Valsartan thesis

transcript

1. Introduction

Dept. of Pharmaceutical Technology CPS, JNTUH 1

1. Introduction: In recent years, it has become evident that development of new drugs alone is not

sufficient for optimal therapy. After drug discovery, drug candidates are usually evaluated for

their efficacy in vitro; a step which usually shows promising results. However, when tested in

vivo, the drug may fail to show any activity because of the following reasons:

• Poor drug solubility.

• Poor absorption, rapid metabolism and/or elimination.

• Widespread of drug distribution to the non-target tissues.

• Fluctuation of drug level in plasma due to erratic absorption after oral administration.

A drug's therapeutic efficacy depends on its pharmacokinetic parameters. These include

fundamental pathways of drug absorption form the administration site into the plasma,

distribution to the tissues where metabolism takes place and elimination from the body. Unless

the drug is given via the intravenous route, it first undergoes absorption, which depends on many

physicochemical parameters related to the drug such as its hydrophobicity, particle size, and

crystallinity. Many leading drugs, that have beneficial roles in treatment of serious chronic

diseases, are lipophilic or poorly-water soluble. For example, anticancer drugs such as etoposide,

camptothecin, and paclitaxel; the leading antifungal drugs such as amphotericin B, fluconazole

and itraconazole; antioxidants such as vitamin A, vitamin E, retinol, lycopene, and P-carotene

are lipophilic (Baskin and Salem, 1997; Basu et al., 1999; Blomhoff, 1994; Garewal, 1997;

Kumpulainen and Salonen, 1996; Prasad et al., 1995; Rosales, 2002; Salonen and Kumpulainen,

1999; Sies and Krinsky, 1994). The delivery of lipophilic drugs is challenging due to their

instability in aqueous biological environments, food interactions, reduced bioavailability, non-

specific targeting, and toxicity. Lipophilic, or poorly-water soluble drugs, must be formulated

and delivered in a safe, efficacious, and cost effective manner. Therefore advanced drug delivery

research, with emphasis on nanotechnology, has surged during the past decade. Nanotechnology

has provided scientists with new techniques for creating novel and advanced drug delivery

technologies. The specific goals of advanced drug deliverysystems are to maximize drug

bioavailability, to enable tissue targeting, and to controldrug release kinetics meanwhile eliciting

minimal immune response.

Dept. of Pharmaceutical Technology CPS, JNTUH

Advanced drugdelivery systems can be classified according to their size into the following

categories:

• Colloidal drug carriers.

• Microparticles.

• Implants.

Of most importance are the colloidal drug carriers (CDCs), which have been used to

improve the pharmacokinetic and pharmacodynamic properties of various types of drug

molecules. For this reason, there is growing interest in CDCs, which can be categorized into

polymeric nanoparticles, liposomes, nanosuspension, lipid-basedformulations (such as self-

emulsifying drug delivery systems (SEDDS) and self- microemulsifying drug delivery systems

(SMEDDS)), solid lipid nanoparticles (SLNs);and nanostructure lipid carriers (NLCs).

1.1. Solid Lipid Nanopartilces (SLNs):

Solid lipid nanoparticles (SLNs) were first introduced by Muller et al. in 1991.Since then

SLNs have attracted increasing interest as a carrier system for therapeutic and cosmetic

applications (Almeida et al., 1997; Muller et al., 2002a; Schwarz et al., 1994; Wissing et al.,

2004). SLNs are considered emerging alternative carriers to colloidal systems for controlled and

targeted drug delivery. They have the colloidal particles of a lipid matrix that remain in solid

state at body temperature. SLNs are aqueous colloidal dispersions with a size in the range of 50-

1000 nm (Castelli et al., 2005), the matrix of which is comprised of biodegradable and

biocompatible solid lipids. SLNs combine the following advantages (Mehnert and Mader, 2001):

• Provision of controlled drug release.

• Protection of incorporated drugs against chemical degradation.

• Biosafety of the carrier.

• Feasibility of large-scale production.

• Physical stability and lack of drug leakage because of the reduced mobility of the incorporated

drugs (Freitas and Muller, 1998).

• Improved bioavailability (Fundaro et al., 2000; Zara et al., 2002).

• Enhanced cytotoxicity against multidrug resistant cancer cells (Wong et al., 2006a; Wong et al.,

2006b).

• SLNs particularly those in the range of 120-200 nm are not taken up by the cells of the Reticulo

Endothelial System (RES) and thus bypass liver and spleen filtration (Chen et al., 2004).

• Possibility of coating or attaching some ligands to SLNs, thereby increasing the scope of drug

targeting (Lockman et al., 2003).

• The feasibility of incorporating both hydrophilic and hydrophobic drugs (Fundaro et al., 2000).

Fig 1: Structure of solid lipid nanopartcile

Table 1: Lipids and surfactants used in SLN production (Adapted from Mehnert and Mader 2001)

Lipids Surfactants/cosurfactants

Triglycerides Acylglycerol

Polyoxyethylene-polyoxypropyleneco-

polymer

Polyoxyethylene sorbitan monofatty acid

esters

TricaprinTrilaurin

TrimyristinTripalmitinTristearin

Softisan® 142

Glycerol monostearateGlycerol monolaurateGlycerol palmitostearate

Poloxamer 188Poloxamer 182Poloxamer 407

Tween® 20Tween®40Tween® 60

Hard fats Fatty acids Phospholipids Bile saltsWitepsol®

W35Witepsol®

H35Witepsol®

H42Witepsol®

Stearic acidPalmitic acidDecanoic acid

Behenic & Butyric acids

Phosphatidylcholine (Epikuron®

170, Epikuron® 200), Egg lecithin

(Lipoid® E 80), Soybean lecithin(Lipoid® S 75, Lipoid® S 100)

Sodium cholateSodium glycocholateSodium taurocholate

Sodium taurodeoxycholate

1.1.1. Factors affecting quality of SLN dispersions

The overall qualities of SLNs are a function of selection of lipid cores, selection of

surfactant(s) and/or co-surfactant(s) used to cover the lipid cores, and the drug solubility in

lipids. Selection of lipid cores Lipid cores used for the production of SLNs for IV administration

should have the following properties (Miiller et al., 2000):

• Ability to produce small particles within nanometer size range.

• Possess sufficient loading capacity for all types of drugs (lipophilic or hydrophilic).

• Suitability for sterilization by autoclaving or by any other means i.e. filtration.

• Must exhibit long-term storage stability in aqueous dispersions with respect toparticle size and drug entrapment.

• Suitability of freeze-drying and/or spray drying for increasing shelf life.

• Toxicologically acceptable with minimum (if any) toxic residues, such as organicsolvents.

• Biodegradable.

1.1.1.1. The choice of lipids for SLNs preparation is critical for the following reasons :

( Wong et al., 2007a):

• To achieve efficient drug loading capacity.

• To achieve stability and, in some cases, sustained or controlled drug release.

• Polymorphism of lipids has an influence on drug payload.

Choice of the lipid cores for SLN preparation is dependent on many factors like their

degree of crystallinity, fatty acid chain length, and drug loading capacity in the lipids. The

loading capacity depends upon solubility of drug in lipid melt, physical and chemical structure of

the lipid matrix and polymorphic state of lipids (Manjunath et al., 2005). Lipids that form highly

crystalline particles with a perfect crystalline lattice, such as triglycerides, cause drug expulsion;

however, lipid mixtures of mono-, di-, and triglycerides containing fatty acids of different chain

length form crystals with many imperfections, which provide more space to accommodate the

drugs. For example, lipids, such as glyceryl monostearate and glyceryl behenate, are known to

possess less ordered crystal lattices; a property that favor successful drug inclusion. Other lipids,

such as beeswax, cetyl palmitate, tripalmitate and solid paraffin, however, have more ordered

crystal packing lipids with limited distance between fatty acid chains, a that cause expelling of

drugs outside the lipid core on storage or immediately after SLNs preparation. High drug loading

capacity into SLNs can be accomplished by disturbing the crystal order structure. This can be

performed by mixing low melting lipids, such as medium chain glyceride oils, with solid lipids.

