A Seminar report on:

Milling of Active Pharmaceutical Ingredients

Submitted to the Institute of Chemical Technology,

Department of Pharmaceutical Sciences & Technology.

Dattaprasad Santosh Jadhav

21/09/2019

Final Year B.Tech Pharma

Under the guidance of:

Prof. P. D. Amin

Institute of Chemical Technology,

Nathalal Parekh Marg, Matunga(East), Mumbai.

 Table of Contents

1. Introduction

2. Types of Mills

3. Factors Influencing Milling

4. Selection of Mill

5. Milling of Norfloxacin using Ball Mill

6. Milling of Bosentan Hydrate using Jet Mill

 MILLING OF ACTIVE PHARMCEUTICAL INGREDIENTS

INTRODUCTION

Few materials used in pharmaceuticals exist in optimum size, and most materials must

be comminuted at some stage during the production of a dosage form. Milling is the mechanical

process of reducing the particle size of solids. Various terms (crushing, disintegration,

dispersion, grinding, and pulverization) have been used synonymously with equipment and the

process.

Milling equipment is usually classified as coarse, intermediate, or fine according to the

size of the milled product. Size is conventionally expressed in terms of mesh (number of

openings per linear inch of a screen). Coarse milling produces particles larger than 20-mesh,

intermediate milling produces particles from 200-20 mesh, and fine milling produces particles

smaller than 200-mesh.

The surface area per unit weight, which is known as the specific surface, is increased

by size reduction.(2) This increased specific surface affects the therapeutic efficiency of

medicinal compounds that possess a low solubility in body fluids by increasing the area of

contact between the solid and dissolving fluid. Thus, a given weight of a finely powdered

medicinal compound dissolves in a shorter time than does the same weight of a coarse powder.

Extraction or leaching from animal glands (liver and pancreas), and from crude

vegetable drugs, is facilitated by comminution. The time required for extraction is shortened

by the increased area of contact between the solvent and the solid and the reduced distance the

solvent has to penetrate into the material. The control of particle size in the extraction process

provides for more complete extraction and a rapid filtration rate when the solution is filtered

from the marc.

 The drying of the wet masses may be facilitated by milling, which increases the surface

area and reduces the distance the moisture must travel within the particle to reach the outer

surface.(3)

In the manufacture of compressed tablets, the granulation of the wet mass results in

more rapid and uniform drying. The dried tablet granulation is then milled to a particle size and

distribution that will flow freely and produce tablets of uniform weight.(4-6)The flowability of

powders and granules in high speed filling equipment and in tablet presses affects product

uniformity.

The mixing or blending of several solid ingredients of a pharmaceutical is easier and

more uniform if the ingredients are approximately the same size. This provides a greater

uniformity of dose. Solid pharmaceuticals that are artificially coloured are often milled to

distribute the colouring agent to ensure that the mixture is not mottled and is uniform from

batch to batch. Even the size of a pigment affects its colour.

Lubricants used in compressed tablets and capsules function by virtue of their ability to

coat the surface of the granulation or powder. A fine particle size is essential if the lubricant is

to function properly(7). The milling of the ointments, creams, and pastes provides a smooth

texture and better appearance in addition to improved physical stability.

The mechanical behaviour of solids, which under stress are strained and deformed, is

shown in the stress-strain curve in Figure 2-6. The initial linear portion of the curve is defined

by Hooke's law (stress is proportional to strain), and Young's modulus (slope of linear portion)

expresses the stiffness or softness in dynes per square centimetre(8-12). The stress-strain curve

becomes nonlinear at the yield point, which is a measure of the resistance to permanent

deformation. With still greater stress, the region of irreversible plastic deformation is reached.
The area under the curve represents the energy of fracture and is an approximate measure of

the impact strength of the material.

Fig. 1:- Stress-Strain curve for solid.(1)

In all milling processes, it is a random matter if and. when a given particle will be fractured.

If a single particle is subjected to a sudden impact and is fractured, it yields a few relatively

large particles and a number of fine particles, with relatively few particles of intermediate size.

If the energy of the impact is increased, the larger particles are of a smaller size and more

numerous, and although the number of fine particles is increased appreciably, their size is not

greatly changed. It seems that the size of the finer particles is related to the internal structure

of the material, and the size of the larger particles is more closely related to the process by
which comminution is accomplished. Size reduction begins with the opening of any small

cracks that were initially present. Thus, larger particles with numerous cracks fracture more

readily than smaller particles with fewer cracks.

In general, fine grinding requires more energy, not only because of the increased new

surface, but also because more energy is needed to initiate cracks. For any particle, there is a

minimum energy that will fracture it; however; conditions are so haphazard that many particles

receive impacts that are not sufficient to fracture them and are eventually fractured by some

excessively forceful blow. As a result, the most efficient mills utilize less than 1% of the energy

input to fracture particles and create new surfaces. The rest of the energy is dissipated in (l)

elastic deformation of unfractured particles, (2) transport of material within the milling

chamber, (3) friction between particles, (4) friction between particles and mill, (5) heat, (6)

vibration and noise, and (7) inefficiency of transmission and motor. If the force of impact does

not exceed the elastic limit (region of Hooke's law), the material is reversibly deformed or

stressed.

When the force is removed, the particle returns to its original condition, and the

mechanical energy of stress in the deformed particle appears as heat. For polymeric materials,

hysteresis is frequent. When a force is released and applied to a polymeric material, an elastic

loop, or hysteresis, occurs in the stress-strain cycle; the area of the loop represents the

dissipation of stress energy (usually heat). A force that exceeds the elastic limit fractures the

particle. Usually, the surfaces of particles are irregular, so that the force is initially taken on the

high portion of the surface, with the result that high stresses and temperatures may be set up

locally in the material. As fracture occurs, the points of application of the force are shifted. The

energy for the new surfaces is partially supplied by the release of stress energy.
 Crystalline materials fracture along crystal cleavage planes; non-crystalline materials

fracture at random. If an ideal crystal were pressed with an increasing force, the force would

be distributed uniformly throughout its structure until the crystal disintegrated into its

individual units. A real crystal fractures under much less force into a few relatively large

particles and several fine particles, with relatively few particles of intermediate size. Crystals

of pure substances have internal weaknesses due to missing atoms or ions in their lattice

structures and to flaws arising from mechanical or thermal stress. A flaw in a particle is any

structural weakness that may develop into a crack under strain. It has been proposed that any

force of milling produces a small flaw in the particle.

The useful work in milling is proportional to the length of new cracks produced. A

particle absorbs strain energy and is deformed under shear or compression until the energy

exceeds the weakest flaw and causes fracture or cracking of the particle. The strain energy

required for fracture is proportional to the length of the crack formed, since the additional

energy required to extend the crack to fracture is supplied by the flow of the surrounding

residual strain energy to the crack. The Griffith theory of cracks and flaws assumes that all

solids contain flaws and microscopic cracks, which increase the applied force according to the

crack length and focus the stress at the atomic bond of the crack apex. The Griffith theory may

be expressed as:

𝑌∈
𝑇=√
𝑐

where T is tensile stress, Y is Young's modulus, € is the surface energy of the wail of the crack,

and c is the critical crack depth required for fracture. A linear relationship between the square

of ten- sile strength of minerals and the critical height for drop weight impact suggests that the

square of tensile strength is a useful criterion of impact fracture.

Types of Mills: -

A mill consists of three basic parts: (1) feed chute, which delivers the material, (2)

grinding mechanism, usually consisting of a rotor and stator, and (3) a discharge chute. The

principle of operation depends on direct pressure, impact from a sharp blow, attrition, or

cutting. In most mills, the grinding effect is a combination of these actions. The most

commonly used mills in pharmaceutical manufacturing are the rotary cutter, hammer, roller,

and fluid-energy Diagrammatic representations of mill these mills are shown in figure below.

Fig 2: Types of mills(1)

 The manner in which an operator feeds a mill markedly affects the product. If the rate

of feed is relatively slow, the product is discharged readily, and the amount of undersize or

fines is minimized. If the mill is choke fed at a fast rate, the material is in the milling chamber

for a longer time, as its discharge is impeded by the mass of material. This provides a greater

reduction of particle size, but the capacity of the mill is reduced, and power consumption is

increased. Choke feed is used when a small amount of material is to be miffed in one operation.

The rate of discharge should be equal to the rate of feed, which is such that the milling parts

can operate most effectively.

