubility but also to increase its stability to withstand higher temperatures encountered during drying [37]. In order to formulate solid dispersion

AC

system, tween-80 and trehalose were dissolved in aqueous solution of 10% v/v formic acid and then PPK was added in the dispersion. Dispersion was mixed for 30 min homogenously on a magnetic stirrer before subjecting to spray drying. Table 2 enlists the composition of solid dispersion system as mentioned in design of experiment (DoE). Different batches of solid dispersion were dried using spray dryer (Spray Dryer Model: SprayMate, Jay Instruments, Navi Mumbai, India) under the following set of conditions: inlet temperature, 120-130°C; recorded outlet temperature was in the range of 60-65°C; and feed rate, 16-20 ml/min (Table 1). 2.7. Evaluation of prepared solid dispersion system 2.7.1. Moisture content determination

8

ACCEPTED MANUSCRIPT Samples were weighed and wet weight of samples were recorded. Wet samples were then dried to a constant weight, at a temperature not exceeding 101°C using hot air oven and

T

subsequently allowed to cool. Cooled samples were weighed again, and dry weight of the

IP

samples were recorded. Moisture content of the samples was calculated using the following

SC R

equation: %W = (A-B)/B × 100

Eq. (1)

%W = Percentage of moisture in the sample,

B = Weight of dry sample (grams)

MA

A = Weight of wet sample (grams), and

NU

Where:

D

2.7.2. Calculation for assay and equivalent weight of PPK to be taken from solid dispersion

TE

for solubility studies

In order to calculate the percentage of PPK present in the solid dispersion, assay of

CE P

formulation was carried out by the following procedure. Solid dispersion (1 g) was taken and dissolved in 10 % v/v formic acid. From this, 5 mL of solution was withdrawn and filtered

AC

through 0.2 µm filter. The solution was diluted to 10 mL using 10 % v/v formic acid and subjected for UV analysis 272.23 nm and % drug content was calculated. The assay of PPK in the solid dispersion was found 85 %. Amount of PPK added in 1g of solid dispersion (before spray drying) was 0.50 g (i.e. 500 mg). Amount of PPK recovered in 1g of solid dispersion (after spray drying) was 0.425 g (i.e. 425 mg) Hence, in order to take PPK equivalent to 0.05g (i.e. 50 mg), 0.118 g (118 mg) of solid dispersion was taken for solubility studies.

9

ACCEPTED MANUSCRIPT 2.7.3. Solubility studies For design of experiments solubility studies were carried out in flask containing 200 ml

T

double distilled water as per the design shown in Table 3.

IP

In order to compare the enhancement in solubility of prepared solid dispersion systems (PPK-

SC R

SD) with respect pure PPK in distilled water as well as different aqueous buffers, PPK (50 mg) and its equivalent powder from optimized batch of solid dispersions (PPK-SD) prepared by spray drying were added separately to a flask containing 200 ml double distilled water;

NU

0.1N HCl pH 1.2, acetate buffer pH 4.6 and 5.0, phosphate buffer pH 6.8, and, alkaline borate

MA

buffer pH 9.6, respectively.

Flasks were shaken on a mechanical shaker for 48 h at room temperature. The solutions were

D

then filtered (Cut-off 0.2µm, Ministart SRP 25, Sartorius) and analyzed. The studies were

TE

performed in triplicate.

CE P

2.7.4. Bulk Drug Properties 2.7.4.1. Angle of repose

The static angle of repose, α, was measured according to the Fixed Funnel and Free Standing

AC

Cone method. A funnel was clamped with its tip 7 cm above a graph paper placed on a flat horizontal surface. The powders were carefully poured through the funnel until the apex of the cone, thus formed, just reached the tip of the funnel. Mean diameters of the base of the powder cones were determined and tangent of the angle of repose calculated using the equation [38, 39]: tan α = 2h/D

Eq. (2)

Where, h is the height of the heap of powder and D is the diameter of the base of the heap of powder. 2.7.4.2. Bulk Density

10

ACCEPTED MANUSCRIPT Apparent bulk density (ρb) was determined by pouring the blend into a graduated cylinder. Bulk volume (Vb) and weight of the powder (M) from optimized batch (PPK-SD) was

ρb = M/Vb

IP

Eq. (3)

T

determined. Bulk density was calculated using the formula [40, 41].

SC R

2.7.4.3. Tapped Density

The measuring cylinder containing a known mass of blend was tapped 100 times. Minimum volume (Vt) occupied by the blend in the cylinder and the weight (M) of the blend (optimized

NU

batch, PPK-SD) was measured. Tapped density (ρt) was calculated using the following

MA

formula [42, 43]. ρt = M/ Vt

D

2.7.4.4. Compressibility Index

Eq. (4)

TE

Carr’s Compressibility index is an indirect measure of bulk density, size and shape, surface area, moisture content and cohesiveness of a material as all these factors can influence the

formula [42, 43].

CE P

compressibility index. Carr’s compressibility index was calculated using the following

AC

CI= Bulk density – Tap density/Bulk density x 100

Eq. (5)

2.8. Powder X-ray diffraction analysis Powder X-ray diffraction (PXRD) patterns of pure PPK and PPK-SD were recorded using an X-ray diffractometer (Bruker axs, D8 Advance) with Cu line as the source of radiation. Standard runs using a 40-kV voltage, a 40-mA current, at a scanning rate of 0.010°min-1 over a 2θ range of 3–45° were used [4]. 2.9. Scanning Electron Microscopy (SEM) SEM was carried out as reported in Renuka et al. (2014). The surface morphology of the pure PPK and PPK-SD were studied by scanning electron microscopy (SEM). A metallic stub with double-sided conductive tape of 12 mm diameter was taken and the samples were 11

ACCEPTED MANUSCRIPT fixed over it. A Supra 35 VP (Oberkochen, Zeiss, Germany) data station with an acceleration voltage of 1.00 kV and a secondary detector was used in the study [6].

T

2.10. Particle size measurement

IP

The particle size of optimized batch of solid dispersion and initial grounded powder was

SC R

measured using Malvern Zetasizer Version 7.11. Both the powders were dispersed in water and diluted 100 times using water only. Each sample was measured at least three times. The

2.11. Differential scanning calorimetry

NU

average values of particle size of 100 time diluted samples were recorded.