The long-term stability of the lipid cores is dependent on their composition. For example,

tribehenin has higher physical stability if compared to tripalmitin. This is usually attributed to the

presence of 15% monoglycerides in tribehenin that possess the surfactant properties (Manjunath

et al., 2005). Presence of these monoglycerides may prevent lipid precipitation and/or crystal

growth. On the contrary, other monoglycerides such as glyceryl monostearate is extremely

unstable and considerable particle growth takes place few days after preparation (Manjunath et

al., 2005) This may be attributed to the presence of 50% of monoglycerides in glyceryl

monostearate, which cause physical destabilization (Jenning and Gohla, 2000; Jenning and

Gohla, 2001). Undoughtedly, the lipid loading capacity and its intended use play a crucial role in

its selection for SLN preparation. For instance, hard fats are not suitable for controlled release

applications because they melt at body temperature (Jenning and Gohla, 2000). Moreover, it has

been shown that the lipid core has a great influence on SLNs' particle size. For instance, the

average particle size of SLN dispersions increased with higher melting point lipids (Siekmann

and Westesen, 1992). The influence of lipid composition on SLNs particle size was also

confirmed. For instance, the average particle size of Witepsol® W35 SLNs was found to be

significantly smaller than the size of Dynasan®! 118 SLNs (Ahlin et al., 1998). These results

may be attributed to the shorter fatty acid chains and the presence of mono- and diglycerides, in

Witepsol® W35, which possess surface-active properties (Ahlin et al., 1998; Mehnert and

Mader, 2001). With regard to the lipid content, concentrations above 5-10% usually results in the

formation of SLNs with microparticles and broader particle size distributions (Siekmann and

Westesen, 1994a). However, other parameters, such as the presence of impurities that vary from

different suppliers, may impact the quality of SLN dispersions, such as particle size, zeta

potential, crystallization tendency; all of which will affect the storage stability (Mehnert and

Mader, 2001).

1.1.1.3. Selection of surfactant(s) and/or co-surfactant(s)

Surfactants in SLNs are used to disperse the molten lipid into aqueous phase and then to

stabilize the lipid/aqueous interface by covering nanoparticles' surfaces after cooling (Wong et

al., 2007a). High surfactant concentrations reduce the lipid/water interfacial tension, resulting in

a decrease in particle size, with a subsequent increase in surface area. Surfactants used in SLN

preparation process should possess the following properties (Manjunath et al., 2005):

• Must be nontoxic, non-irritant, and compatible with other excipients used in SLN preparation

(such as other surfactants, drugs and lipids).

• Capable of producing low particle size.

• Provide stability to SLNs by covering lipid surfaces.

• Effective to produce SLNs at low concentrations.

Several factors should be considered when choosing surfactants for SLN preparation:

For instance, the concentration of surfactant should be optimum in order to decrease the

interfacial tension at the lipid/water interface and cover the surface of the nanoparticles. It was

evident from literature that low surfactant concentrations result in particle aggregation with a

consequent increase in particle size. Nonetheless, excess amount of surfactant should be avoided

to prevent the decrease in entrapment efficiency, burst drug release, and toxicity (Miiller et al.,

2000). The effect of surfactant concentration on the particle size of SLNs has been extensively

studied and report elsewhere (Miiller et al., 1995; Zur Mtihlen 1996). For example, it was

reported that concentrations down to 5% (w/w) of either sodium cholate or poloxamer 188 were

able to produce good quality Compritol® SLN dispersions (Zur Miihlen 1996). SLNs prepared

using lower surfactant concentrations; however, produce dispersions with broader particle

distribution contained higher amounts of microparticles (Siekmann and Westesen 1994b).

Nonionic surfactants (such as poloxamers and Tween®) stabilize SLNs dispersions, producing

products with particles larger than those obtained with ionic surfactants (Manjunath et al., 2005).

The combination of nonionic surfactants wit lecithin has been shown to produce dispersions of

large particle size because of the formation of mixed surfactant films at the interface (Cavalli et

al., 1998). These mixed surfactants have dual role in SLNs dispersions. First, they are capable of

covering the surface efficiently; second, they can produce enough viscosity to promote SLN

stability (Cavalli et al., 1998). The effect of combination of surfactants on SLNs' particle size

was extensively evaluated. It was found that SLNs stabilized with mixtures of Lipoid

S75/poloxamer 188 or tyloxapol/lecithin has lower particle size and higher storage stability

(Siekmann and Westesen 1994b). It was also demonstrated that using ionic

surfactant/cosurfactant blends of Epikuron® 100, taurodeoxycholate and monooctylphosphate to

prepare stearic acid based SLNs produced considerably smaller particles compared to a nonionic

system composed of Tween® 80 and butanol (Cavalli et al., 1998). Some times surfactants are

not capable of covering the newly created interfaces during lipid recrystallization, leading to

particle aggregation and increase in the particle size of SLNs. In this case it is very important to

employ a secondary surfactant, or so called co-surfactant, such as glycocholate (ionic) as well as

tyloxapol (nonionic polymer), in order to cover the interfaces in a much shorter time than

phospholipids do. These types of water-soluble polymer surfactants are able to form micelles

having higher diffusion/mobility capability and may serve as reservoirs too (Siekmann and

Westesen, 1997).

1.1.2. Preparation methods of SLNs:

1.1.2.1. High-pressure homogenization (hot and cold)

High-pressure homogenization (HPH), using a high pressure homogenizer, has emerged

as a powerful technique for the preparation of SLNs (Miiller et al., 1995; Zur Miihlen and

Mehnert, 1998; Zur Miihlen et al., 1998). This method was first used for production of

nanoemulsions for total parenteral nutrition. For SLN production by HPH, dispersions are forced

under high pressure, within 100-2000 bar range, through a narrow gap of very small size. This

produces shear stress and cavitational forces to the moving dispersions with a consequent

decrease in their particle sizes. HPH could be performed under hot or cold conditions as depicted

in Figure 6. In both techniques, the lipid is first molten, and then the drug is either dissolved or

dispersed in the drug lipid melt. In the hot homogenization technique, lipids are molten at

temperature 5-10°C above their melting points. Drugs are then either dissolved or dispersed in

the molten lipid. Afterwards, the drug-loaded lipid is dispersed by high shear homogenizer at

high temperature in a hot aqueous surfactant solution to form pre-emulsion. The high

temperature decreases the lipid phase viscosity in the hot surfactant solution, yielding small

particle sizes (Lander et al., 2000). The formed emulsion is subsequently homogenized, by high-

pressure homogenizer, at a temperature above the melting point of the lipid, and the

homogenization process is usually repeated at number of cycles until the desired particle size is

obtained. However, it is recommended not to use more than 3-5 cycles at 500- 1500 bar because

it may initiate particle coalescence, resulting in particle size increase (Siekmann and Westesen

1994b). Eventually, SLNs are formed by cooling the nanoemulsion to room temperature. In the

cold homogenization technique, the drug is first dissolved or dispersed in the lipid melt.