Most mills used in pharmaceutical operations are designed so that the force of gravity

is sufficient to give free discharge generally from the bottom of the mill. For ultrafine grinding,

the force of gravity is re- placed by a fluid carrier. A current of steam, air, or inert gas removes

the product from the attrition, fluid-energy, or high-speed hammer mill. The powder is removed

from the fluid by cyclone separators or bag filters.

If the milling operation is carried out so that the material is reduced to the desired size

by passing it once through the mill, the process is known as open-circuit milling. A closed-

circuit mill is one in which the discharge from the milling chamber is passed through a size-

separation device or classifier, and the oversize particles are returned to the grinding chamber

for further reduction of size. Closed-circuit operation is most valuable in reduction to fine and

ultrafine size.

1) Hammer Mill

The hammer mill is an impact mill using a high-speed rotor (up to 10,000 rpm) to which

a number of swinging hammers are fixed. The material is fed at the top or centre, thrown out

centrifugally, and ground by impact of the hammers or against the plates around the periphery
of the casing. The clearance between the housing and the hammers contributes to size

reduction. The material is retained until it is small enough to fall through the screen that forms

the lower portion of the casing. Particles fine enough to pass through the screen are discharged

almost as fast as they are formed.

The hammer mill can be used for almost any type of size reduction. Its versatility makes

it popular in the pharmaceutical industry, where it is used to mill dry materials, wet filter-press

cakes, ointments, and slurries. Comminution is effected by impact at peripheral hammer speeds

of up to 7600 meters per minute, at which speed most materials behave as if they were brittle.

Brittle material is best fractured by impact from a blunt hammer; fibrous material is best

reduced in size by cutting edges. Some models of hammer mills have a rotor that may be turned

180 degrees to allow use of either the blunt edge for fine grinding or the knife edge for cutting

or granulating.

In the preparation of wet granules for compressed tablets, a hammer mill is operated at

2450 rpm with knife edges, using circular or square holes of a size determined by what will

pass without clogging (1.9 to 2.54 cm). In milling the dried granulation, the mill is operated at

1000 or 2450 rpm with knife edges and circular holes in the screen (0.23 to 0.27 cm). Hammer

mills range in size from 5 to 500 horsepower, the smaller mills are especially useful for

developmental and small-batch milling.

A hammer mill can be used for granulation and close control of the particle size of

powders. The size of the product is controlled by selecting the speed of the hammers and the

size and type of the screen. Speed is crucial. Below a critical impact speed, the rotor turns so

slowly that a blending action rather than comminution is obtained. This results in overloading

and a rise in temperature. Microscopic examination of the particles formed when the mill is

operating below the critical speed shows them to be spheroidal, indicating not an impact action,
but an attrition action, which produces irregularly shaped particles. At very high speeds, there

is possibly insufficient time between hammers for the material to fall from the grinding zone.

In wet milling of dispersed systems with higher speeds, the swing hammers may lay back with

an increased clearance; for such systems, fixed hammers would be more effective.

A circular hole design is the strongest screen and the most difficult to keep from

clogging. It is recommended for the grinding of fibers. The herringbone design consists of a

series of slotted holes repeated across the surface of the screen at an angle of 45 degrees to the

length of the screen. A herringbone design is preferred for grinding crystalline material and for

continuous operation. A herringbone design with the width of the slot equal to the diameter of

a round hole grinds more coarsely than the round hole. A herringbone design should not be

used for fibrous material, as it is possible for the fibers to align themselves along the slots and

pass through with inadequate size reduction.

A cross slot at right angles to the path travelled by the hammer is not used in fine

grinding because it clogs readily; a cross slot is recommended for milling slurries. The jump-

gap screen is a series of bars so arranged that the particle approaches a ramp, which deflects

the particle into the chamber away from the opening of the screen. The jump-gap screen is for

abrasive and clogging materials. Hammer mills are compact with a high capacity. Size

reduction of 20 to 40 microns may be achieved; however, a hammer mill must be operated with

internal or external classification to produce ultrafine particles. Because inertial forces vary

with mass as the inverse cube of the diameter, small particles with a constant velocity impact

with much less kinetic energy than larger ones, and the probability that particles less than a

certain size will fracture decreases rapidly. In addition, small particles pass through the screen

almost as fast as they are formed. Thus, a hammer mill tends to yield a relatively narrow size

distribution. Hammer mills are simple to install and operate. The speed and screen can be
rapidly changed. They are easy to clean and may be operated as a closed system to reduce dust

and explosion hazards.

2) Ball Mill

The ball mill consists of a horizontally rotating hollow vessel of cylindric shape with

the length slightly greater than its diameter. The mill is partially filled with balls of steel or

pebbles, which act as the grinding medium. If pebbles are used, it is known as a pebble mill; if

rods or bars are used, it is known as a rod mill. The rod mill is particularly useful with sticky

material that would hold the balls together, because the greater weight of the rods causes them

to pull apart. The tube mill is a modified ball mill in which the length if about four times that

of the diameter and in which the balls are some- what smaller than in a ball mill. Because the

material remains in the longer tube mill for a greater length of time, the tube mill grinds more

finely than the ball mill. The ball mill may be modified to a conical shape and tapered at the

discharge end. If balls of different size are used in a conical ball mill, they segregate according

to size and provide progressively finer grinding as the material flows axially through the mill.

Recently, small-scale vibration ball mills, which produce particles of a few microns,

have been introduced. 60 These oscillate 1500 to 2500 cycles per minute through an amplitude

of approximately 4 mm. Most ball mills utilized in pharmacy are batch- operated; however,

there are available continuous ball mills, which are fed through a hollow trunnion at one end,

with the product dis- charged through a similar trunnion at the opposite end. The outlet is

covered with a coarse screen to prevent the loss of the balls. In a ball mill rotating at a slow

speed, the balls roll and cascade over one another, providing an attrition action. As the speed

is increased, the balls are carried up the sides of the mill and fall freely onto the material with

an impact action, which is responsible for most size reduction. Ball milling is a combination of
impact and attrition. If the speed is increased sufficiently, the balls are held against the mill

casing by centrifugal force and revolve with the mill. The critical speed of a ball mill is the

speed at which the balls just begin to centrifuge with the mill.

The charge of balls can be expressed in terms of percentage of volume of the mill (a

bulk volume of balls filling one half of a mill is a 50% ball charge). To operate effectively, a

ball charge from 30 to 50% of the volume of the mil] is required.

The amount of material to be milled in a ball mill may be expressed as a material-to-

void ratio (ratio of the volume of material to that of the void in the ball charge). The efficiency

of a ball mill is increased as the amount of material is increased until the void space in the bulk

volume of ball charge is filled; then, the efficiency of milling is decreased by further addition

of material.

Increasing the total weight of balls of a given size increases the fineness of the powder.

The weight of the ball charge can be increased by increasing the number of balls or by using a

ball composed of a material with a higher density. Since optimum milling conditions are

usually obtained when the bulk volume of the balls is equal to 50% of the volume of the mill,

variation in weight of the balls is normally effected by the use of materials of different densities.

Thus, steel balls grind faster than porcelain balls, as they are three times more dense. Stainless

steel balls are also preferred in the production of ophthalmic and parenteral products, as there

js less attrition and less subsequent contamination with particulate matter.

In dry milling, the moisture should be less than 2%. With batch processing, dry ball

milling produces a very fine particle size. With wet milling, a ball mill produces 200-mesh

particles from slurries containing 30 to 60% solids. From the viewpoint of power consumption,

wet grinding is more efficient than dry grinding. A slower speed is used in wet milling than in
dry milling to prevent the mass from being carried around with the mill. A high viscosity

restricts the motion of the grinding medium, and the impact is reduced.

Wetting agents may increase the efficiency of milling and the physical stability of the

product by nullifying electrostatic forces produced during comminution. For those products

containing

wetting agents, the addition of the wetting agent at 'the milling stage may aid size reduction

and reduce aggregation.

In addition to being used for either wet or dry milling, the ball mill has' the advantage

of being used for batch or continuous operation. In a batch operation, unstable or explosive

materials may be sealed with an inert atmosphere and satisfactorily ground. Ball mills may be

sterilized and sealed for sterile milling in the production of ophthalmic and parenteral products.

The installation, operation, and labor costs involved in ball milling are low. Finally, the ball

mill is unsurpassed for fine grinding of hard, abrasive materials.