In order to check the degradation of polypeptide-k in the optimized formulation, DSC was

MA

carried out for pure PPK and optimized formulation. About 1 mg of pure polypeptide-k and optimized formulation were crimped separately in an aluminium pan. Each sample was

D

heated from 0 to 3000C at a heating rate of 10 0C/min under a stream of nitrogen at a flow rate of 50 ml/min. An empty aluminium pan was used as reference. The melting points (Tm)

TE

were determined using TA-Universal Analysis 2000 software (version 4.7A).

CE P

2.12. Fourier Transform Infrared Spectroscopy (FT-IR) Further structural degradation of polypeptide-k in the optimized formulation was checked through Fourier Transform Infrared Spectroscopy (FTIR). The KBr sample discs were

AC

prepared using 1 mg of pure polypeptide-k and optimized formulation were compressed individually into discs under pressure of 10,000 to 15,000 pounds per square inch. The infrared spectrum was recorded in the wave number range of 4000-400 cm-1 using FTIR spectrophotometer and the characteristic bands of pure polypeptide were compared with optimized formulation. 3. Results and discussions 3.1. Selection of solubilizer Solubilities of PPK achieved by using different solubilizers are shown in Fig. 1. As maximum solubility of PPK was observed with tween-80 (544.86 µg/mL), it was selected as the solubilizer for preparing solid dispersion of PPK. 3.2. Evaluation of solid carriers 12

ACCEPTED MANUSCRIPT The ratio of various carriers to drug was varied as - 0.025-1, 0.050-1, 0.075-1, 0.10, 0.125 w/w, and evaluated for angle of repose. The results are shown in Fig.2. It appeared that with

T

an increase in the content of carriers, angle of repose value was increasing. For trehalose the

IP

angle of repose value was found least at all the ratios as compared to other carriers, whereas,

SC R

for sorbitol the angle of repose value was found maximum. It was also observed that with increase in concentration of carriers, agglomeration of particles also increased. The increase in angle of repose with increase in amount of carriers in the formulation could be due to

NU

increase in particle agglomeration. Since, trehalose has shown comparatively better flow

MA

properties, it was selected for final formulation development. It was also observed from Fig.2 that there was no much change in angle of repose value of formulations containing trehalose

D

as carrier with increase in amount of trehalose.

TE

Furthermore, to justify selection of trehalose as suitable carrier exploratory studies has been carried out, wherein, trehalose, mannitol and sorbitol at constant ratio (keeping carrier to drug

CE P

ratio 0.075-1.0 w/w) were evaluated. The value of carrier to drug ration was taken 0.075-1 w/w for comparison because at this value angle of repose value for all the three carriers used

AC

were nearby and comparable. Hence, by comparing other parameters like moisture content and particle judicious The parameters of angle of repose, moisture content and particle agglomeration of the prepared dispersion were studied. Among the three solid carriers, trehalose was found to yield best flow properties [44]. Angle of repose of PPK solid dispersion with trehalose was found to be 26.22°. Dispersion had least moisture content and did not exhibit any agglomeration or lump formation. The results are shown in Table 4. 3.3. Effect of independent factor on moisture content (Y1), product yield (Y2), solubility (Y3) and angle of repose (Y4) A total of 25 experiments were carried out to study the effect of formulation and processing parameters on moisture content (Y1), product yield (Y2), solubility (Y3) and angle of repose

13

ACCEPTED MANUSCRIPT (Y4) of solid dispersion system. Response data for all experimental runs of Box–Behnken experimental design is presented in Table 3.

T

The responses obtained for this study are well modelled by a linear function of the

IP

independent variables; hence first order polynomial equation was used for approximating the

SC R

function as shown in Eq. (6). Y = β + β1X1 + β2X2 + β3X3 + β4X4 + €

Eq. (6)

Where, € represents noise or error, X represents independent variable, Y represents response

NU

and β represents coefficient. The value of various responses like moisture content (Y1),

MA

product yield (Y2), solubility (Y3) and angle of repose (Y4) ranges from 0.01 to 0.1 %, 34.48 to 66.18 %, 4.82 to 8.02 mg/ml and 16.12 to 26.22°, respectively. Ratio of maximum to

D

minimum for the four responses, Y1, Y2, Y3 and Y4 is 10, 1.92, 1.66 and 1.63 respectively.

TE

Since a value above 10 indicates the requirement of power transformation, in all the four cases power transformation was not applied. Analysis of variance (ANOVA) was applied to

CE P

determine the significance and magnitude of the effects of the main variables and their interactions. Regression model thus obtained was used to generate the counter plots for

AC

independent factors. ANOVA table confirms the adequacy of the model (i.e. F < 0.05) as shown in Table 5. It also identifies the significant factors that affect the responses Y1-Y4 of solid dispersion.

The perturbation plots for all factors and responses are shown in Figs. 3 A-D. It is pertinent to add here that a steep slope or curvature in a plot shows that the response is sensitive to that particular factor. A relatively flat line shows lack of dependence of response on the said factor [4]. Results show that ratio of tween-80 to PPK (B), inlet air temperature (C) and feed flow (D) are the major factors influencing moisture content (Y1) of the drug product (Fig. 3A). However, for product yield (Y2), the critical factors include ratio of trehalose (A) to PPK and feed flow rate (D) (Fig. 3B). Product yield was also found to be influenced by inlet

14

ACCEPTED MANUSCRIPT air temperature (C). However, the influence was not very significant. As shown in Fig. 3C, there was a significant influence of the ratio of tween-80 to PPK (B) on the solubility of PPK

T

(Y3). Factors A, C and D also influenced solubility, however, the effects were not significant.

IP

Fig. 3D indicates that ratio of trehalose to PPK (A) and ratio of tween-80 to PPK (B)

SC R

influenced angle of repose (Y4) to a greater extent. The effect of inlet air temperature (C) and feed flow rate (D) on angle of repose (Y4) was found to be negligible. Final mathematical model in terms of coded factors as determined by Design-Expert software

NU

is shown below in Eqs. 7-10 for responses Y1, Y2, Y3 and Y4, respectively.

MA

Y1 (Moisture content) = + 0.040 – 8.33 * A + 0.010 * B – 0.014 * C + 1.00 * D

Eq. (7)

Y2 (Product Yield) = + 50.75 + 3.05 * A – 1.03 * B - 0.47 * C – 10.65 * D

Eq. (8)

Y3 (Solubility) = + 6.17 – 0.21 * A + 1.12 * B + 0.024 * C – 0.12 * D

Eq. (9) Eq.