However, in contrast to the hot homogenization procedure, the drug-lipid blend is rapidly cooled

using either liquid nitrogen or dry ice, producing a solid solution of the drug in the lipid matrix

(Manjunath et al., 2005). The formed powder is then ground by means of ball or mortar milling

to produce 50-100 (im particles. Eventually, the microparticles are dispersed in a previously

chilled aqueous surfactant solution forming pre-suspension that is subsequently homogenized at

room temperature. The main disadvantages of the cold HPH method when compared to hot

homogenization include the production of SLNs with larger particle sizes with broader size

distribution (Zur Muhlen et al., 1996) and the specific need to regulate the temperature during

the homogenization process. Temperature control is mandatory to avoid lipid melting and

subsequent drug degradation. In contrast, the main disadvantages of the hot HPH method include

temperature-induced drug degradation, drug distribution into the aqueous surfactant solution

during homogenization, and the formation of nanoemulsion and/or supercooled melts, instead of

SLNs, that can persist for weeks or even months (Mehnert and Mader, 2001).

1.1.2.2. Microemulsion technique

Microemulsion is defined as clear, thermodynamically stable and heterogeneous system

that is composed of inner lipid phase dispersed in an aqueous solution of surfactant and co-

surfactant blend. This method was first developed by Gasco and coworkers (Gasco, 1993). The

procedure of SLNs preparation starts with melting of lipid and subsequent dispersion of drug in

the molten lipid matrix. Then, the surfactant, and co-surfactant blend is dissolved in water, and

the aqueous solution is heated to the same temperature as the lipid phase containing the drug.

Thereafter, the aqueous surfactant solution is added to the lipid melt while stirring until a

transparent microemulsion is formed. Eventually, the produced microemulsion is dispersed in

cold water (2-10 °C) under mild mechanical stirring. The SLNs are subsequently formed by

rapid recrystallization of oil droplets in the cold aqueous solution. Surfactants and co-surfactants

used in this method include lecithin, bile salts, sodium taurodeoxycholate and butanol. High

shear homogenization followed by sonication High shear homogenization and sonication are

dispersing techniques, which were used to prepare SLNs (Domb, 1993). In this method, the drug

is dissolved or dispersed in lipid melt, which may be enhanced by the use of organic solvent

followed by evaporation of the solvent. The drug loaded lipid melt is then added to a hot aqueous

surfactant solution while homogenized by high shear homogenizer to produce coarse emulsion.

Thereafter, the coarse emulsion is further homogenized using ultrasonic homogenizer to obtain a

nanoemulsion, which is subsequently cooled to produce SLNs. Clozapine SLNs were prepared

by this method (Venkateswarlu and Manjunath, 2004). The effect of different process

parameters, including emulsification time, stirring rate and cooling conditions on the particle size

and the zeta potential of SLNs were previously reported (Ahlin et al., 1998). The lipids used in

this study were trimyristin, tripalmitin, tristearin, a mixture of mono-, di- and triglycerides

(Witepsol® W35, Witepsol® H35) and glyceryl behenate. Poloxamer 188 was used as a

surfactant at 0.5% (w/v). The average particle size of the SLNs was in the range from 100-200

nm. Higher stirring rates did not significantly change particle size, but slightly improved particle

size distribution as determined by polydispersity index. The main disadvantages of this method

include presence of microparticulates due to the inhomogeneous power developed by the

sonicator and the possibility of metal contamination by the probe, which may compromise the

SLNs dispersion quality.

1.1.2.3. Solvent emulsification/evaporation

A preparation of SLNs by this method has been reported in literature (Siekmann and

Westesen, 1996a; Sjostrom and Bergenstahl, 1992). The procedure, given in, begins by

dissolving the lipid in water immiscible organic solvent, such as chloroform, toluene, or

cyclohexane with subsequent emulsification in a surfactant aqueous solution to form an o/w

emulsion. SLN dispersion is subsequently produced upon solvent evaporation and precipitation

of the lipid in the aqueous medium. The average particle size produced by this method depends

on the lipid concentration. For instance, smaller particles would be obtained with lipid

concentrations up to 5% (w/v). Increasing lipid content above this level would produce

dispersions of higher viscosity, and the homogenization efficiency would diminish.

The advantage of this technique is the avoidance of the thermal degradation of drugs.

However, complete removal of solvent is hardly possible. The organic solvent residues might be

problematic due to their toxicity. Additionally, the limited solubility of lipids (e.g. tripalmitin) in

organic solvents necessitates the use of relatively diluted SLNs dispersions (e.g. 0.5-2.5%

(w/w)).

1.1.2.4. Solvent injection method

The first step in this method is to dissolve the lipid in water miscible solvents, such as

acetone, ethanol, isopropanol, and methanol. The resulting solution is then injected into water,

resulting in lipid crystallization. SLNs will be then obtained by centrifugation. SLNs having

particles of 80-300 nm were prepared by this method (Schubert and Muller-Goymann, 2003).

1.1.2.5. W/O/W double emulsion method

This method is designated for the formulation of hydrophilic drugs in the form of

liposheres (Cortesi et al., 2002). Briefly, the aqueous drug solution is dispersed, under vigorous

stirring, into a molten lipid phase containing lipophilic stabilizers (hydrophobic surfactants) to

form a primary w/o emulsion. This step is followed by dispersing the w/o emulsion into a large

volume of aqueous solution containing hydrophilic surfactant to produce w/o/w double emulsion.

SLNs are subsequently formed by cooling the emulsion, and the product can be separated by

centrifugation or ultrafiltration.

1.1.2.6. Choice of preparation method is dependent on the following factors:

• The desirable particle size of SLNs.

• The surfactants/co-surfactants added in the formulation.

• The intended route of administration.

• The drug stability (if it is thermolabile or not).

Effect of preparation procedure on particle size of SLNs As reported, it is possible to

obtain SLNs with particle size in the range from 30 to 180 nm by ultrasonication (using probe

sonicator) (Siekmann and Westesen, 1996). However, it is difficult to disperse higher lipid

concentrations by probe sonication, and therefore HPH is applied for effectively dispersing SLNs

with high lipid content. By using HPH, a reduction in the average particle size from 474 to 155

nm was obtained after the first homogenization cycle (800 bars). Increasing homogenization

cycles produced SLNs with lower particle sizes, which explain the dependence of the particle

size on the homogenization pressure and the number of cycles (Muller et al., 1995; Schwarz,

1995). For example, for poloxamer 188 stabilized systems, the optimal dispersion was obtained

with 500 bars at three cycles (Schwarz, 1995). Solvent-emulsification is sometimes considered

superior to melt-homogenization with respect to its ability to produce SLNs with small particle

sizes. This might be explained by the lower homogenization efficiency required for a lipid that is

dissolved in an organic solvent compared to a lipid melt. Additionally, the mobility of surfactant

molecules would be higher in organic solvents than in lipid melt, which promote immediate

coverage of the lipid molecules upon dispersion. In literature, the solvent

emulsification/evaporation process was compared to the melt-homogenization method

(Siekmann and Westesen, 1996). It was found that solvent emulsification method yielded

significantly smaller particles than melt-homogenization at the same production conditions when

lecithin/sodium glycocholate was used to stabilize tripalmitin dispersions. Effect of

surfactants/co-surfactants on particle size during preparation of SLNs It is important to consider

the surfactant composition for production of SLNs with small particle sizes. For instance, it was

found that SLNs stabilized by phospholipids and nonionic surfactants and prepared by melt-

homogenization procedure produced smaller particles than those prepared by solvent-

emulsification method (Siekmann and Westesen, 1996). This was attributed to the formulation

composition rather than preparation procedure. The influence of the surfactant concentration on

the SLNs particle size prepared by high shear homogenizer was investigated (Ahlin et al., 1998).