3) Fluid-Energy Mill

In the fluid-energy mill or micronizer the material is suspended and conveyed at high

velocity by air or steam, which is passed through nozzles at 100 to 150 pounds per square inch

(psi). The violent turbulence of the air and steam reduces the particle size chiefly by

interparticular attrition. Air is usually used because most pharmaceuticals have a low melting

point or are thermolabile. As the compressed air expands at the orifice, the cooling effect

counteracts the heat generated by milling.

 The material is fed near the bottom of the mill through a venturi injector (A). As the

compressed air passes through the nozzles (B), the material is thrown outward against the wall

of the grinding chamber (C) and other particles. The air moves at high speed in an elliptical

path carrying with it the fine particles that pass out of the discharge outlet (D) into a cyclone

separator and a bag col- lector. The large particles are carried by centrifugal force to the

periphery, where they are further exposed to the attrition action. The design of the fluid-energy

mill provides internal classification, which permits the finer and lighter particles to be

discharged and the heavier oversized particles, under the effect of centrifugal force, to be

retained until reduced to a small size.

Fluid-energy mills reduce the particle to 1 to 20 microns. The feed should be permitted

to approximately a 20- to IDD-mesh size to facilitate milling. A 2-inch laboratory model using

20 to 25 cubic feet per minute of air at 100 psi mills 5 to 10 grams per minute. In selecting

fluid-energy mills for production, the cost of a fluid-energy source and dust collection

equipment must be considered in addition to the cost of the mill.

4)Cutting Mill

Cutting mills are used for tough, fibrous materials and provide a successive cutting or

shearing action rather than attrition or impact. The rotary knife cutter has a horizontal rotor

with two to 12 knives spaced uniformly on its periphery turning from 200 to 900 rpm and a

cylindric casing having several stationary knives. The bottom of the casing holds a screen that

controls the size of the material discharged from the milling zone. The feed size should be less

than 1 inch thick and should not exceed the length of the cutting knife. For sizes less than 20-

mesh, a pneumatic product-collecting system is required. Under the best operating conditions,

the size limit of a rotary cutter is 80-mesh.

 A disc mill consists of two vertical discs; each may rotate in opposite directions

(double-runner disc mill), or only one may rotate (single-runner disc mill), with an adjustable

clearance. The disc may be provided with cutting faces, teeth, or convolutions. The material is

premilled to approximately 40-mesh size and is usually sus- pended in a stream of air or liquid

when fed to the mill.

5) Colloid Mill

A colloid mill consists of a high-speed rotor (3000 to 20,000 rpm) and stator with

conical milling surfaces between which is an adjustable clearance ranging from 0.002 to 0.03

inches, as indicated by the schematic diagram in Figure 2-15. The rotor speed is 3000 to 20,000

rpm. The material to be ground should be premilled as finely as possible to prevent damage to

the colloid mill.

In pharmacy, the colloid mill is used to process suspensions and emulsions; it is not

used to process dry materials. The premilled solids are mixed with the liquid vehicle before

being introduced into the colloid mill. Interfacial tension causes part of the material to adhere

to, and to rotate with, the rotor. Centrifugal force throws part of the material across the rotor

onto the stator. At a point between the rotor and stator, the motion imparted by the rotor ceases,

and hydraulic shearing force exceeds the particle-particle attractive forces holding the

individual particles in an aggregate. The particle size of milled particles may be smaller than

the clearance, because the high shear is the dispersing force. In emulsification, a clearance of

75 microns may produce a dispersion with an average particle size of 3 microns. The milled

liquid is discharged through an outlet in the periphery of the housing and may be recycled.
 Rotors and stators may be smooth-surfaced or rough-surfaced. With a smooth-surfaced

rotor and stator, there is a thin, uniform film of material between them, and it is subjected to

the maximum amount of shear. Rough-surfaced mills add intense eddy currents, turbulence,

and impaction of the particles to the shearing action. Rough-surfaced mills are useful with

fibrous materials because fibers tend to interlock and clog smooth-faced mills.

A colloid mill tends to incorporate air into a suspension. Aeration may be minimized

by use of a vertical rotor, which seals the point at which the rotor shaft enters the housing, and

keeps the rotor and stator in contact with the liquid. The wasted energy of milling, which

appears as heat, may raise the temperature of a liquid by as much as 40º. The passage of

cooling water through the mill jacket may reduce the temperature by as much as 20º. Sanitary

design mills, which may be sterilized, are available.

Factors Influencing Milling: -

The properties of a solid determine its ability to resist size reduction and influence the choice

of equipment used for milling. The specifications of the product also influence the choice of a

mill. The grindability of coal is expressed in terms of numbers of revolutions of a standardized

ball mill required to yield a product of which 80% passes a 200-mesh screen. Although a similar

expression could be applied to pharmaceutical materials, no quantitative scale has been adopted

to express hardness. It is perhaps as useful to speak of hard, intermediate, and soft materials.

Hard materials (iodine, pumice) are those that are abrasive and cause rapid wear of mill parts

immediately involved in size reduction.

The physical nature of the material determines the process of comminution. Fibrous

materials (glycyrrhiza, rauwolfia) cannot be crushed by pressure or impact; they must be cut.

Friable materials (dried filter cake, sucrose) tend to fracture along well-defined planes and may
be milled by an attrition, impact, or pressure process. The presence of more than 5% water

hinders comminution and often produces a sticky mass upon milling. This effect is more

pronounced with fine materials than with larger particles. At concentrations of water greater

than 50%, the mass becomes a slurry, or fluid suspension. The process then is a wet milling

process, which often aids in size reduction. An increase in moisture can decrease the rate of

milling to a specified product size. Glauber's salt and other drugs possessing water of

crystallization liberate the water at low temperatures, causing clogging of the mill. Hygroscopic

materials (calcium chloride) rapidly sorb moisture to the extent that the wet mass sticks and

clogs the mill.

The heat during milling softens and melts materials with a low melting point. Synthetic

gums, waxes, and resins become soft and plastic. Heat-sensitive drugs may be degraded or even

charred. Pigments (ochre and sienna) may change their shade of color if the milling temperature

is excessive. Unstable compounds and almost any finely powdered material may ignite and

explode if the temperature is high.

Other product specifications influence the choice of a mill. The shape of the milled

particles may be important. An impact mill produces sharp, irregular particles, which may not

flow readily. When specifications demand a milled product that will flow freely, it would be

better to use an attrition mill, which produces free-flowing spheroidal particles.

Milling may alter crystalline structure and cause chemical changes in some materials.

Wet milling may be useful in producing a suspension that contains a metastable form causing

crystal and caking. For example, when cortisone acetate crystals are allowed to equilibrate with

an aqueous vehicle, subsequent wet milling provides a satisfactory suspension. Starch,

amylose, and amylopectin may be broken down by a vibratory mill to a wide molecular weight

range. Powdered povidone breaks down into lower molecular weight polymers during ball
milling. Pure CIT and C16-fatty acids may be decarboxylated and converted to the hydrocarbon

containing one less carbon atom by ball milling with wet sand. Milling well-dried

microcrystalline cellulose from 1 to 25 hours decreases its crystallinity.65 Excessive shear of

a colloid mill may damage polymeric suspending agents so that there is a loss of viscosity.66

A decrease in particle size of crystals in a hammer mill was reported to increase the rate of

crystal growth during storage, owing to alterations in crystal lattice and the formation of active

sites. Specifically, crystals of phenobarbital (initial size of 310 microns) milled to 22.7 microns

grew to 38.9 microns after 4 weeks at 6000; however, crystals milled to 31.5 microns showed

little growth on storage.

Selection of a Mill: -

In general, the materials used in pharmaceuticals may be reduced to a particle size less

than 40-mesh by means of ball, roller, hammer, and fluid-energy mills. The choice of a mill is

based on (l) product specifications (size range, particle size distribution, shape, moisture

content, physical and chemical properties of the material); (2) capacity of the mill and

production rate requirements; (3) versatility of operation (wet and dry milling, rapid change of

speed and screen, safety features); (4) dust control (loss of costly drugs, health hazards,

contamination of plant); (5) sanitation (ease of cleaning, sterilization); (6) auxiliary equipment

(cooling system, dust collectors, forced feeding, stage reduction); (7) batch or continuous

operation; and (8) economical factors (cost, power consumption, space occupied, labour cost).

It is suggested that the equipment manufacturer be consulted and its pilot laboratory be

utilized, as there exists a wide variety of mills differing in details of design and modifications.