TE

D

Y4 (Angle of repose) = + 21.91 – 2.18 * A + 1.51 * B – 0.20 * C - 0.49 * D (10)

effect [4].

CE P

A positive sign represents the synergistic effect, while a negative sign indicates antagonistic

AC

In the case of response Y1 (Moisture content), negative coefficients of A and C of the model indicate a decrease in moisture content on increasing trehalose to PPK ratio (A) and higher level of inlet air temperature (C), respectively (Eq. (7)). On the other hand, positive coefficients of B and D point towards increase in moisture content with increase in ratio of tween-80 to PPK and feed flow rate, respectively. Decrease in moisture content with increase in trehalose content is attributed to better moisture adsorbing capacity of trehalose, which accounts for better flow of solid dispersion. Decrease in moisture content with increase in inlet air temperature is directly related to the loss of water from the formulation with increase in temperature. Increase in moisture content with increase in tween-80 concentration is linked to the liquid nature of tween-80 which completely wets the formulation. Moisture content

15

ACCEPTED MANUSCRIPT was found to be increased with increase in feed flow rate. Increased feed flow rate introduces relatively larger volume of solution in the drying chamber resulting in inefficient heat transfer

T

to liquid droplets. Increase in moisture content in turn, increases the chances of aggregation

IP

of particles in the dispersion.

SC R

In case of response Y2 (product yield), negative coefficients of B, C and D of the model refer to decreased product yield at higher level of tween-80 to PPK ratio and higher level of inlet air temperature and higher feed flow, respectively (Eq. (8)). However, the positive coefficient

NU

of A indicates the increase in product yield with increase in ratio of trehalose to PPK. Initial

MA

trials carried out to evaluate the important variables that could affect the product yield indicated sharp decline in product yield when drying was carried out without trehalose.

D

Addition of trehalose increased the yield upto 50%. Similar results were obtained after

TE

applying design of experiments. Thus, addition of trehalose to the formulation was found to increase overall product yield, decrease angle of repose and thus positively affected the flow

CE P

of prepared dispersion. In addition, it also acted as a cryoprotectant. The decrease in product yield (Y2) due to higher inlet temperature (C) and feed rate (D) was observed. This is

AC

probably due to slow heat and mass transfer with higher feed flow rate. Moreover, when higher feed flow rates were used, part of the feed passed straight to the chamber without atomization resulting in a higher of process waste and a lower process yield [45, 46]. Similar results were observed by Toneli et al. (2006), who have reported spray drying of inulin. An increase in the mass production was observed with decreasing pump speeds, which is lower feed flow rate [45]. Increase in factors B and C increased the aqueous solubility (Y3) of PPK (Eq. (9)). Tween-80 decreased the interfacial tension between poorly soluble drug (PPK) and aqueous medium, resulting in increased solubility of PPK. With increase in temperature, the interaction of tween-80 and PPK might have increased in the drying chamber and the resulting product so

16

ACCEPTED MANUSCRIPT formed would exhibit higher solubility. Decrease in solubility with increase in ratio of trehalose to PPK (A) could be attributed to the fact that the increase in the trehalose would

T

reduce the interaction between solubilizer (tween-80) and PPK. At lower concentrations of

IP

trehalose, more tween-80 will be absorbed on the surface of PPK. As trehalose competes with

SC R

tween-80 for adsorption on the surface of PPK, increasing trehalose ultimately results in decrease adsorption of tween-80 on the surface of PPK, thereby reducing its solubility.

ultimately decreased the solubility of PPK.

NU

Increase in feed flow (D) reduced the interaction time between tween-80 and PPK, which

MA

Angle of repose (response Y4) decreased with increase in trehalose to PPK ratio (A) and inlet air temperature (B). However, as shown in Fig. 3D, the influence of inlet air temperature and

D

feed flow rate was negligible on flow properties. Hence, it can be inferred that trehalose plays

TE

a major role in improving the flow of spray dried powder. There are a number of reports in literature that indicate the potential of trehalose in improving flow properties of powders over

CE P

other cryoprotectants [20, 47, 48]. Reducing sugars like lactose monohydrate have an impact on the stability of proteins and peptides as they tend to interact with the functional groups of

AC

proteins and peptides [49, 50]. In such cases polyhydric alcohols like sorbitol, mannitol or erythritol prove to be better alternatives. However, they fail to produce efficient dispersion of drugs [51]. Trehalose is a disaccharide and is classified under non reducing sugars. It is reported to increase the stability of proteins and peptides by reducing their degradation. It also increases powder flow by inhibiting agglomeration and aids efficient dispersion of drug in the formulation [20]. The use of trehalose with polysorbates (e.g. tween-80) is reported to further enhance the stability of proteins and peptides [37]. It also reduces the aggregation of powder, which could be one of the reasons for decrease in angle of repose with increase in trehalose concentration in the formulation. Angle of repose lesser than 30 indicates good flow of powders [44].

17

ACCEPTED MANUSCRIPT 3.4. Optimization of parameters using graphical optimization method A total of 25 trials were performed to optimize the parameters for preparing solid dispersion

T

of PPK. Factors A–D were used in such a manner that final moisture content (Y1) was in the

IP

range of 0.01 to 0.1 %, product yield (Y2) in the range of 34.48 to 66.18 %, solubility (Y3) in

SC R

the range of 4.82-8.02 mg/mL and angle of repose (Y4) was in the range of 16.12 to 26.22°, respectively. For a batch size of 5 g, the values of Y1-Y4 were predicted in the required range, where A (ratio of trehalose to PPK) was 0.075 to 1 w/w , B (ratio of tween-80 to PPK)

NU

was 0.15 to 1 w/w, C (inlet air temperature) was 125°C and D (feed flow rate) was 18

MA

mL/min . By using these values of factors three different batches of solid dispersions of PPK were prepared. Fig. 4 represents an overlay plot showing the optimized parameters suggested

D

by DoE software to get the responses in required range.

TE

3.5. Evaluation of physicochemical and bulk drug properties of optimized batch The optimized batch of PPK-SD was evaluated for moisture content, angle of repose, bulk

CE P

and tapped density and compressibility index. The results are shown in Table 6. All the parameters were found to be within pharmacopoeial limits.