In this study, it was found that the mean particle size decreased with increasing surfactant

concentration up to 2-3% (w/w). Further increase produced large particles. On the contrary,

when using the same ingredients to produce SLNs by HPH resulted in a continuous decrease in

particle size with an increase in lecithin concentrations (Ahlin et al., 1998). These results showed

the difference between the dispersing capacity of HPH and high shear homogenizer.

Inhomogeneous power distribution is observed in high-shear homogenizers. On the other hand,

high pressure homogenizers attain the highest power densities and the most homogenous power

distribution due to the small size of the homogenizing gap (25-30|im) (Mehnert and Mader

2001). Additionally, the increase in the surface area during HPH occurs very rapidly.

1.1.3. Secondary SLNs production steps

After the preparation of SLNs, they may be further processed to improve their quality.

These steps include sterilization and/or lyophilization.

1.1.3.1. Sterilization

The next step after the production of SLNs includes the sterilization of the prepared

dispersions. Sterilization is essential especially if SLNs are taken by parenteral or ophthalmic

routes. The common sterilization methods for pharmaceutical dosage forms are autoclaving

(steam sterilization), filtration, and y-irradiation. Some times, sterilization by filtration is not

practical because the possibility of membrane clogging if the particles are greater than 0.2 nm.

Sterilization by autoclaving at 121 °C for 15-20 minutes is the most popular and convenient

method; however, it always creates the following concerns:

• Temperature-induced drug degradation.

• Formation of supercooled lipid melts with uncontrolled recrystallization of molten lipid,

resulting in the loss of controlled release properties (Manjunath et al., 2005).

• Possibility of particle size aggregation

1.1.3.2. Lyophilization

SLNs are aqueous dispersions. The presence of water may create undesirable storage

stability issues resulting in drug degradation or particle size agglomeration due to Ostwald

ripening (Mehnert and Mader, 2001). Therefore, it may be necessary to convert SLN aqueous

dispersions into dry product by freeze-drying or called lyophilization. The basic principle of this

technique is to freeze the sample at approximately -60 to -80 °C, and then subliming the ice into

water vapors under lower pressure. However, lyophilization usually is accompanied by changes

in the properties of the surfactant layer around the lipid particles, and an increase in the particle

concentration, which favor particle aggregation (Mehnert and Mader, 2001). Therefore,

lyohilization may result in close packing of lipid nanoparticles resulting in poor reconstitution in

water. To overcome this problem, cryoprotectants (e.g. trehalose, glucose, mannose, mannitol,

sorbitol, sucrose, lactose), or so-called lyoprotectants, are used during the lyophilization process.

The mechanism of these cryoprotection include the formation of a hydrophilic sheath interacting

with the polar head groups of surfactants around lipid particles (Mobley ans Schreier 1994),

which is easily reconstituted by simple shaking with water. The freezing process should be

optimized because it critically affects the quality of lyophilizate, such as the crystal structure. For

instance, rapid cooling leads to production of small and amorphous lyophilizates; whereas, slow

freezing leads to the formation of large crystals (Mehnert and Mader, 2001). Generally, in order

to obtain a good quality lyophilizate, samples should be of low lipid content, up to 5% (w/v), and

mixed with the cryoprotector trehalose (Cavalli et al., 1997; Heiati et al., 1998). Slow freezing in

ultrafreezer (at -70 to -80 °C) is superior to rapid cooling using liquid nitrogen; furthermore,

some modifications in thermal treatment to SLN dispersions (e.g. 2 hours at -22°C followed by a

2-hour temperature decrease to -40°C) was found to improve the lyophilizate quality

(Zimmermann et al., 2000).

1.1.3.3. Spray cooling :( Jannin et al., 2008)

Spray cooling also referred to as spray congealing is a process whereby the molten

formula is sprayed into a cooling chamber. Upon contact with the cooling air, the molten

droplets congeal and re-crystallize into spherical solid particles that fall to the bottom of the

chamber and subsequently collected as fine powder. The fine powder may then be used for

development of solid dosage forms — tablets or direct filling into hard shell capsules.

1.1.3.4. Spray drying: (K.Manjunath, J.Suresh Reddy and V.Venkateswarlu).

This an alteranative method to lyophilization to convert aqueous dispersion of SLN’s in

to dry product.Spray drying is defined as a process by which a liquid solution is sprayed into a

hot air chamber to evaporate the volatile fraction, i.e. the organic solvent or the water contained

in an emulsion. The process yields solid microparticles. The same equipment described for spray

cooling can be used for spray drying, the main difference relating to the temperature of the air

circulating in the atomizer chamber.

1.1.4. Types and proposed structures of SLNs

Classical SLNs

SLN structure was postulated based on the difference in melting points between the drug

and the lipid matrix (Zur Mtihlen et al., 1998). The three hypothetical models for classical SLN:

drug-enriched core, drugenriched shell, and solid solution are shown in Figure 2 (Müller, R. H.

Mäder, K., and Gohla, S., 2000). In core-enriched model, the drug concentration in the molten

lipid is near its solubility limit (Zur Miihlen and Mehnert, 1998). The drug in this case will

precipitate before lipid crystallization during cooling step because of drug supersaturation, and

subsequently surrounded by a lipid shell (Figure 2) (Müller, R. H. Mäder, K., and Gohla, S.,

2000). .An example of this model is prednisolone-loaded SLNs, which have inner crystalline

drug core and an outside amorphous shell (Zur Miihlen et al., 1996). In contrast, in the drug-

enriched shell model, the lipid crystallization precedes drug precipitation. The drug

concentration is below its saturation solubility; thus, upon cooling, the lipid crystallizes, and the

drug partitions into the lipid phase forming drug-rich layer covering the lipid core (Figure 2). If

drug precipitation and lipid crystallization occur simultaneously, the drug will be molecularly

dispersed in the lipid matrix

Fig: 2. Proposed structural models for drug loading profiles in lipid nanoparticles (Müller, R. H. Mäder, K., and Gohla, S., 2000).

1.1.5. Drug loading in SLNs

Drug loading implies drug localization in the solid lipid matrix. During the preparation of

classical SLNs and NLCs, heating and subsequent cooling processes affect distribution of the

drug between the lipid and the aqueous phases. Drug molecules may be accommodated in the

interspaces of the fatty acids chains of the lipid crystal lattice, resulting in biphasic release

kinetics. Factors determining the loading capacity of a drug in the solid lipid are (Kaur et al.,

2008a):

• Drug solubility or miscibility in the molten lipid.

• Chemical and physical structure of solid lipid matrix.

• Polymorphic state of the lipid material.

Typically, drug solubility is higher in the molten lipid than in the solid lipid (Kaur et al.,

2008a): Solubilizers can be added to enhance drug solubility in the lipid melt. Alternatively,

lipids that contain mono- and diglycerides may be used to promote drug solubilization. High

loading capacity can be achieved by adding liquid oils in SLNs to produce NLCs. Loading

capacity can also be affected by the chemical nature of the lipid. For instance, lipids with highly

crystalline particles form perfect lattice leading to drug expulsion (Westesen et al., 1993). Lipids

that are mixtures of mono-, di- and triglycerides and/or containing fatty acids of different chain

lengths form crystals with many imperfections offering more space to accommodate the drugs.

Polymorphic transformations of lipid have a crucial role in drug loading. Generally, lipid

nanoparticles recrystallize preferentially in the P'-modification, which transforms to the stable P-

form (Westesen et al., 1993). The formation of the more stable modification leads to a decrease

in the number of imperfections in the lattice and thereby promoting drug expulsion. Triglyceride

lipids with long chain fatty acids undergo transformation more rapidly than those with short

chain fatty acids (Westesen et al., 1993). Therefore, with a gradual transformation from P'- to P-

forms, a controlled drug delivery would be maintained (Jenning and Gohla 2001). In PLN, the

physicochemical compatibility between the drug-polymer complex and the solid lipid phase is

the primary factor that predicts drug loading (Wong et al., 2007a).