The industrial pharmacist should evaluate the pilot study personally to observe the temperature

of the inlet and outlet air, the temperature of the milled material, and the size reduction
performance at different mill speeds. A size-frequency analysis should be made on samples

from each condition of operation. Samples should be recycled to find if there is build-up in the

milling chamber. The pilot evaluation is important because laboratory procedures of size

reduction do not duplicate milling conditions in production mills.

 Milling of Norfloxacin using Ball Mill

Almost more than 90% drugs are given through oral route. Solubility of orally

administered drugs in aqueous medium is of prime importance for their sufficient and

reproducible bioavailability along with pharmacokinetic profile and absorption parameters(14).

Drugs with poor water solubility belong to class II and IV of Biopharmaceutical Classification

System (BCS). The poor solubility is a cause of lower bioavailability and poses a major

challenge for formulation and dosage form design. Even if formulated in a dosage form, they

have to be administered in large doses. Thus, improving solubility results in reduced dose,

lesser side effects, decreased gastric irritancy, improved bioavailability and reduced effect of

food on absorption. Bioavailability of these can be enhanced by improving solubility and

dissolution characteristics. Various formulation techniques to improve bioavailability have

been developed in literature, including salt formation, addition of co-solvents, micellar

solubilization, micro emulsions, solid dispersion technology, nanosuspensions and

micronization and milling with additives.

Norfloxacin is a broad-spectrum, synthetic antibacterial of fluoroquinolones class.

Along with its broader antibacterial activity it is occasionally used to treat common and

complicated bacterial infections of urinary tract, prostate and gonorrhoea(15). Norfloxacin is 1-

ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolonecarboxylic acid with chemical

formula C16H18FN3O3. Norfloxacin is a white to pale yellow crystalline powder with a

molecular weight of 319.34 and a melting point of about 221OC. It is very slightly soluble in

water having solubility only 0.37 mg/mL (370 ppm). It belongs to the same class of

biopharmaceutics classification system which has characteristics as mentioned in above

paragraph.
 Different approaches have been adopted to increase the solubility of norfloxacin

including inclusion complex formation with beta cyclodextrin , norfloxacin gastro retentive

drug delivery system , hydrotropic solubilization technique , crystal modification , solid

dispersion by fusion method , prodrug formation , solid dispersion by solvent evaporation

method , binary blends with co-solvents and hydrochloride derivatives of norfloxacin but no

remarkable improvements in solubility of norfloxacin has been achieved, so the issue of its

poor aqueous solubility is still to be resolved. However, particle size reduction approach has

not been used for the enhancement of aqueous solubility of norfloxacin by using co-milling

technique.

Milling is used to achieve particle size reduction by maximizing the specific surface of

the beached material and obtaining sufficient mixing with the expenditure of minimum energy

and time . Milling is used to increase the dissolution rate of poorly soluble drugs, but this is

counter-productive if the newly created surfaces are hydrophobic and poorly wettable when

poorly soluble drugs are milled. This means that the surface needs to be hydrophilized to

increase dissolution rate with the addition of hydrophilic polymers or surfactants in the milling

process . This process has been termed as co-milling. Co-milling (or milling of two or more

materials concurrently) alternatively referred to as co-grinding is a simple and effective method

of improving the physicochemical properties such as solubility, stability of various active

pharmaceutical ingredients (APIs). Although it is a highly energy intensive process, co-milling

has been found to be economical, does not require sophisticated instruments and is environment

friendly without the use of organic solvents.

Physical parameters are considered to be more beneficial and easy to control than

chemical and molecular modifications of compounds. Powder flow ability is an important

requirement for manufacturing process of pharmaceuticals such as flow from hoppers,

transportation, mixing, compression and packaging. So, it becomes essential to understand the
flow behaviour of powders prior to mixing, tableting and capsule filling and these powder flow

properties are affected by size, shape, surface morphology and packing ability. For this reason

in this study flow properties were also estimated in order to determine either these are affected

by this size reducing technique or not.

EXPERIMENT

Materials

Norfloxacin (Cherished Pharmaceuticals Pvt. Ltd., Lahore.), low substituted

hydroxypropyl cellulose (Klucel®) EXF 80 kDa (CCL Pharmaceuticals, Pakistan), Polyvinyl

pyrrolidone (PVP K30) (MP Biomedicals, LLC., USA), sodium hydroxide and glacial acetic

acid (Analar, BDH Laboratory, England), Potassium dihydrogen phosphate (Riedelde- Haen,

Germany) and distilled water (locally prepared). All the chemicals used in the research were

of analytical grade.

Preparation of physical mixtures (PMs)

Physical mixtures of norfloxacin with polyvinyl pyrrolidone (PVP) and low substituted

hydroxypropyl cellulose (HPC-L) were prepared in the weight ratio of 1 : 1, 1 : 2, 1 : 3 and 2 :

1. Drug and excipient were weighed accurately according to the weight ratios as stated above,

ground and were mixed for 15 min using porcelain mortar and pestle. Physical mixtures of

norfloxacin-PVP and norfloxacin-HPC-L were labelled as PMPVP 1 : 1, PMPVP 1 : 2, PMPVP

1 : 3, PMPVP 2 : 1 and PMHPC-L 1 : 1, PMHPC-L 1 : 2, PMHPC-L 1 : 3, PMHPC-L 2 : 1,

respectively.
Preparation of separately milled physical mixtures (MPMs)

For preparation of milled physical mixtures, drug and excipients were first milled

individually for 30 min in a planetary ball mill (NQM 0.4) at rotational speed of 20 Hz. A total

4 g of powder with 80 stainless steel balls having diameter of 7 mm and 5 stainless steel balls

having diameter of 10 mm were charged in a cylindrical chamber of the mill to fill one third of

the volume of the chamber. Then, the milled norfloxacin and respective carriers were mixed in

4 different weight ratios by grinding in a mortar and pestle for 5 min. Milled physical mixtures

of norfloxacin-PVP and norfloxacin-HPC-L were labelled as MPMPVP 1 : 1, MPMPVP 1 : 2,

MPMPVP 1 : 3, MPMPVP 2 : 1 and MPMHPC-L 1 : 1, MPMHPC-L 1 : 2, MPMHPC-L 1 : 3,

MPMHPC-L 2 : 1, respectively.

Preparation of co-milled mixtures (CMs)

Co-milled samples were prepared using carriers, polyvinyl pyrrolidone (PVP) and low

substituted hydroxypropyl cellulose (HPC-L) and drug in 4 ratios mentioned above. Milling

were performed in planetary ball mill (NQM 0.4) using the same conditions given in MPMs

section above. Co-milled mixtures of norfloxacin-PVP and norfloxacin-HPCL were labeled as

CMPVP 1 : 1, CMPVP 1 : 2, CMPVP 1 : 3, CMPVP 2 : 1 and CMHPC-L 1 : 1, CMHPC-L 1

: 2, CMHPC-L 1 : 3, CMHPC-L 2 : 1, respectively.

Optimization of selected formulations

Formulations showing the best flow properties and maximum aqueous solubility among

all milled physical mixtures (MPM) and co-milled mixtures (CM) were selected for
optimization analysis. One milled physical mixture and one co-milled mixture with both the

polymers showing the best properties were selected as optimized formulations. Selected

formulations were milled at 30 Hz and 40 Hz speed for 30 min using the same number of balls

with their specifications mentioned in previous section.

Determination of optimum wavelength (ë max)

The standard solutions of 50 μg/mL of norfloxacin in distilled water, acetate buffer pH

4 and phosphate buffer pH 6.8 separately were scanned over a UV range of 200-600 nm using

UV spectrophotometer (UV-1602, BMS, UK). The attained spectrum was observed for the

maximum absorbance value obtained at the specific wavelength which was selected for the

measurement of unknown samples in the study (λ max).

Determination of flow properties

For determination of flow properties different parameters such as bulk density, tapped

density, compressibility index, Hausner’s ratio and angle of repose were determined by

following the method given in USP30NF25.

Solubility studies

Solubility of different preparations of norfloxacin was determined in distilled water and

phosphate buffer pH 6.8 separately, i.e., un-milled norfloxacin base, milled norfloxacin and all

the formulations(16). Classical shake flask method proposed by Higuchi and Corons was
adopted to determine the solubility. Accurately weighed 100 mg of norfloxacin was added to

the conical flask containing 50 mL of distilled water. The flasks were stoppered and stirred on

a mechanical shaker for 48 h and allowed to stand for 24 h after shaking. Samples were taken

using disposable syringe after 24 h of sedimentation and filtered using 0.45 micron syringe

filter to get the clear solution (Membrane solutions, LLC, China). Solutions with any type of

turbidity due to dispersion of solid were not used for further experimentation. Samples were

diluted appropriately and the absorbance was taken on the λ max determined above. All the

experiments were performed in triplicate. Unknown concentration was calculated by using

calibration curve.