AC

3.6. Solubility studies of optimized batch (PPK-SD) In order to evaluate the improvement in solubility of PPK by solid dispersion technique, pure PPK and PPK-SD were subjected to solubility studies. The result of these studies is shown in Fig. 5. A marked increase in solubility of PPK-SD was observed in water as well as all the aqueous buffers as compared to pure PPK. In water about 50.6 % enhancement in solubility was observed. About 21.65, 72.11, 33.33, 23.29, 38.38 and 65.42 % increase in solubility for PPK-SD over pure PPK was observed in pH 1.2, 4.6, 5.0, 6.8, 7.4 and 9.6 buffers respectively. 3.7. Powder X-ray diffraction (PXRD) studies

18

ACCEPTED MANUSCRIPT The PXRD patterns of pure PPK and solid dispersions (PPK-SD) are shown in Figs. 6a and 6b. Diffraction pattern of the solid dispersion indicates changes in the crystalline nature of the

T

drug. The diffraction pattern of pure PPK shows a sharp peak at 21.66° with 100 % relative

IP

intensity. For PPK-SD samples, appearance of sharp peak at 21.60° with 100 % relative

SC R

intensity indicated that no polymorphic changes occurred in PPK during the spray drying process. Minor decrease in peak intensity is attributed to decrease in the particle size of drug during spray drying (which was further confirmed by SEM analysis) as well as complete

MA

3.8. Scanning Electron Microscopy (SEM)

NU

miscibility of PPK with trehalose and tween-80.

Fig. 7a shows the crystalline structure of pure PPK with average diameters of 8.57 μm. Fig.

D

7b shows solid dispersions of PPK (PPK-SD), which revealed irregular structure due to the

TE

porous nature of the carrier with the fine particles of the drug deposited on it. Moreover, a reduction in the particle size of PPK was also observed in PPK-SD formulation. Thus,

CE P

reduced particle size, increased surface area and closer contact between the hydrophilic carriers and the drug, could be the major attributing factors for enhanced solubility of PPK in

AC

solid dispersions (PPK-SD) [52]. In previous contributions also it has been proven that the solubility of poorly soluble drugs have been improved by formulation of solid dispersion systems wherein the reduced particle size without agglomeration of fine particles increased the exposed surface area of the drug [53, 54]. In addition, the carrier material may contribute to increase the solubility/dissolution rate through its solubilizing and wettability-enhancing properties [53]. There are several researches in which different methods have been reported to reduce the particle size of drugs such as micronization, recrystallization, freeze drying and spray drying [53]. However, such fine particles produced by these techniques may cause aggregation and agglomeration of fine particles. In addition, poor wettability of fine powders may reduce the solubility/dissolution rate [55]. Through the application of solid dispersion in

19

ACCEPTED MANUSCRIPT combination with spray drying/freeze drying one can achieve fine particles without or, minimum agglomeration [53]. Judicious selection of ratio of carriers and surfactants (like

T

tween-80 used in the present study) in the formulation one can control the particle size of

IP

prepared solid dispersion of a poorly soluble drug [3, 11, 12].

SC R

3.9. Particle size analysis

The average particle size of optimized batch of solid dispersion and initially grounded powder of PPK was found to be 25.31 nm (0.02531µm) and 1222 nm (1.222µm),

NU

respectively. The polydispersity index (PDI) of optimized batch of solid dispersion (A) and

MA

initially grounded powder of PPK (B) is shown in Figs.8a and 8b. The particle size analysis revealed that the particle size of optimized batch of solid dispersion is about 50 times smaller

D

than that of initially grounded powder of PPK. Hence the findings of SEM analysis is well

TE

supported by particle size analysis. It is pertinent to note here that the PDI value of prepared

distribution.

CE P

solid dispersion (Fig.8a) is found to be 0.489 which is an indication of very good particle size

3.10. DSC analysis

AC

The DSC thermograms of pure PPK showed the single melting peak of drug at 103.310C. The melting peak of optimized formulation containing PPK (PPK-SD) was observed at 102.260C. There were no additional melting peaks observed in the DSC thermograms before as well as after the melting peak of PPK-SD. Moreover, no shift in the melting peak was observed which could evidence about degradation or, polymorphic behaviour of PPK during melting. This confirmed that the PPK-SD did not degrade during spray drying process. The overlay of DSC thermograms of pure PPK and PPK-SD is shown in Fig. 9. 3.11. Fourier Transform Infrared (FTIR) Spectroscopy The vibrational frequency peaks for pure PPK and optimized formulation (PPK-SD) are shown in Fig. 10. Since PPK contains 18 amino acids linked with peptide linkage, Fig.10

20

ACCEPTED MANUSCRIPT shows the presence of vibrational peaks of acidic carbonyl group (- C = O) at 1650 – 1660 cm-1, amino group at 3284 cm-1. It can be clearly observed from the figure that vibrational

T

frequencies of all major peaks are matching for pure PPK and optimized formulation

IP

containing PPK (PPK-SD). This confirms the DSC findings that PPK did not get degraded

SC R

during spray drying process which could be due to the presence of trehalose. 4. Conclusion

NU

The study presents the application of quality by design in optimization of solid dispersion formulation and process variables of polypeptide-k (PPK-SD) prepared by spray drying

MA

technique. It was observed that ratio of trehalose to PPK significantly affected the product flow properties (angle of repose) and yield. The ratio of tween-80 to PPK was found to

D

significantly affect moisture content, solubility and angle of repose of the developed solid

TE

dispersion system, whereas, inlet air temperature significantly influenced moisture content and feed flow significantly affected moisture content and product yield. Enhanced solubility

CE P

in water as well as aqueous buffers of different pH was achieved in the optimized formulation. Hence, it can be concluded that prepared solid dispersion system with enhanced

AC

solubility can further enhance oral bioavailability of polypeptide-k, when administered orally. However, it has yet to be proven by in-vivo studies. Acknowledgements

We are highly thankful to Science and Engineering Research Board, Department of Science and Technology, New Delhi, India for providing financial support for this project under Fasttrack Young Scientist Scheme (SB/YS/LS – 102/2013). Conflict of interest Declared none. References

21

ACCEPTED MANUSCRIPT [1]. P. Khanna, Polypeptide-k extracted from Momordica charantia and a process for extraction. Momordica charantia L., Its method of preparation and uses. US 6, 831, 162

T

(2004).