1.1.6. Drug release from SLNs

Drug release rate from is determined by the physicochemical properties of the lipid

material (Scholer et al., 2002), choice of surfactant composition and ratio (Kabanov and

Alakhov, 2002; Scholer et al., 2001), particle size and the inner structure of SLNs. In most cases,

drug release kinetics exhibits a biphasic feature: a burst release followed by a sustained or

prolonged release (Wong et al., 2007a). Drug release is also influenced by its localization in the

solid lipid. For example, in drug-enriched core model, sustained release profiles are usually

obtained. On the other hand, in both drug-enriched shell and solid solution models fast drug

release kinetics are expected.

During Homogenization During Cooling

Fig: 3. Proposed redistribution of drug from molecularly dispersed state to enriched shell state, postulated as a cause of drug burst release phenomena observed in lipid nanoparticles (Müller, R. H. Mäder, K., and Gohla, S., 2000).

1.1.7. Characterization of SLNs

SLNs and NLCs may be characterized with respect to particle size and zeta potential,

particles morphology and/or shape, crystallinity and lipid modification, mobility of molecules

within nanoparticles, drug entrapment efficiency (EE), and in vitro drug release for the

assessment of drug release from SLNs.

1.1.7.1. Measurement of particle size and zeta potential:

Photon Correlation Spectroscopy (PCS) and Laser Diffraction (LD)

PCS, also called dynamic light scattering, measures the fluctuation of the scattered light

intensity caused by particle movement (Mehnert and Mader, 2001). Therefore, it estimates the

particle hydrodynamic radius. PCS, however, can not detect large microparticles more than 3 ^m.

LD method, on the other hand, is used to measure particle size from few nanometer up to several

millimeter. This method is based on measuring the degree of light diffraction from the surface of

particles. Zeta potential is defined as the difference in potential between the actual particle

surface and the dispersion medium (bulk medium). The zeta potential value primarily depends on

two parameters: Surface charge of the particle and the presence of adsorbed layers at the

interface. For example, presence of ionic surfactants, either anionic or cationic, greatly

influences the zeta potential and hence overall SLNs stability by providing electric repulsion

between particles. Improved stability is expected if zeta potential is greater than +30 mV (for

cationic surfactants) or lower than -30 mV (for anionic surfactants) (Lai et al., 2006). Non ionic

surfactants, such as polyethylenepolypropylene block co-polymers (poloxamer 188), stabilize the

particles by serving as steric stabilizers, thereby preventing particle flocculation and coalescence

(Porter, 1994).

1.1.7.2. Observation of particle morphology and/or shape in SLNs Scanning Electron

Microscopy (SEM) and Transmission Electron Microscopy (TEM):

They provide information about the morphology of lipid nanoparticles and can be utilized

for the estimation of particle size. While the same instrument provides SEM and TEM, they

utilize different principles for particle observation. In SEM, backscattered or secondary electrons

transmitted from the specimen surface are observed; whereas, in TEM the electron beam

transmitted through the sample is detected. In TEM, it is possible to use visualization

enhancement tools, such as staining with phosphotungestic acid. In either method, however,

SLNs might be coated with gold for improved visualization and particle size determination.

Alternatively, cryo-imaging (such as cryo-SEM or cryo-TEM) in which specimen is quickly

frozen in order to reduce the morphological distortion important for structural observations, may

be used for the visualization of SLNs. It was reported that SLNs made from well-defined lipids

of high purity (e.g. pure triglycerides) might have cubical or platelet-like patterns, and the

chemically homogenous lipids tend to form more perfect crystals with the typical platelet-like

pattern (Siekmann and Westesen, 1992; Westesen and Wehler, 1993). Measurement of

crsytallinity, lipid modification, and other colloidal structure that might coexist with SLNs After

crystallization, solid lipids might undergo polymorphic changes or modifications resulting in

instability associated with drug incorporation and release. Alternatively, lipids might not

crystallize at room temperature, thereby producing supercooled melts, which may aggregate to

form liposomes or some other colloidal structures. For instance, Dynasan® 112 SLNs could

remain as a supercooled melt for several months, which generally happens to SLNs due to the

small size of the particles and the presence of surfactants that retard lipid crystallization

(Westesen and Bunjes, 1995). Lipid crystallinity, lipid modification, and co-existence of other

colloidal structures can be detected by Differential Scanning Calorimetry (DSC), and X-ray.

Diffraction (XRD), Infrared and Raman spectroscopy, and Proton Nuclear Magnetic Resonance

('H-NMR) spectroscopy.

1.1.7.3. Differential Scanning Calorimetry (DSC):

This technique can be widely applied to investigate the crystalline status of the lipid.

Different lipid polymorphs possess different melting points and enthalpies. Therefore, the

presence of drugs inside the lipid core as amorphous or crystalline lattice, the presence of

supercooled lipids, and the interaction of lipid components with other SLNs ingredients could be

evaluated by DSC.

1.1.7.4. X-ray diffraction (XRD):

Detecting the crystalline state of lipids and/or the presence of drug either in amorphous or

crystalline state is a challenge. Therefore, complementary to DSC, XRD was used to collect

information about formulation parameters (such as crystallization temperature) and to detect

phase separation that might occur during SLN preparation. Time resolved XRD studies, for

example, was used to study the kinetic phenomena associated with the polymorphic transition of

lipids (Bunjes and Koch, 2005).

1.1.7.5. Determination of entrapment efficiency:

In the cooling step, during the production of SLNs, drug expulsion might occur.

Therefore, after SLNs preparation, it is important to measure the amount of drug incorporated,

which is a measurement of the solid lipid efficiency to encapsulate the drug. The entrapment

efficiency can be calculated from the following equation:

Amount of drug entrapped in SLNs

Entrapment efficiency (%) = × 100

Theoretical total amount of drug added to SLNs

The amount of drug incorporated is calculated by subtracting the free drug from the total

amount of drug added to the SLNs. In order to determine the free drug, it must be separated from

the drug loaded into SLNs by ultracentrifugation, centrifugation filtration, or gel permeation

chromatography. In centrifugation filtration, filter assembly is used. This assembly consists of

sample chamber and a recovery chamber. Both chambers are separated by a filter with specific

molecular weight cutoff (e.g. molecular weight cutoff (MWCO) is 100,000 Da). After

centrifugation, the supernatant aqueous solution containing the free drug is analyzed by HPLC,

spectrophotometry, or spectrofluorophotometry. The quantity of the entrapped drug can be then

estimated using the equation given above. In gel permeation chromatography, Sephadex® and

Sepharose® gels are used to remove the free drug from the SLNs.

1.1.7.6. In vitro drug release from SLNs:

1.1.7.6.1. Dialysis tubing to from a bag

In this method, SLNs nanodispersion is placed in dialysis tubing, which can be

hermetically sealed to from a dialysis bag having a molecular weight limit of 12,000-14,000. The

bag allows the transport of free drug while hindering the passage of SLNs with the encapsulated

drug. The bag is then placed in a continuously stirred and suitable dissolution medium, usually a

buffer, at 37 °C. In addition to stirring, sink conditions can be maintained by the addition of

0.5% polysorbate 80, sodium dodecyl sulfate, or 30% ethanol. Aliquots are withdrawn from the

receiving compartment at different time intervals, centrifuged and analyzed for drug content.