Dissolution studies

In vitro dissolution studies were performed using USP dissolution apparatus type II

(Curio, Pakistan) according to the conditions specified in USP under norfloxacin dissolution

testing . The milled, un-milled norfloxacin and the prepared formulations equivalent to 500 mg

weight were dried for 2 h using tray dryer till moisture level less than 5% was achieved. The

obtained mass was passed through sieve number 120 and properly lubricated with minimum

quantities of talc and compressed into tablets by using static compression force of 400 kg/cm2

using single punch tableting machine. Tablets were placed in dissolution vessel filled with 750

mL of acetate buffer pH 4.0. This dissolution medium was maintained at 37 ± 0.5OC and stirred

constantly at 50 rpm. Samples were collected at 5, 10, 20, 30, 45, 60 and 90 min and replaced

with equal amount of fresh media already maintained at 37 ± 0.5OC. Samples were filtered

through 0.45 μm syringe filter. The filtered samples were serially diluted and were measured

for absorbance at the selected wavelength UV spectrophotometer. The samples were tested in

triplicate and mean percentage drug release was calculated at different time intervals using
calibration curve. Dissolution of the optimized formulations was also performed in distilled

water to evaluate the dissolution behaviour of drug in aqueous medium as the main objective

of this study was to enhance the water solubility of drug. Dissolution was performed using the

same apparatus with 900 mL dissolution medium (distilled water) in each vessel maintained at

37 ± 0.5OC and stirred constantly at 50 rpm. The same method was used for withdrawing

samples and calculation of their percentage drug released as mentioned above.

RESULTS

Norfloxacin and other powder mixtures were characterized by compressibility, bulk

and tapped density, angle of repose, solubility and dissolution. The data on flow properties,

solubility and dissolution were calculated for comparative analysis of norfloxacin in bulk with

un-milled and milled physical mixtures and co-milled mixtures.

Flow properties

The results of flow properties are summarized in Table 1. The highest value of bulk

density was observed with norfloxacin-polyvinyl pyrrolidone co-milled mixture. Increasing the

amount of PVP decreased the densities in physical mixtures, while increased the bulk densities

in the milled physical mixtures and co-milled mixtures and tapped densities in the co-milled

mixtures. The highest bulk and tapped densities among HPC-L mixtures were seen with the

physical mixtures containing the least amount of HPC-L in the mixture. The lowest bulk and

tapped densities among HPC-L mixtures were seen with the co-milled samples containing the

highest amount of HPC-L in the mixture. Increasing the amount of HPC-L decreased the bulk

and tapped densities in all three types of mixtures. Better flow was observed with un-milled

norfloxacin than milled norfloxacin. Addition of different concentration of polymers to

 norfloxacin improved the flow properties of both milled and unmilled norfloxacin. Co-milled

norfloxacin with PVP proved better flow characters when milled in equal quantities.

Angle of repose for unmilled norfloxacin was calculated to be 19.80O. Milled

norfloxacin exhibited increased value for angle of repose. Minimum value of angle of repose

was seen with CMPVP1 : 1 indicating the best flow. In physical mixtures and milled physical

mixtures increased amount of HPCL increased the angle of repose but after co-milling higher

concentrations of HPC-L reduced the angle of repose exhibiting better flow.

Table 1. Flow properties of norfloxacin un-milled, milled and its different formulations

Formulations Bulk Tapped Compressibility Hausner’s Angle of

density density index ratio repose [O]

Norfloxacin 0.67 ± 0.014 0.80 ± 16.25 1.19 19.80 ± 0.014

0.015

Milled Norfloxacin 0.38 ± 0.016 0.57 ± 33.33 1.50 34.64 ± 0.017

0.014

PMPVP 1 : 1 0.57 ± 0.017 0.66 ± 0.023 14.33 1.16 22.65 ± 0.021

PMPVP 1 : 2 0.52 ± 0.013 0.60 ± 13.33 1.15 21.85 ± 0.017

0.017

PMPVP 1 : 3 0.50 ± 0.024 0.57 ± 12.43 1.14 19.30 ± 0.015

0.015

PMPVP 2 : 1 0.58 ± 0.027 0.72 ± 20.00 1.25 23.89 ± 0.012

0.016

MPMPVP 1 : 1 0.44 ± 0.035 0.63 ± 30.15 1.43 36.68 ± 0.031

0.019
 MPMPVP 1 : 2 0.45 ± 0.012 0.62 ± 0.025 27.41 1.38 34.60 ± 0.016

MPMPVP 1 : 3 0.45 ± 0.017 0.55 ± 18.18 1.22 12.26 ± 0.042

0.042

MPMPVP 2 : 1 0.35 ± 0.015 0.52 ± 32.69 1.48 36.86 ± 0.031

0.021

CMPVP 1 : 1 0.57 ± 0.019 0.62 ± 08.06 1.08 11.30 ± 0.015

0.018

CMPVP 1 : 2 0.58 ± 0.051 0.65 ± 10.76 1.12 30.96 ± 0.017

0.027

CMPVP 1 : 3 0.59 ± 0.032 0.67 ± 11.94 1.13 32.56 ± 0.023

0.021

CMPVP 2 : 1 0.52 ± 0.043 0.61 ± 14.75 1.17 34.96 ± 0.042

0.037

PMHPC-L 1 : 1 0.58 ± 0.024 0.64 ± 0.026 09.37 1.10 22.93 ± 0.058

PMHPC-L 1 : 2 0.46 ± 0.019 0.61 ± 24.59 1.32 30.75 ± 0.041

0.023

PMHPC-L 1 : 3 0.34 ± 0.014 0.47 ± 25.53 1.38 32.96 ± 0.037

0.012

PMHPC-L 2 : 1 0.58 ± 0.045 0.65 ± 0.049 10.76 1.12 24.82 ± 0.015

MPMHPC-L 1 : 1 0.42 ± 0.013 0.54 ± 22.22 1.28 29.51 ± 0.029

0.012

MPMHPC-L 1 : 2 0.36 ± 0.016 0.51 ± 29.41 1.41 30.96 ± 0.017

0.017
MPMHPC-L 1 : 3 0.34 ± 0.042 0.50 ± 0.037 32.00 1.47 35.78 ± 0.025

MPMHPC-L 2 : 1 0.42 ± 0.031 0.61 ± 31.14 1.45 35.21 ± 0.019

0.028

CMHPC-L 1 : 1 0.47 ± 0.027 0.63 ± 0.025 25.39 1.34 30.25 ± 0.036

CMHPC-L 1 : 2 0.34 ± 0.051 0.45 ± 0.046 24.44 1.32 19.98 ± 0.027

CMHPC-L 1 : 3 0.31 ± 0.016 0.39 ± 20.51 1.25 16.85 ± 0.014

0.015

CMHPC-L 2 : 1 0.49 ± 0.039 0.62 ± 20.96 1.26 29.24 ± 0.031

0.031

Determination of solubility

The solubility of un milled norfloxacin in water and phosphate buffer pH 6.8 was found

to be 0.383 ± 0.017 and 0.64 ± 0.016 mg/mL, respectively (Table 2). The solubility results of

norfloxacin-PVP mixtures (Table 2) had shown the highest increase in solubility for the co-

milled norfloxacin mixture at 1 : 1 ratio in water followed by milled physical mixture in 1 : 1

ratio. Unmilled norfloxacin showed very little solubility i.e., 0.383 mg/mL (19.15%) in water

after separate milling and when mixed with PVP, it showed 59.2% solubility and after co-

milling of both drug and polymer aqueous solubility increased to 74.65%. The solubility

analysis of norfloxacin HPC-L physical mixtures (Table 2), exhibited that maximum increase

in solubility was achieved with milled physical mixture in 2 : 1 ratio i.e., 55.35% in water.