IP

[2]. Y.Y. Sirn, L. C. Lok, Z. Ahmad, M. N. Hakim, Improved Blood Glucose Level

SC R

Associated with Polypeptide-K (Diabegard®), A Polypeptide Isolated from the Seeds of Momordica charantia Supplementation: Evaluation of 6 Cases, Int. J. Pharm. Sci. Rev. Res. 25 (2014)147-150.

NU

[3]. S.K. Singh, K.K. Srinivasan, K. Gowthamarajan, D.S. Singare, D. Prakash, N.B.

MA

Gaikwad, Investigation of preparation parameters of nanosuspension by top-down media milling to improve the dissolution of poorly water-soluble glyburide, Eur. J. Pharm.

D

Biopharm. 78 (2011) 441–446.

TE

[4]. K.V. Mahesh, S.K. Singh, M. Gulati, A comparative study of top-down and bottom-up

436-449.

CE P

approaches for the preparation of nanosuspensions of glipizide, Powder Technol. 256 (2014)

[5]. S. K. Singh, K. K. Srinivasan, D. S. Singare, K. Gowthamarajan, D. Prakash,

AC

Formulation of ternary complexes of glyburide with hydroxypropyl-β-cyclodextrin and other solubilizing agents and their effect on release behavior of glyburide in aqueous and buffered media at different agitation speeds, Drug Dev. Ind. Pharm. 38 (2012) 1328-1336. [6]. Renuka, S. K. Singh, M. Gulati, I. Kaur, Characterization of solid state forms of glipizide, Powder Technol. 264 (2014) 365-376. [7] K. Löbmann, C. Strachan, H. Grohganz, T. Rades, O. Korhonen, R. Laitinen, Coamorphous simvastatin and glipizide combinations show improved physical stability without evidence of intermolecular interactions, Eur. J. Pharm. Biopharm. 81 (2012) 159-169.

22

ACCEPTED MANUSCRIPT [8] N. Chieng, J. Aaltonen, D. Saville, T. Rades, Physical characterization and stability of amorphous indomethacin and ranitidine hydrochloride binary systems prepared by

T

mechanical activation. Eur. J. Pharm. Biopharm. 71 (2009) 47–54.

IP

[9] M. Allesø, N. Chieng, S. Rehder, J. Rantanen, T. Rades, J. Aaltonen, Enhanced

SC R

dissolution rate and synchronized release of drugs in binary systems through formulation: amorphous naproxen–cimetidine mixtures prepared by mechanical activation, J. Control. Rel. 136 (2009) 45–53.

NU

[10] S. K. Singh, K. K. Srinivasan, K. Gowthamarajan, D. Prakash, N. B. Gaikwad, D. S.

MA

Singare, Influence of formulation parameters on dissolution rate enhancement of glyburide using liquisolid technique, Drug Dev. Ind. Pharm. 38 (2012) 961–970.

D

[11] S. K. Singh, K. K. Srinivasan, K. Gowthamarajan, D. Prakash, N. B. Gaikwad,

TE

Investigation of preparation parameters of solid dispersion and lyophilization technique in order to enhance the dissolution of poorly soluble glyburide, J. Pharm. Res. 4 (2011) 2718-

CE P

2723.

[12] D.S. Singare, S. Marella, K. Gowthamarajan, G.T. Kulkarni, R. Vooturi, P.S. Rao,

AC

Optimization of formulation and process variable of nanosuspension: an industrial perspective, Int. J. Pharm. 402 (2010) 213–220. [13] B.V. Eerdenbrugh, G. Van den Mooter, P. Augustijns, Top-down production of drug nanocrystals: nanosuspension stabilization, miniaturization and transformation into solid products, Int. J. Pharm. 364 (2008) 64–75. [14] B. C. Hancock, G. Zografi, Characteristics and significance of the amorphous state in Pharmaceutical systems, J. Pharm. Sci. 86 (1997) 1–12. [15] G.G.Z. Zhang, D. Law, E.A. Scmitt, Y. Qiu, Phase transformation considerations during process development and manufacture of solid oral dosage forms, Adv. Drug Deliv. Rev. 56 (2004) 371–390.

23

ACCEPTED MANUSCRIPT [16] J. Szejli, Cyclodextrin Technology. Dordrecht: Kluwer Academic Publishers, Netherlands (1988).

T

[17] K. Uekama, F. Hirayama, T. Irie, Cyclodextrin drug carrier systems. Chem. Rev. 98

IP

(1998) 2045–2076.

SC R

[18] T. Loftsson, M.E. Brewster, Pharmaceutical applications of cyclodextrins. Drug solubilization and stabilization, J. Pharm. Sci. 85 (1996) 1017–1025. [19] O.E. Cassidy, C. Rouchotas, Comparison of surface modification and solid dispersion

NU

techniques for drug dissolution, Int. J. Pharm. 195 (2000) 1–6.

MA

[20] H. M. Mansour, Z. Xu, A. J. Hickey, Dry powder aerosols generated by standardized entrainment tubes from alternative sugar blends: 3. Trehalose dehydrate and D-mannitol

D

carriers. J. Pharm. Sci. 99 (2010) 3430-3441.

TE

[21] J. S. Patton, M. J. Bossard, Drug delivery strategies for proteins and peptides from discovery and development to life cycle management, Pharm. Dev. Technol. 4 (2004) 73-77.

CE P

[22] J. S. Patton, R. M. Platz, Pulmonary delivery of peptides and proteins for systemic action, Adv. Drug Deliv. Rev. 8 (1992) 179-228.

AC

[23] L. A. Nikghalb, G. Singh, G. Singh, K. F. Kahkeshan, Solid Dispersion: Methods and Polymers to increase the solubility of poorly soluble drugs, J. Appl. Pharm. Sci. 2 (10) 170175.

[24] S. A. Mogal, P. N. Gurjar, D. S. Yamgar, A. C. Kamod, Solid dispersion technique for improving solubility of some poorly soluble drugs, Der Pharm. Lett. 4 (2012) 1574-1586. [25] R. Kumar, S. Duggal, U. R. Lal, P. Khanna, M. Gulati, U. Shankar, Preparation and Characterization of Aquasomes Loaded with Antidiabetic Polypeptide from Momordica charantia seeds, In: P. Khanna (Ed.), Polypeptide K and Biterin Oil, Melrose Books, Cambridgeshire, Vol. 1, 2011, ISBN: 9781907732218.

24

ACCEPTED MANUSCRIPT [26] V. Patil, A. K. Chauhan, R. P. Singh, Optimization of the spray-drying process for developing guava powder using response surface methodology. Powder Technol. 253 (2014)

T

230–236.