1.1.7.6.2. Reverse dialysis

In this technique, the small dialysis bag is filled with one mL of the dissolution medium

and then placed in SLN dispersion. The sample inside the bag is analyzed for drug content. This

technique is usually applied for potent compounds.

1.1.7.6.3. Franz diffusion cell

There are two types of Franz diffusion cells: side-by-side and vertical diffusion cells.

Both are utilized for the assessment of drug release from SLNs. Franz diffusion cell consists of a

donor compartment in which SLN sample is placed, and a receptor compartment containing the

dissolution medium, usually buffer, to which the drug will diffuse. The two compartments are

separated by a cellophane membrane of suitable molecular weight cutoff size. The temperature in

both compartments is maintained at 37 °C. Sink conditions are attained by the addition of 0.5%

polysorbate 80, sodium dodecyl sulfate, or the addition of 30% ethanol. Aliquots are withdrawn

from the receptor compartment at different time intervals, centrifuged and analyzed for drug

content.

1.1.7.6.4. In vitro Dissolution Testing

A number of biorelevant dissolution test media and experimental methodologies have

found application in assessing drug release from both lipid-based and conventional oral

formulations (Dressman et al., 2005 & 2007). Unlike conventional dosage forms, from which the

drug substance simply dissolves in the aqueous dissolution test media, lipid-based formulations

release the drug from an oily solution which is often immiscible with water.

1.1.8. Problems encountered during SLN preparation:

The following are problems that might appear during or after SLNs preparation:

• Existence of supercooled melts.

• Presence of some lipid modifications.

• Change in the shape of lipid nanodispersions with a possibility of gelation.

• Coexistence of several colloidal species.

1.1.8. 1. Existence of supercooled melts

Supercooling is defined as the temperature difference between the melting and

crystallization points. It describes a phenomenon whereby the lipid fails to crystallize although

the sample is stored at a temperature below the melting point of the lipid. This occurs most often

when SLNs are prepared by the application of heat such as melthomogenization. Supercooling

range can reach 30-40°C. In this case, lipid dispersions are considered nanoemulsions rather than

1.1.8. 2. Coexistence of several colloidal species

During the preparation of SLNs, other colloidal species might be formed. These include

micelles, liposomes, and surfactant monomers. For example, when sodium dodecyl sulfate (SDS)

is used, it might produce micelles or mixed micelles. Lecithin, on the other hand, could form

liposomes (Siekmann and Westesen, 1998). The problem of these colloidal species is that they

are considered as alternative sites for drug incorporation. Lipophilic drug molecules might be

relocated into the micelles rather than into the solid lipid matrix of SLNs, resulting in the

hydrolysis of unstable drugs in the aqueous environment, or it might result in burst drug release.

1.1.8. 3. Gelation phenomena

Gelation can be defined as the transformation of SLNs from low-viscosity dispersions

into a viscous gel, which involves the loss of the colloidal particle size (Mehnert and Mader,

2001). The possible mechanism for gelation is the increase in the surface area of the particles due

to platelets formation, which could not be covered by sufficient surfactant molecules (Siekmann

and Westesen, 1994a). Gelation may occur when the lipid phase undergoes structural changes

resulting in a decrease in the zeta potential with a consequent particle growth (Freitas and

Miiller, 1998). There are different factors that potentiate gelation, such as high lipid

concentrations and high ionic strengths (Bunjes et al., 1996; Freitas and Miiller, 1999a). Rapid

crystallization of the lipid may promote gelation (Bunjes et al., 1996). One of the stability

indicators of SLNs is their zeta potential. Stable samples have a zeta potential greater than -25

mV, while SLNs having -15 mV zeta potential would produce gel (Freitas and Miiller, 1999a).

Other environmental factors may also contribute to SLNs stability and gelation. For instance, it

was found that high temperatures and the exposure to light and mechanical stress, which increase

the kinetic energy and particles' collision, promote SLN gelation (Freitas and Miiller, 1998).

Oxygen may facilitate fatty acids oxidation with subsequent gelation. Gelation can be retarded or

prevented by the addition of surfactants or coemulsifying surfactants, such as glycocholate

(Westesen and Siekmann, 1997). Storage in dark place at 8°C may prevent particle growth

(Freitas and Muller, 1998). Samples stored under nitrogen were shown to be more stable than

samples exposed to air due to the inhibition in the lipid degradation (Freitas, 1998).

1.1.9. General stability aspects and storage stability of SLNS:

Typically, the shelf life of SLNs formulation should be at least one year (Wong et al.,

2007a). The criteria for the assessment of long-term physical stability of SLN include particle

size and size distribution, zeta potential, drug content, drug entrapment efficiency, and drug

expulsion outside the lipid matrix during storage. All of these parameters are influenced by the

lipid type, sterilization process, and lyophilization. Temperature and light are considered as the

most important factors affecting SLN stability, and therefore for optimum long-term stability of

SLNs, vials containing SLNs should be stored at controlled temperature and protected from light.

1.1.9.1. Zeta potential:

Optimum zeta potential for physically stable dispersions should be, in general, higher

than -60 mV. A decrease in zeta potential may lead to particle agglomeration and rapid growth in

particle size, which usually occurs when SLNs are stored at 50 °C (Freitas and Miiller, 1998).

Although in some cases long term storage at 20 °C does not result in SLN aggregation, storage

of SLNs at 4 °C is generally more favorable (Freitas and Miiller, 1998).

1.1.9.2. Particle size and size distribution:

Particle size is a critical safety factor for SLNs parenteral administration. In addition, it

greatly affects SLNs biodistribution and clearance by the reticuloendothelial system (RES). The

degree of polydispersity can affect particle size growth and the overall drug release. The factors

that influence change in particle size and/or zeta potential include type of lipid, sterilization

process, lyophilization, storage temperature, light, and packing materials.

1.1.9.3. Drug expulsion outside lipid matrices:

Lipid polymorphism greatly affects SLNs stability. For illustration, during storage of

SLNs, or even in the cooling step during SLNs preparation by melt emulsification, the lipid may

crystallize or produce supercooled melts. If the lipid favors crystallization into a specific

polymorph that has low intermolecular distance within its matrices (i.e. with perfect crystalline

structure), subsequent drug expulsion leakage may occur.

1.1.9.3.1. Factors affecting drug expulsion outside lipid matrices

1.1.9.3.1.1. Type of lipid:

Waxes usually lead to slower particle growth and aggregation than glycerides (Jenning

and Gohla, 2000). Monoglycerides-containing lipids, such as Dynasan® 116 and Compritol®

888 ATO, are usually more stable than those do not contain monoglycerides. Ionic surfactants

stabilize SLNs better than non-ionic (Wong et al., 2007a).

1.1.9.3.1.2. Sterilization process:

Sterilization, which is required for SLNs administration by the parenteral route, may

affect the physical stability of SLNs, particularly particle size and zeta potential. Steam

sterilization (autoclaving) may affect the physical stability, depending on the lipids, surfactants,

and drugs used in SLNs preparation. In one study, autoclaving was found to have insignificant

effect on the particle size, polydispersity index (PI), and zeta potential of SLNs (Cavalli et al.,

1997). On the contrary, and in another study, a 2-3 fold increase in SLNs particle size, a shift of

zeta potential from positive to negative, and a slight drop in drug loading was reported after

sterilization of SLNs (Muller et al., 2006; Penkler et al., 2003).