There was no considerable increase in solubility for co-milled mixtures in buffer while physical

mixtures and milled physical mixtures showed greater solubility values in buffer than co-milled

samples.
 Table 2. Solubility of norfloxacin un-milled, milled and its different formulations

Solubility in water Solubility in buffer

Formulations mg/mL Mole fraction ◊ mg/mL Mole fraction ◊

105 105

Norfloxacin 0.383 ± 0.017 2.156 0.642 ± 0.016 3.615

Norfloxacin milled 0.670 ± 0.016 3.773 0.729 ± 0.015 4.106

PMPVP 1 : 1 0.682 ± 0.034 3.845 0.742 ± 0.023 4.179

PMPVP 1 : 2 0.670 ± 0.026 3.773 0.694 ± 0.031 3.911

PMPVP 1 : 3 0.623 ± 0.019 3.510 0.666 ± 0.043 3.752

PMPVP 2 : 1 0.738 ± 0.017 4.156 0.811 ± 0.021 4.570

MPMPVP 1 : 1 1.184 ± 0.025 6.691 0.748 ± 0.018 4.216

MPMPVP 1 : 2 1.046 ± 0.038 5.891 0.742 ± 0.019 4.179

MPMPVP 1 : 3 0.857 ± 0.015 4.829 0.739 ± 0.016 4.167

MPMPVP 2 : 1 0.923 ± 0.017 5.201 0.815 ± 0.017 4.594

CMPVP 1 : 1 1.493 ± 0.014 8.412 0.631 ± 0.015 3.557

CMPVP 1 : 2 0.967 ± 0.029 5.447 0.612 ± 0.012 3.447

CMPVP 1 : 3 0.963 ± 0.035 5.423 0.590 ± 0.041 3.325

CMPVP 2 : 1 0.869 ± 0.042 4.897 0.593 ± 0.032 3.337

PMHPC-L 1 : 1 1.009 ± 0.013 5.686 0.768 ± 0.019 4.326

PMHPC-L 1 : 2 0.844 ± 0.026 4.754 0.731 ± 0.028 4.118

 PMHPC-L 1 : 3 0.836 ± 0.021 4.706 0.668 ± 0.017 3.765

PMHPC-L 2 : 1 0.937 ± 0.018 5.280 0.857 ± 0.015 4.826

MPMHPC-L 1 : 1 0.895 ± 0.031 5.042 0.913 ± 0.029 5.143

MPMHPC-L 1 : 2 1.008 ± 0.012 5.679 0.924 ± 0.018 5.204

MPMHPC-L 1 : 3 0.961 ± 0.019 5.414 1.024 ± 0.023 5.766

MPMHPC-L 2 : 1 1.107 ± 0.017 6.236 0.863 ± 0.014 4.863

CMHPC-L 1 : 1 0.904 ± 0.051 5.089 0.573 ± 0.017 3.228

CMHPC-L 1 : 2 0.942 ± 0.016 5.304 0.595 ± 0.015 3.349

CMHPC-L 1 : 3 0.950 ± 0.034 5.352 0.599 ± 0.041 3.374

CMHPC-L 2 : 1 0.814 ± 0.027 4.587 0.567 ± 0.025 3.191

Dissolution studies

The intrinsic dissolution showed 52.87% drug release from the unmilled drug in acetate

buffer after 4 h and 60.48% from the milled drug (Fig. 1) and in distilled water it was 28.61

and 45.12%, respectively (Fig. 2). The blends of norfloxacin with HPC-L displayed that the

maximum increase in dissolution was with unmilled physical mixtures as compared to milled

and co-milled mixtures. The highest dissolution among physical blends was seen with PMHPC-

L 1 : 1.
 (13)

(13)

Optimization of formulations

Formulations showing best flow properties and maximum aqueous solubility among all

milled physical mixtures (MPM) and co-milled mixtures (CM) were selected for optimization

studies.
 Flow properties of optimized formulations

Optimized formulations showed poor flow ability as compared to the formulations

milled at 20 Hz in the preliminary studies (Table 3). However, optimized mixtures of

norfloxacin with polymers showed better flow ability than milled norfloxacin alone.

Table 3. Flow properties of optimized formulations

Formulations Bulk Tappe Compressibilit Hausner Angle of

density d y index ratio repose [O]

densit

Norfloxacin 0.67 ± 0.014 0.80 ± 16.2 1.19 19.80 ± 0.013

0.015 5

Milled norfloxacin 0.39 ± 0.017 0.59 ± 33.8 1.49 41.74 ± 0.021

0.017 9

MPMPVP 1 : 1 0.42 ± 0.013 0.60 ± 28.3 1.39 34.82 ± 0.018

0.016 3

MPMHPC-L 2 : 1 0.46 ± 0.029 0.60 ± 23.3 1.30 33.69 ± 0.017

0.027 3

CMPVP 1 : 1 0.35 ± 0.016 0.48 ± 27.0 1.37 32.61 ± 0.015

0.014 8

CMHPC-L 1 : 3 0.37 ± 0.026 0.51 ± 27.4 1.37 21.80 ± 0.024

0.019 5
 Solubility studies of optimized formulations

By increasing the speed of milling from 20 to 30 Hz aqueous solubility of norfloxacin

alone increased from 33.5 to 50.35% but further increase in speed from 30 to 40 Hz resulted in

decrease in solubility from 50.35 to 47.1%. There was considerable increase in the aqueous

solubility of the optimized formulations as compared to formulations prepared at 20 Hz (Table

4). Among optimized formulations maximum aqueous solubility was seen with CMPVP1: 1

up to 79.55%.

Table 4. Solubility of optimized formulations

Solubility in water Solubility in buffer

Formulations mg/mL Mole fraction ◊ mg/mL Mole fraction ◊

105 105

Norfloxacin 0.383 ± 0.017 2.156 0.642 ± 0.016 0.383 ± 0.017

Norfloxacin milled at 40 0.942 ± 0.015 5.305 0.974 ± 0.021 0.942 ± 0.015

Hz

Norfloxacin milled at 30 1.007 ± 0.018 5.672 0.837 ± 0.015 1.007 ± 0.018

Hz

MPMPVP 1 : 1 1.288 ± 0.016 7.249 0.463 ± 0.032 1.288 ± 0.016

CMPVP 1 : 1 1.591 ± 0.014 8.961 0.840 ± 0.017 1.591 ± 0.014

MPMHPC-L 2 : 1 1.118 ± 0.015 6.296 0.582 ± 0.016 1.118 ± 0.015

CMHPC-L 1 : 3 1.094 ± 0.019 6.161 0.788 ± 0.028 1.094 ± 0.019

Dissolution of optimized formulations

Milled norfloxacin alone and with polymers showed increased release of drug than

unmilled norfloxacin in both acetate buffer pH 4.0 (Fig. 3) and distilled water (Fig. 4). Among

optimized formulations, CMPVP 1: 1 showed maximum release within 30 min in acetate buffer

but in water drug release was slower than in buffer. The initial high drug release was observed

at the 30 min time point and reduced at subsequent time points. Slow dissolution was observed

subsequently till the equilibrium concentration was reached.

(13)
 (13)

CONCLUSION

The main objective is to increase aqueous solubility of drug without affecting its flow

properties was achieved successfully. Flow properties, solubility and dissolution profile of

norfloxacin improved after co-milling with polyvinyl pyrrolidone and hydroxylpropyl

cellulose. Un milled norfloxacin was very little soluble in water i.e., 0.383 mg/mL (19.15%),

after optimization using high speed milling with hydrophilic polymers, the polymer blend

CMPVP 1 : 1 showed maximum increase in aqueous solubility i.e., 1.591 mg/mL (79.55%)

followed by MPMPVP 1 : 1 i.e., 1.288 mg/mL (64.4%), MPMHPC-L 2 : 1 i.e., 1.118 mg/mL

(55.9%) and CMHPC-L 1 : 3 i.e., 1.094 mg/mL (54.7%). Positive effect of milling on solubility

even after simple compaction of powders into tablet form was observed. So it can be concluded

that co-milling with both polymers can be used as a tool to enhance aqueous solubility of

norfloxacin. Process of comilling could help us to improve the aqueous solubility of BCS class

II drugs having properties similar to norfloxacin. As HPC-L mixtures showed slow release
behaviour so it can be considered for sustained release formulations and PVP can be considered

for designing immediate release formulations.

 Milling of Bosentan Hydrate using Jet Mill

Bosentan hydrate (BST) is a competitive dual endothelin receptor antagonist that is

nonselective for both endothelin A and endothelin B receptors. It decreases both pulmonary

vascular resistance and systemic vascular resistance and hence increases cardiac output

without increasing the heart rate(17,18). BST hydrate was approved by the US Food and Drug

Administration (FDA) in 2001 under the brand name of Tracleer® (Bosentan), which is

orally administered to alleviate the symptoms of pulmonary arterial hypertension (PAH).