IP

[27] R. Murugesan, V. Orsat, Spray drying for the production of nutraceutical ingredients - a

SC R

review, Food Bioprocess Technol. 8 (2011) 1–12.

[28] F. Wu, X. Li, Z. Wang, H. Guo, Z. He, Q. Zhang, X. Xiong, P. Yue, Low-temperature synthesis of nano-micron Li4Ti5O12 by an aqueous mixing technique and its excellent

NU

electrochemical performance, J. Power Sources 202 (2012) 374-379.

MA

[29] F. Wu, Z. Wang, X. Li, H. Guo, P. Yue, X. Xiong, Z. He, Q. Zhang, Characterization of spherical-shaped Li4Ti5O12 prepared by spray drying, Electrochimica Acta 78 (2012) 331-

D

339.

TE

[30] A. Gharsallaoui, G. Roudaut, C.O. Voilley, R. Saurel, Applications of spray-drying in microencapsulation of food ingredients: an overview, Food Res. Int. 40 (2007) 1107–1121.

CE P

[31] S. C. Ee J. Bakar, K. Muhammad, D. M. Hashim, N. Adzahan, Spray-Drying Optimization for Red Pitaya Peel (Hylocereus polyrhizus), Food Bioprocess Technol. 6

AC

(2013) 1332–1342.

[33] A. P. Mestry, A. S. Mujumdar, B. N. Thorat. Optimization of Spray Drying of an Innovative Functional Food: Fermented Mixed Juice of Carrot and Watermelon. Drying Technol. 29 (2011) 1121-1131. [33] M. Selvamuthukumaran, F. Khanum, Optimization of spray drying process for developing seabuckthorn fruit juice powder using response surface methodology, J. Food Sci. Technol. 51 (2014) 3731- 3739. [34] A. D. Elbein , Y. T. Pan , I. Pastuszak, D. Carroll. New insights on trehalose: a multifunctional molecule. Glycobiology. 13 (2003) 17R-27R.

25

ACCEPTED MANUSCRIPT [35] R. D. Lins, C. S. Pereira, P. H. Hünenberger. Trehalose-protein interaction in aqueous solution, Proteins. 55 (2004) 177-186.

T

[36] Jain N.K., I. Roy, Effect of trehalose on protein structure, Protein Sci. 18 (2009) 24–36.

of

Stable

Protein

Formulations

Pharmaceutical

Biotechnology.

Kluwer

SC R

Design

IP

[37] J. Lee, Spray drying of proteins. In: J. F. Carpenter, M. C. Manning (Eds.). Rational

Academic/Plenum Publishers, New York, Volume 13, 2002, pp 135-158. [38] G. K. Jain, N. Jain, G. S. Banker, N. R. Anderson, K. Marshall, J. A. Seitz, S. P. Mehta,

NU

J. L. Yeager, Tablets. In: Khar RK, Vyas SP, Ahmad FJ, Jain GK (Eds). Lachman/Lieberman’s

MA

The Theory and practice of Industrial Pharmacy. CBS Publishers and Distributors, India, 2014, pp 449-545.

D

[39] F. O. Ohwoavworhua, E. O. Ogah, O. O. Kunle, Preliminary investigation of

TE

physicochemical and functional properties of alpha cellulose obtained from waste paper- a

CE P

potential pharmaceutical excipient, J. Raw. Mat. Res. 2 (2005) 84-93. [40] A. Martin, P. Bustamante, A. H. C. Chun, Micromeritics, In: P. J. Sinko (Ed), Physical

AC

pharmacy, Lippincott Williams & Wilkins, USA, 2011, pp. 442-68. [41] M. K. Modasiya, I. I. Lala, B. G. Prajapati, V. M. Patel, D. A. Shah, Design and characterization of fast disintegrating tablets of Piroxicam, Int. J. Pharm. Technol. Res. 1 ( 2009) 353-57. [42] R. L. Carr. Classifying flow properties of solids, Chem. Eng. 72 (1965) 69-72. [43] Indian Pharmacopoeia, The Controller of Publication, Ministry of Health and Family Welfare, India, 2010, pp 751-54. [44] C. V. S. Subrahmanyam, Dissolution, In: M. K. Jain, (Ed.), Textbook of Physical Pharmaceutics, Vallabh Prakashan, Delhi, India, Vol. 1, 2000, pp 222-224.

26

ACCEPTED MANUSCRIPT [45] J. Toneli, K. J. Park, F. Murr, A. Negreiros, Spray drying optimization to obtain inulin powder. In Proceedings of the 15th International Drying Symposium, Budapest, Hungary,

T

2006.

IP

[46] L. H. Tee, A. C. Luqman, K. Y. Pin, A. A. Rashih, Y. A. Yusof, Optimization of spray

SC R

drying process parameters of Piper betle L. (Sirih) leaves extract coated with maltodextrin., Journal of Chemical and Pharmaceutical Research, 4 (2012) 1833-1841.

NU

[47] H. Hamishehkar, J. Emami, A. R. Najafabadi, K. Gilani, M. Minaiyan, H. Mahdavi, A. Nokhodchi, (2010). Influence of carrier particle size, carrier ratio and addition of fine ternary

Powder Technol. 201 (2010) 289-295.

MA

particles on the dry powder inhalation performance of insulin-loaded PLGA microcapsules.

D

[48] D. Cline, R. Dalby, Predicting the quality of powders for inhalation from surface energy

TE

and area. Pharm. Res. 19 (2002) 1274-1277.

CE P

[49] S. Li, T.W. Patapoff, D. Overcashier, C. Hsu, T.H. Nguyen, R. T. Borchardt, Effects of reducing sugars on the chemical stability of human relaxin in the lyophilized state. J. Pharm. Sci. 85 (1996) 873-877.

AC

[50] D. C. Dubost, J. A. Zimmerman, M. J. Bogusky, A. B. Coddington, S. M. Pitzenberger, Characterization of a solid state reaction product from a lyophilized formulation of a cyclic heptapeptide: a novel example of an excipient-induced oxidation, Pharm. Res. 13 (1996) 1811-1814. [51] S.K. Tee, C. Marriott, X. M. Zeng, G. P. Martin, The use of different sugars as fine and coarse carriers for aerosolised salbutamol sulphate, Int. J. Pharm. 208 (2000) 111-123. [52] A.A. Abd. Elbary, H. F. Salem, M. E. Maher, In vitro and in vivo Evaluation of Glibenclamide using Surface Solid Dispersion (SSD) Approach, Br. J. Pharmacol. Toxicol. 2 (2011), 51- 62.