1.1.9.3.1.3. Lyophilization (freeze drying):

Lyophilization is usually utilized to prevent SLN aggregation or drug hydrolysis due to

the aqueous medium. It is carried out using cryoprotectants, such as trehalose, glucose, lactose,

mannose, mannitol, or sucrose, to stabilize SLNs and to prevent lipid adhesion after freeze

drying by forming a hydrophilic protective sheath (Shahgaldian et al., 2003). Optimization of the

lyophilization process parameters, such as freezing velocity and application of different

lyophilization cycles, is essential to produce reconstitutable SLNs that are suitable for

intravenous injection (Zimmermann et al., 2000). However, a few studies have shown particle

size increase by freeze drying (Schwarz and Mehnert, 1997). In these studies, the reconstituted

SLNs attained sufficiently small and stable particle sizes that could be administered orally.

1.1.9.4. Storage temperature, light, and packing materials:

During storage, the stability of SLNs might be affected by strong light, high temperature,

and packing material, all of which will decrease particles' zeta potential and induce particle

aggregation (Freitas and Miiller, 1998). For instance, it was found that by storing SLNs in

siliconized vials at 8 °C in the dark, a significant stability over 3 years for Compritol® SLNs

dispersions was achieved (Freitas and Miiller, 1998).

1.1.10. Possible administration routes of SLNs and their in vivo fate:

1.1.10. 1. Oral administration

SLNs can be formulated as tablets, pellets or capsules for oral drug delivery. Some times,

the acidity and high ionic strength of the microclimate of the stomach favors particle

aggregation. However, oral administration of SLNs has the advantages of reproducible

bioavailability (less variability in drug plasma levels) and prolonged drug plasma levels.

Bioavailability enhancement and reproducibility after oral administration could be demonstrated

by Cyclosporine A, which was formulated into SLNs for oral administration (Miiller et al., 2006;

Penkler et al., 2003).

1.1.10. 1.1. Mechanism of oral absorption:

Mechanism of oral absorption enhancement and reproducible bioavailability, the

increased absorption and reproducible bioavailability, observed in certain drugs formulated in

SLNs after oral administration, might be attributed to adhesiveness of nanoparticles to the GIT

membrane (Muchow et al., 2008). Adhesiveness due to the small particle size and large surface

area of SLNs leads to fast and specific drug release at the site of absorption (Liversidge and

Cundy, 1995). Furthermore, lipid nanoparticles are ultrafme dispersions, and thus they have the

potential to enhance oral bioavailability. For instance, after the digestion of lipids in the gut, the

formation of surface active monoand diglycerides may produce micelles that can entrap drugs

within their cores leading to drugs solubilization (Figure 4) (Muller and Keck, 2004). The

solubilized drug molecules may interact with the biological bile salts to produce mixed micelles

with a consequent increase in drug absorption (Muller and Keck, 2004). It was also reported that

in SLNs the fatty acid chain length may affect the site of absorption (Porter and Charman,

2001a). For instance, fatty acids with C-14 to C-18 chains promote lymphatic drug absorption

(Porter and Charman, 2001a). By this specific drug delivery to the lymphatics, it is possible to

avoid first pass metabolism, and thereby enhancing the oral bioavailability of drugs (Porter and

Charman, 200la). Schematic representation of the mechanisms of drug absorption from SLNs

and the promoting effect of lipids (Miiller and Keck, 2004) Camptothecin was prepared in SLNs

using stearic acid and a blend of lecithin and poloxamer 188 as stabilizers (Yang et al., 1999). In

addition to protecting the drug against hydrolysis, offered by SLNs, the plasma profile of SLNs

exhibited a first burst drug release attributed to the free drug with a subsequent controlled drug

release that was attributed to prolonged gut uptake of SLNs (Yang et al., 1999).

Fig: 4. Drug solubilizatin in the GIT

1.1.10. 1.2. Potential effect of lipids and lipidic excipients on drug absorption :

There are three primary mechanisms by which lipids and lipophilic excipients affect drug

absorption, bioavailability and disposition after oral administration. Lipids can affect drug

absorption (Porter et al., 2007):

By enhancing drug (D) solubilization in the intestinal milieu through alterations to the

composition and character of the colloidal environment — for example, vesicles, mixed

micelles and micelles,

By interacting with enterocyte-based transport and metabolic processes, thereby

potentially changing drug uptake, efflux, disposition and the formation of metabolites

(M) within the enterocyte,

By altering the pathway (portal vein versus intestinal lymphatic system) of drug transport

to the systemic circulation — which in turn can reduce first-pass drug metabolism as

intestinal lymph travels directly to the systemic circulation without first passing through

the liver. Cellular junctions are represented by green ovals, and a representative transport

protein is depicted by a blue oval.

Fig: 5 Effect of lipids on drug absorption

1.1.10. 2. Parenteral administration:

SLNs could be injected either intravenously, intramuscularly, or subcutaneously. When

injected intravenously, SLNs could be used to target drugs to specific tissue or organ. However,

the particle size must be below 5 (am to avoid embolism. The hydrophobic surfaces of SLNs

cause rapid clearance from the circulation by the RES and uptake by the liver, spleen and other

parts of the RES. In order to produce particles with longer circulation times, SLNs should be 100

nm or less in diameter with a hydrophilic surface in order to reduce clearance by macrophages

(Storm et al., 1995). Thus, to facilitate drug targeting to tumor tissue, "stealth SLNs" could be

prepared by pegylation, or by incorporating polyethylene glycol, to form a hydrophilic sheath

around SLNs. Such modification creates chains at the particle surface, which will repel plasma

proteins and thereby evading the RES and increasing tumor accumulation (Chen et al., 2001).

SLNs may also act as a sustained or depot release after subcutaneous administration in which

case drug release rate is controlled by the nature of the lipid, surfactant, particle size, and inner

structure of SLNs.

1.1.10. 3. Topical administration:

SLN’s posses a number of advantages for the topical route of administration due to small

particle size.SLN’s ensure close contact to stratum corneum and there by increases penetration of

encapsulated drug in to the viable skin.Sustained release of the drug from SLN’s supplies the

drug to the skin over a prolongrd period and there by reduces systemic absorption.SLN’s showed

occlusive properties as a result of film formation on the skin, which reduces transdermal water

loss. Increase of water content in the skin reduces the symptoms of atopic eczema and also

improves the appearance of healthy human skin .Occlusion also favours the drug penetration in

the skin (K.Manjunath, J.Suresh Reddy and V.Venkateswarlu).

1.1.10. 4. Rectal administration:

It is the preffered route of administration in pediatric patients due to ease of application.

Advantage of submicron emulsions and SLN’s over conventional rectal solution is that organic

solvents present in commercial preparations can be avoided. But lower relative bioavailability

was observed for SLN’s compared to solution;the reason repoeted for this is lack of efficient

diffusion through lipid matrix. Therefore the lipid matrix solid at room temperature is not an

advantageous system for rectal delivery of drugs, even if delivered as submicron dispersions.

Thus low melting point lipids were to be selected for formulations of rectal delivery drugs.to

achieve prolonged release of drug as well as higher absorption and bioavailabilites

(K.Manjunath, J.Suresh Reddy and V.Venkateswarlu).

1.1.10. 5. Ocular administration

SLN’s appear as a promising delivery system for occlular administration of pilocarpine

and tobramycin. It was observed that when tobramycin loaded SLN’s were administered

topically there is significantly higher bioavailability in the aqueous humor when compared with

the standard commercial eye drops. The increased tobramycin availability in aqueous humour

might be due to entrapment and prolonged retention of SLN’s in the mucin layer covering the

corneal epithelium and /or enhancement of corneal penetration of drug (K.Manjunath, J.Suresh

Reddy and V.Venkateswarlu).