Since PAH is a disease of pulmonary circulation and not of the whole circulatory

system, the oral dosage of BST hydrate that is currently marketed has several limitations and

produces side effects associated with its systemic circulation. The side effects of the systemic

circulation include systemic hypotension, deterioration in the right ventricular performance,

reduction in the right coronary blood flow, increase in the shunt function, and reduced

oxygenation. Furthermore, orally administered BST hydrate also exhibits hepatotoxicity via

the first-pass hepatic metabolism. Thus, there is an unmet clinical need to explore the

possibilities of delivering anti-PAH drugs right into the diseased pulmonary circulation and

also to develop a drug delivery system that is capable of releasing medication only in the

vicinity of the diseased part of the pulmonary circulation.

Targeted pulmonary delivery of BST hydrate has several advantages, including

avoidance of the hepatic first-pass metabolism, a higher therapeutic concentration locally in

the pulmonary system and, thus, a reduction in the systemic side effects. The targeted

pulmonary delivery through inhalation is an effective noninvasive method for local

therapeutic delivery of active pharmaceutical ingredients

 Of the various types of targeted pulmonary delivery formulations and devices, dry

powders for inhalation are attractive since these offer several advantages, including a high

stability in the dry state, easy handling, and portability. Actually, dry powder inhalations

(DPIs) significantly decrease the daily treatment burden compared to a liquid formulation due

to the absence of cleaning and disinfection requirements

The efficiency of DPIs is typically determined by the aerosol performance of particles

and is also significantly influenced by the physicochemical properties of the formulation,

including the particle size distribution, density, surface morphology, shape, interparticulate

forces, and solid state

Jet milling is a size reduction process by particle–particle and particle–wall collisions

under high-velocity jets of compressed gases, and this allows for micronizing without

contamination as well as the milling of heat-sensitive materials. The mechanical parameters

for milling, namely the jet pressure and particle nature, strongly affect the particulate and

physicochemical properties of the particles(19-21)

The objectives of this study were to prepare BST microparticles as DPIs using

different jet-milling processes under various parameters, to comprehensively characterize the

particulate and physicochemical properties and to indirectly evaluate their suitability as DPIs

by determining the aerosol dispersion performance and drug dissolution behaviour of the

BST microparticles.
Materials and Methods

Materials

BST hydrate (molecular weight: 569.63 g/mol) was obtained from Hanmi Pharmaceutical Co.

Ltd. (Seoul, Korea). High-performance liquid chromatography (HPLC)-grade ethanol and

acetonitrile were used (Honeywell Burdick & Jackson®, Muskegon, MI, USA), and all other

reagents were either analytical grade or HPLC grade.

Preparation of BST Microparticles

Jet-milling method

The jet-milled BSTs microparticles (JM-BSTs) were prepared using an air jet mill (A-O JET

MILL; JS Tech Co. Ltd., Sacheon, Korea). As listed in Table 1, JM-BSTs were prepared under

three different grinding air pressures of 0.2, 0.3, and 0.4 MPa, designated as JM 0.2, JM 0.3,

and JM 0.4, respectively. In all cases, the other jet-milling parameters were set as follows:

feeding rate of 250%, feeding vibration of 40 Hz and pushing air pressure of 0.5 MPa. Nitrogen

air was used for milling, and the JM-BSTs were then kept in a glass vial containing silica gel

at -20°C until used.

HPLC analysis

The BST contents in the SD and JM microparticle formulations were analysed using a validated

HPLC (Ultimate 3000 series HPLC system; Thermo Fisher Scientific, Waltham, MA, USA)

consisting of a pump, an autosampler, a column compartment, and a diode array detector

operating at ultraviolet 220 nm with a Luna L11 250 ×4.60 mm, 5 μm column (Phenomenex,

Torrance, CA, USA). The mobile phase consisting of acetonitrile and buffer (0.1%

triethylamine solution, pH 2.5) at a ratio of 45:55 (v/v) was eluted at a flow rate of 1.5 mL/min.
The column temperature was maintained at 35°C, and the volume of each injected sample was

10 μL

Physicochemical characterization of BST microparticles

Scanning electron microscope (SEM)

The BST microparticles were visually imaged via SEM (ZEISS-GEMINI LEO 1530; Carl

Zeiss AG, Oberkochen, Germany). The samples were placed onto carbon tape and were then

coated with platinum using a Hummer VI sputtering device, reaching 200 Å coating thick. A

voltage of 3 kV and magnifications of 5,000× and 20,000× were used.

Particle size distribution and surface charge

The particle size distribution and surface charge of the BST microparticles were determined

using a dynamic light scattering technique (Zetasizer Nano ZS; Malvern Instruments, Malvern,

UK, measurement range of 0.3 nm–10.0 μm). Three milligrams of the samples were added to

10 mL of distilled water, and the suspensions were vortexed for 20 s and allowed to equilibrate

for 1 h. The mean particle size and size distribution that were measured are expressed as the Z-

average and polydispersity index, while the surface charge was expressed as the zeta potential

(ZP).
Water content

The water content of the BST microparticles was quantified via Karl Fischer titration (736 GP

Titrino; Metrohm, Herisau, Switzerland). A known amount of sample was dissolved in

methanol and titrated with Hydranal®-Composite as a reagent. Karl Fischer titration was

standardized using water before quantifying the water content of the samples.

True density

A pycnometer (AccuPyc 1330; Micromeritics Instrument Corporation, Norcross, GA, USA)

was used to determine true density of the BST microparticles under helium gas. The

temperature was maintained at 27°C, and each sample was run 10 times.

Surface area

The surface areas of the BST microparticles were measured using an accelerated surface area

and porosimetry analyzer (ASAP 2000; Micromeritics Instrument Corporation). The samples

were degassed at 90°C for 90 min under nitrogen prior to analysis, and the surface area was

calculated according to the Brunauer–Emmet–Teller (BET) equation.

Differential scanning calorimeter (DSC )

The thermal behavior and phase transition of the BST microparticles were measured using a

DSC (DSC 2910; TA Instruments, New Castle, DE, USA). Each sample was placed in DSC

aluminum sample pans that were then sealed and heated from 30°C to 180°C at a heating scan

rate of 10°C/min.
Powder X-ray diffraction (PXRD)

The PXRD patterns of the BST microparticles were measured using an X-ray diffractometer

(XDS 2000; Scintag, Cupertino, CA, USA). The scanning range of 2θ was from 5° to 60° with

a step size of 0.009°/2θ at ambient temperature with a Cu radiation source (40 kV, 40 mA).

Fourier transform infrared (FT-IR ) spectroscopy

The infrared spectra were recorded using the FT-IR spectroscopy (IFS 66v/S; Bruker Optics,

Ettlingen, Germany) by following the potassium bromide technique, and the spectroscopic

wavelength range from 2,400 to 4,000 cm-1 was investigated.

RESULTS

Particle and surface morphology of JM-BST microparticles

Images were obtained using an SEM, as shown in Figure 7, to obtain the qualitative information

of the shape and surface morphology of the BST microparticles. The raw BST had irregular

non-spherical morphology, polydisperse size range, and crystalline particle state. Changes in

crystallinity and in the morphology for JM 0.4 were not observed, but the particles appeared to

be smaller and narrower in size than the raw BST. In addition, the quite small particles of JM

0.4 showed a tendency for aggregation. The drug solution concentrations of the SD-BSTs and
the grinding air pressure of the JM-BSTs did not significantly influence the morphologic

properties.

Fig 7: SEM micrographs of bosentan microparticles(22)

(A & B) Raw Bonsentan Hydrate;(E & F) Jet Milled 0.4 Mpa
Magnification for samples was 5,000x & 20,000x
Physicochemical properties of JM-BST microparticles

Particle size distribution

Table 1 shows data pertaining to particle size and size distribution, suggesting that the Z-

average of the SD-BSTs ranged from 2.2 to 2.7 μm. The JM-BSTs showed a Z-average within

the range from 1.7 to 2.8 μm. In particular, the JM 0.4 exhibited a considerable reduction in

particle size.

Surface charge

Table 1 also presents the ZP describing the surface charge. The ZP was from -41.4±0.6 to -

42.6±0.5 mV for JM-BSTs, which varies significantly according to the different preparation

methods used (P,0.05, ANOVA/Tukey).