27

ACCEPTED MANUSCRIPT [53] S. Singh, R.S. Baghel, L. Yadav, A review on solid dispersion, Int. J. Pharm. Life Sci. 2 (2011) 1078-1095.

T

[54] A.B.T.M. Serajuddin, Solid Dispersion of Poorly Water-Soluble Drugs: Early Promises,

IP

Subsequent Problems, and Recent Breakthroughs, J. Pharm. Sci. 88 (1999) 1058 – 1066.

SC R

[55] D.W. Bloch, P.P. Speiser, Solid dispersions – fundamentals and examples, Pharm Acta

AC

CE P

TE

D

MA

NU

Helv. 62 (1987) 23-27.

28

ACCEPTED MANUSCRIPT List of Figures Fig.1. Solubility results of PPK in various solubilizers. Fig.2. Angle of repose value to evaluate flow properties of formulations prepared by various carriers.

SC R

Fig.5. Solubility of pure PPK and PPK-SD in various media.

IP

Fig.4. Overlay plot for optimized parameters of solid dispersions of PPK.

T

Fig.3. Perturbation plot showing influence of individual factors on moisture content (Y1) (Fig.3A.), product yield (Y2) (Fig.3B.), solubility (Fig.3C.) and angle of repose (Fig. 3D.).

Fig.6. X-ray diffraction peaks of pure PPK (Fig. 6a) and PPK solid dispersion (PPK-SD) (Fig. 6b). Fig.7. SEM images of pure PPK (Fig. 7a) and PPK-solid dispersion (PPK-SD) (Fig. 7b). Fig.8. Particle size analysis of optimized batch of solid dispersion (8a) and initially grounded powder (8b).

NU

Fig.9. DSC thermograms of pure PPK and optimized formulation.

AC

CE P

TE

D

MA

Fig.10. Overlay of IR Spectra showing vibrational frequencies of pure PPK and PPK-SD.

29

ACCEPTED MANUSCRIPT

500 400

IP

T

300 200 100 0 1

PEG 400

PEG 600

Tween 20

Tween 40

Tween 60

Span 40

Span 60

Span 80

PEG 4000

PEG 6000

Tween 80

Span 20

PG

Pluronic F-68

MA

PEG 200

NU

Solubilizing agents

SC R

Amount of PPK soluble (µg/mL)

600

AC

CE P

TE

D

Fig.1. Solubility results of PPK in various solubilizers.

30

ACCEPTED MANUSCRIPT 60

40

T

30 20

IP

Angle of repose (Ө)

50

0

Carriers

SC R

10

Trehalose 0.05-1

Trehalose 0.075-1

Trehalose 0.1-1

Trehalose 0.125-1

Sorbtol 0.025-1

Sorbtol 0.05-1

Sorbitol 0.075-1

Sorbitol 0.1-1

Sorbitol 0.125-1

Mannitol 0.025-1

Mannitol 0.075-1

Mannitol 0.1-1

Mannitol 0.125-1

NU

Trehalose 0.025-1

MA

Mannitol 0.05-1

AC

CE P

TE

D

Fig.2. Angle of repose value to evaluate flow properties of formulations prepared by various carriers.

31

ACCEPTED MANUSCRIPT

3B.

MA

NU

SC R

IP

T

A3A.

3D.

AC

CE P

TE

D

3C.

Fig.3. Perturbation plot showing influence of individual factors on moisture content (Y1) (Fig.3A.), product yield (Y2) (Fig.3B.), solubility (Fig.3C.) and angle of repose (Fig. 3D.).

32

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

Fig.4. Overlay plot for optimized parameters of solid dispersions of PPK.

33

ACCEPTED MANUSCRIPT

200

IP

T

150

100

50

0 pH 1.2

pH 4.6 Pure PPK

pH 5

pH 6.8

PPK-SD

pH 7.4

pH 9.6

NU

Water

SC R

Solubility of PPK in mg/L

250

AC

CE P

TE

D

MA

Fig.5. Solubility of pure PPK and PPK-SD in various media.

34

ACCEPTED MANUSCRIPT Counts

6a.

IP

T

1000

SC R

500

0

20 30 Position [°2Theta] (Copper (Cu))

NU

10 Counts

6b.

MA

1000

40

0

20 30 Position [°2Theta] (Copper (Cu))

40

AC

10

CE P

TE

D

500

Fig. 6. X-ray diffraction peaks of pure PPK (Fig. 6a) and PPK solid dispersion (PPK-SD) (Fig. 6b).

35

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

Fig.7a. SEM of Pure PPK

Fig.7b. SEM of PPK-SD Fig.7. SEM images of pure PPK (Fig. 7a) and PPK-solid dispersion (PPK-SD) (Fig. 7b).

36

ACCEPTED MANUSCRIPT

CE P

AC

Fig.8b

TE

D

MA

NU

SC R

IP

T

Fig.8a

Fig.8. Particle size analysis of optimized batch of solid dispersion (8a) and initially grounded powder (8b).

37

SC R

IP

PPK-Solid dispersion (PPK-SD)

T

ACCEPTED MANUSCRIPT

102.26°C

MA

103.31°C

NU

Pure PPK

AC

CE P

TE

D

Fig.9. DSC thermograms of pure PPK and optimized formulation containing PPK (PPK-SD).

38

IP

T

ACCEPTED MANUSCRIPT

CR

Pure PPK

MA N

US

Vibrational peaks of pure PPK and PPK-Solid dispersion (PPK-SD). Vibrational Major Functional Vibrational Major frequencies of groups present in frequencies of Functional groups major peaks pure the pure PPK major peaks PPK- present in the PPK (cm-1) SD (cm-1) PPK-SD 1556.61 -NH bend 1539.25 -NH bend 1653.05 Carbonyl peak of 1651.12 Carbonyl peak of acid (-COOH) acid (-COOH) 2929.97 -CH stretch 2929.97 -CH stretch 3284.88 -NH stretch 3284.88 -NH stretch

PPK-Solid dispersion

CE P

TE D

(PPK-SD)

AC

Fig.10. Overlay of IR Spectra showing vibrational frequencies of pure PPK and PPK-SD.

39

ACCEPTED MANUSCRIPT List of Tables Table 1. Variables for Box-Behnken study. Table 2. Formula composition of PPK solid dispersion system.

T

Table 3. Factor level and response data for Box Behnken design for spray dried batches of Polypeptide-k solid dispersion system.

IP

Table 4. Evaluation solid carriers.

SC R

Table 5. ANOVA summary report of responses obtained from Box-Behnken design.

Table 6. Physicochemical and physicomechanical properties of optimized batch polypeptide-k solid dispersion

AC

CE P

TE

D

MA

NU

(PPK-SD).

40

ACCEPTED MANUSCRIPT Table 1. Variables for Box-Behnken study.

Inlet air temperature (°C)

C (X3)

Feed flow rate (ml/min)

D (X4)

T

B (X2)

Design Level Coded -1 0 +1 -1 0 +1 -1 0 +1 -1 0 +1

IP

Solubilizer to drug ratio (w/w)

Uncoded 0.050-1.0 0.075-1.0 0.100-1.0 0.10-1.0 0.15-1.0 0.20-1.0 120 125 130 16 18 20

SC R

Coded A (X1)

AC

CE P

TE

D

MA

NU

Independent Factors Uncoded Carrier to drug ratio (w/w)

41

ACCEPTED MANUSCRIPT

Table 2. Formula composition of PPK solid dispersion system.

IP

T

Quantity/batch (g) 5 0.25-0.5 0.50-1

AC

CE P

TE

D

MA

NU

SC R

Ingredients Polypeptide-k Trehalose Tween-80

42

ACCEPTED MANUSCRIPT Table 3. Factor level and response data for Box Behnken design for spray dried batches of Polypeptide-k solid dispersion system.

120 120 125 130 130 125 125 125 125 120 125 125 120 125 125 125 130 125 130 125 130 120 130 120 125

18 18 16 18 16 18 16 20 18 18 18 16 16 18 20 20 18 18 18 16 20 18 18 20 20

0.02 0.04 0.1 0.04 0.02 0.07 0.05 0.09 0.04 0.09 0.07 0.03 0.07 0.08 0.08 0.07 0.06 0.05 0.01 0.03 0.02 0.05 0.02 0.07 0.09

Response 2 Product yield (g)

Response 3 Solubility (mg/mL)

T

Response 1 Moisture (%)

MA

NU

SC R

IP

Factor 4 D: Feed flow (mL/min)

D

0.1 0.05 0.075 0.075 0.075 0.1 0.075 0.075 0.075 0.075 0.05 0.1 0.075 0.1 0.1 0.075 0.075 0.05 0.1 0.05 0.075 0.075 0.05 0.075 0.05

Factor 3 C: Inlet temperature (Degree)

TE

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Factor 2 B: Drug to Tween 80 ratio (w/w) 0.15 0.15 0.2 0.1 0.15 0.1 0.1 0.2 0.15 0.2 0.1 0.15 0.15 0.2 0.15 0.1 0.2 0.2 0.15 0.15 0.15 0.1 0.15 0.15 0.15

CE P

Factor 1 A: Drug to trehalose ratio (w/w)

53.18 47.18 57.59 51.24 60.12 55.12 62.10 38.68 52.18 53.45 52.18 66.18 62.10 47.68 43.12 40.14 53.45 49.68 54.44 51.82 53.17 36.88 52.16 46.44 40.12

5.88 6.78 7.54 4.9 6.76 4.9 5.01 7.23 6.42 6.96 6.42 5.32 6.1 8.02 5.22 4.82 7.16 7.48 5.76 5.88 6.61 4.96 5.91 6.13 5.11

Response 4 Angle of repose (degree)

18.18 24.12 23.18 21.18 20.18 16.12 26.22 24.18 22.41 26.18 22.41 19.52 23.11 22.10 19.47 20.16 23.18 24.56 19.22 22.18 20.82 19.16 24.21 20.48 23.34

AC

Run

43

ACCEPTED MANUSCRIPT

Table 4. Evaluation solid carriers (Keeping carrier to drug ratio 0.075-1.0 w/w).

*↑ - Less

Moisture Content (%) 0.04 0.12 0.09

Particle agglomeration Nil ↑* ↑↑**

T

Angle of repose (°) 26.22 31.54 38.46

IP

Solid Carrier Trehalose Mannitol Sorbitol

AC

CE P

TE

D

MA

NU

SC R

**↑↑ - Comparatively higher

44

ACCEPTED MANUSCRIPT

Table 5. ANOVA summary report of responses obtained from Box-Behnken design.

*R2 – A value above 0.50 indicates very good regression of the model.

***P value 0.020 < 0.0001 < 0.0001 0.0002

T

Regression parameters **Fcal 3.12 62.32 14.59 8.37

IP

Moisture content (Y1) Product yield (Y2) Solubility (Y3) Angle of repose (Y4)

*R2 0.76 0.91 0.71 0.58

SC R

Responses

**Fcal – A value above 3.00 and P value less than 0.05 indicates that the developed is highly significant.

AC

CE P

TE

D

MA

NU

***P value – A value less than 0.05 indicates that the developed is highly significant.

45

ACCEPTED MANUSCRIPT

Table 6. Physicochemical and physicomechanical properties of optimized batch polypeptide-k solid dispersion (PPKSD).

Solubility in

Angle of repose

Bulk Density 3

water (mg/ml)

(°)

(g/cm )

0.038

5.92

21.32

0.47

(g/cm )

index (%)

0.59

20.3

AC

CE P

TE

D

MA

NU

*(Mean ± SD) – Mean of triplicate studies

Compressibility

3

SC R

content (%)

Tapped Density

IP

Moisture

T

(Mean ± SD)*

46

ACCEPTED MANUSCRIPT Graphical abstract

Trehalose Tween-80

SC R

IP

T

Polypeptide-k

Pure PPK

CE P

TE

D

250 200 150 100 50 0

Spray dryer (SprayMate)

Spray Dried Polypeptide-k solid dispersion (PPK-SD)

PPK-SD

AC

Solubility of PPK in mg/L

MA

NU

Spray Drying

47

ACCEPTED MANUSCRIPT

Research Highlights

CE P

TE

D

MA

NU

SC R

IP

T

Solid dispersion of polypeptide-k (PPK) was formulated through spray drying. Optimization of formulation was done using Box Behnken design. Optimized batch has higher solubility in different media used compared to pure PPK.

AC

  

48