Water content

The water content of all prepared BST microparticles was analytically quantified via Karl

Fischer titration, and the results are listed in Table 1. The water content of the JM-BSTs was

from 3.7% to 3.8%, which indicates that this method kept the raw BST water content

True density

The true density of the BST microparticles was measured using a pycnometer, and the results

are listed in Table 1. All BST microparticles showed a density of 1.3 g/cm3 that is equal to the
true density of raw BST. The true density was not affected by the preparation methods and

formulation since the particles were composed of a single material.

Surface area

The surface area was measured using the nitrogen adsorption BET method, and Table 1 shows

the surface area of the BST microparticles, in which the data are seen to be consistent with the

observations from the SEM images. Significant differences can be seen in the surface area after

the jet-milling process. the surface area of the JM-BSTs was 3.2±0.1–3.8±0.1 m2/g. Jet milling

resulted in small, rough, and irregular particles. In practice, the surface area is seriously

affected by the degree of roughness and the size of the particles.

TABLE 1

Formulation Z-average(d,µm)(PDI) ZP(mV) Water% True Surface

density(g/cm3) area(m2/g)

Raw BST 3.1(1.0) -43.4±1.5 3.5 1.3 2.9±0.0

JM 0.2 2.6(0.7) -42.4±2.0 3.7 1.3 3.2±0.1

JM0.3 2.8(0.9) -41.4±0.6 3.8 1.3 3.4±0.1

JM0.4 1.7(0.7) -42.6±0.5 3.9 1.3 3.8±0.1

Crystallinity characterization of JM-BST microparticles

DSC

Figure shows the DSC thermograms that were used to characterize the thermal behaviour of

the BST microparticles. The raw BST exhibits two endotherm peaks at 113.6°C and 125.9°C,

which is consistent with the results previously reported in the literature.26 The first peak
represents the water elimination of hydrate, and the second sharp peak is the melting point of

drug, which indicates that a high crystalline solid state is achieved. The JM-BSTs did not show

a significant influence in the thermal behaviour, with a sharp endothermic peak at ~125°C

representing the melting point

Fig 8:DSC of JM BST Microparticles(22)

PXRD

The PXRD patterns of the BST microparticles are shown in Figure 5. The diffractograms of

the SD-BSTs had no specific diffraction peaks due to complete phase transforming to an

amorphous solid state, whereas the raw BST and JM-BSTs had approximately identical

diffractograms with sharp and strong diffraction peaks at the main angles (2θ) of 9.28, 15.55,
16.69, and 18.64°, indicating that both had a high crystallinity and that jet milling did not

influence the solid state of the drug.

Fig 9: PXRD of JM BST Microparticles(22)

FT-IR spectroscopy

The results of the FT-IR for the BST microparticles are depicted in Fig below to determine the

functional groups that are present in the BST microparticles’ structure. The raw BST spectra

were in agreement with that from a previous study. In comparison to the raw BST, the FT-IR

spectra of the SD-BSTs showed a disappearance in the peaks at ~3,650–3,600, ~3,400–3,500,

and ~3,000–3,100 cm-1. These peaks may respectively correspond to the O–H stretching, N–
H stretching, and C–H stretching in the particle structure. As expected from the DSC and

PXRD, the FT-IR spectra of the JM-BSTs showed no appearance or disappearance of any of

the characteristic peaks, which confirmed the absence of chemical changes in the structure

resulting from the jet-milling process.

Fig 10: FTIR- Spectra of JM BST Microparticles(22)

In vitro drug dissolution study

The dissolution profiles of the BST microparticles obtained via a Franz diffusion cell are shown

in Figure 8. The drug concentrations were measured, and the data are plotted as the cumulative

percentage of drug dissolved over 9 h. The Franz diffusion cell was used to wet the BST

microparticles deposited on the membrane and then to dissolve and diffuse them into the
surrounding media. It mimicked the diffusion-controlled air–liquid interface of the lung after

administration of the inhaled drug. The JM-BSTs showed a relatively constant drug dissolution

rate during the 9 h period, and these results were not influenced by the grinding nozzle pressure

of the jet milling. However, for all BST microparticles, the cumulative percentage of the drug

dissolution at 9 h ranged from 28.8% to 39.9% and did not vary significantly according to the

preparation methods.

Fig 11: In-vitro drug dissolution study(22)

Conclusion

There were significant differences in the physicochemical properties of the BST microparticles

depending on the preparation methods that were used. The JM-BSTs were irregular and rough

with a crystal solid state. These properties directly influenced the aerosol dispersion and the

dissolution behaviour of the SD-BSTs and JM-BSTs as DPIs. In conclusion, the JM-BSTs are

ideal DPIs, and high grinding air pressures with jet milling may produce an improved

deposition in the deep lung region for pulmonary delivery due to the smaller aerodynamic

diameter and aggregate strength owing to their physicochemical properties. The DPIs in the

JM 0.4 sample exhibited suitable properties for clinical delivery and have the possibility to

deliver a higher drug concentration locally in the lung and thus reduce the systemic side effects

and improve the therapeutic effect for PAH treatment. Further studies are needed to explore

the BST microparticles delivering to lung target region and therapeutic effects for PAH in

animals.
References/ Bibliography

1. The theory and practice of industrial pharmacy by Lachman and Lieberman

2. Kraml, M., Dubue, J., and Gaudry, R: Antibiot. Chemother., 12:232, 1962.

3. Garrett, E. R, Schumann,E. L, and Grostic, M. F : J . Am. Pharm. Assoc., Sci Ed.,

48:684, 1959

4. Gold, G., Duvali, R. N, Palermo, B. T and Slater, J. G. J. Pharm. Sci., 57:668, 1968.

5. Danish, F. Q, and Parrott, E. L,: J. Pharm. Sci, 60:548,1971.

6. Sumner, E. D., Thompson, H.O Poole, W. K and Grizzle, J. E: J Pharm Sci.,

55:1441,1966.

7. Danish, F. Q., and Parrott, E. L: J. Pharm. Sci, 60:752, 1971.

8. Piret, E, L : Chem. Eng. Prog., 49:56, 1953.

9. Heywood, H,: J. I . C. Chem Eng Soc., 6:26,1951

10. Austin, L. G, and Klimpel, R. R: Ind. Eng . Chem., 56:18, 1964.

11. Bond, F. C : Min. Eng., 4:484, 1952

12. Riley, R. V .: Chem. Proc. Eng., 46:189,1965

13. MARIA MIR, KHEZAR HAYAT, TALIB HUSSAIN, MUHAMMAD KHURRAM

WAQAS and NADEEM IRFAN BUKHARI.BALL MILL BASED CO-MILLING: A

PROMISING WAY TO ENHANCE AQUEOUS SOLUBILITY OF POORLY

SOLUBLE DRUGS EMPLOYING NORFLOXACIN AS MODEL DRUG. Acta

Poloniae Pharmaceutica-Drug Research, Vol. 75 No. 1 pp. 155-168, 2018

14. Lindenberg M., Kopp S., Dressman J.: Eur. J.Pharm. Biopharm 58, 265 (2004).

15. Sabitha Reddy P., Greeshma Haritha M.,Ravindra Reddy K.: Pharma Science Monitor

3,118 (2012).

16. Gavhane Y.N., Yadav A.V.: Int. J. Pharm.Pharm. Sci. 5, 414 (2013).
17. Rubin LJ, Badesch DB, Barst RJ, et al. Bosentan therapy for pulmonary arterial

hypertension. N Engl J Med. 2002;346(12):896–903.

18. Farber HW, Loscalzo J. Pulmonary arterial hypertension. N Engl J Med.

2004;351(16):1655–1665.

19. Djokić M, Kachrimanis K, Solomun L, Djuriš J, Vasiljević D, Ibrić S. A study of jet-

milling and spray-drying process for the physicochemical and aerodynamic dispersion

properties of amiloride HCl. Powder Technol. 2014;262:170–176.

20. Palaniandy S, Azizli KAM, Hussin H, Hashim SFS. Effect of operational parameters

on the breakage mechanism of silica in a jet mill. Miner Eng. 2008;21(5):380–388.

21. Midoux N, Hošek P, Pailleres L, Authelin J. Micronization of pharmaceutical

substances in a spiral jet mill. Powder Technol. 1999;104(2):113–120.

22. Hyo-Jung Lee,Ji-Hyun Kang,Hong-GooLee,Dong-Wook Kim,Yun-Seok Rhee,Ju-

Young Kim,Eun-Seok Park,Chun-Woong Park. Preparation and physicochemical

characterization of spray-dried and jet-milled microparticles containing bosentan

hydrate for dry powder inhalation aerosols