1.1.11. Concluding remarks about SLNs

SLNs represent an innovative and alternative approach for the administration of

challenging drug molecules by overcoming the solubility, permeability, physical stability, and

toxicity problems associated with these drugs. In contrast to polymeric nanoparticles, SLNs

ingredients are particularly safe and free from cytotoxicity problems. Furthermore, large-scale

production of SLNs is feasible by high-pressure homogenization. SLN-based systems are not

only limited to lipophilic compounds, rather, hydrophilic and charged agents can be efficiently

encapsulated. Therefore, SLNs were shown to be an effective delivery of a vast variety of drug

molecules including analgesics, anticancer, antianxiety, antibiotics, and antiviral agents.

Table 2: List of some molecules incorporated in to SLN’s

Molecule Therapeutic useDoxorubicin Various cancers

PrednisoloneInflammation and arthritis

Tetracaine Ophthalmic treatment

Etomidate Anesthetic

2. LITERATURE

REVIEW

2. LITERATURE REVIEW

2.1. Review Articles,

Wolfgang Mehnert, Karsten Mader presented overview about the selection of the ingredients

used in formulation, different ways of SLN’s production and applications. Aspects of SLN’s

stability , sterilization by lyophilization and spray drying of SLN’s are discussed.Drug

incorporation complexity of SLN’s dispersion,physical state of lipid,analytical methods for

characterization and stability of SLN’s.Administration of SLN’s by various routes , invivo fate

of SLN’s are presented in this article.

K.Manjunath, J.Suresh Reddy and V.Venkateswarlu discussed various lipid

matrices,surfactants,and other excipients used in formulation along with preparation methods,

sterilization and lyophilization of SLN’s ,entrapment efficiency of drug carrier and its effect on

physical parameters ,drug release,and release mechanisms of various compositions,

characterization and stability of SLN’s.Various invivo studies carried out by different research

groups ,administration of SLN’s by various routes ,passive and active targeting using SLN’s are

also presented in this article.

S.A.Wissing, O.Kayser, R.H.Muller describes the use of nanoparticles based on solid lipids for

parenteral application of drugs and structural differences among different types of nanoparticles

such as solid lipid nanoparticles (SLN,nanostructured lipid carriers (NLC) and lipid drug

conjugate (LDC) along with that they described different production methods including

suitability for large scale production,stability issues,drug incorporation mechanisms in to tha

particles,biological activity of parenterally applied SLN and biopharmaceutical aspects as well as

toxicity aspects of SLN.

Anusha Rupenagunta, I.Somasundaram, V.Ravichandiran, J.Kausalya, B.Senthilnathan

reviewed the recent advances ,various method of preparation,methods of evaluation,various

routes of administration,stability and pharmaceutical applications of SLN were discussed and

solid lipid nanoparticles (SLN’S) have been proposed as suitable colloidal carriers for delivery of

drugs with limited solubility.

LITERATURE REVIEW REVIEW

Katja Jores, Wolfgang Mehnert, Markus Drechsler, HeikeBunjes, Christoph

Johann,Karsten Mader investigated the structures of SLN and NLC based on glyceryl behenate

and medium chain triglycerides were characterized by photon correlation spectroscopy (PCS)

and laser diffraction (LD), field flow fractionation (FFF) with multi-angle light scattering

detection (MALS),and cryo transmission electron microscopy (cryo TEM).

2.2. Research Articles

Kopparam Manjunath & Vobalaboina Venkateswarlu incorporated Nitrendipine in to SLN

prepared by hot homogenization followed by ultra sonication method using various triglycerides

(trimyristin,tripalmitin and tristearin),soy phosphatidylcholine 95%,polaxamer 188 and charge

modifiers (dicetyl phosphate,DCP and stearyl amine,SA) . Dispersion was investigated for

particle size and charge, they studied pharmacokinetics after intravenous (i.v.) and intraduodenal

(i.d.) administration to conscious male wistor rats and tissue distribution studies were carried out

in Swiss albino mice after intravenous (i.v.) administration and compared to Nitrendipine

suspension.

Roberta Cavalli, Otto Caputo, Maria Rosa Gasco describes the development of stealth and

non-stealth solid lipid nanospheres (SLN’s) as colloidal carriers for paclitaxel by using lipid

materials such as tripamitin and phosphatidyl choline. These particulates were evaluated for

particle size, drug release and finally sterilized and freeze-dried.They conducted thermal analysis

(DSC), stability studies.

YiFan Luo, DaWei Chen, LiXiang Ren, XiuLi Zhao, Jing Qin prepared the vinpocetine

loaded Glyceryl monostearate nanoparticles with narrow size distribution by ultra-Solvent

emulsification .To increase the lipid load this process was conducted at 50oC, and these

nanoparticles were evaluated for particle size and size distribution, drug loading capacity, drug

entrapment efficiency (EE%), zeta potential and drug release by using a dialysis bag method.

Michele Trotta, Francesca Debernardi, Otto Caputo prepared the Glyceryl monostearate

nanoparticles with narrow size distribution by Solvent emulsification-diffusion technique using

butyl lactate or benzyl alcohol solvents and lecithin, taurodeoxycholic acid sodium salt, as

surfactants. To increase the lipid load this process was conducted at 47+2oC.

LITERATURE REVIEW

Kopparam Manjunath & Vobalaboina Venkateswarlu incorporated Clozapine in to SLN

prepared by hot homogenization followed by ultra sonication method using various triglycerides

(trimyristin,tripalmitin and tristearin),soy phosphatidylcholine 95%,polaxamer 188 and charge

modifiers (dicetyl phosphate,DCP and stearyl amine,SA) . Dispersion was investigated for

particle size and charge, they studied pharmacokinetics after intravenous (i.v.) and intraduodenal

(i.d.) administration to conscious male wistor rats and tissue distribution studies were carried out

in Swiss albino mice after intravenous (i.v.) administration.

Zaida Uraban-Morlan, Adriana Ganem-Rondero,Luz Maria Melgoza-Contreras,Jose Juan

Escobar-Chavez,Maria Guadalupe Nava-Arzaluz,David Quintanar-Guerrero prepared

cyclosporine loaded solid lipid nanoparticles by the emulsification- diffusion method using

Glyceryl behenate (Compritol ATO 888) and lauroyl macrogol glycerides (Gelucire 44/14) as

matrix materials and evaluated for particle size, zeta potential ,XRD,SEM analysis.

Severine Jaspart, Pascal Bertholet, Geraldine Piel, Jean-Michel Dogne, Luc Delattre,

Brigitte Evrard studied sustained release profile of salbutamol acetomide (SA) loaded soli lipid

microparticles, which are produced by hot emulsion technique followed by high shear

homogenization and investigated for particle size, XRD,SEM analysis.

Annete zur Muhlen, Cora Schwarz, Wolfgang Mehnert incorporated the model drugs

tetracaine, etomidate and prednisolone in to solid lipid nanoparticles by high pressure

homozgenization for parenteral drug administration using Compritol 888 ATO and Dynasan 112

as matrix material and these particulate systems are investigated for particle size, drug load

capacity, effect of drug incorporation on the structure of the lipid matrix and the release profiles

and mechanism.

Chrysantha Freitas, Rainer H.Muller assess the destabilizing factors by using a poloxamer

188 stabilized Compritol SLN formulation and stability was investigated as function of storage

temperature,light exposure and packing material (untreated and siliconized vials of glass quality

LITERATURE REVIEW

3. OBJECTIVE

OF THE STUDY

3. OBJECTIVE OF THE STUDY

The objective of the present study is to develop Solid lipid nanopaticles (SLN’s) for

Valsartan to increase its saturation solubility in low pH conditions and dissolution velocity for

enhancing bioavailability while reducing variability in systemic exposure.

Valsartan is poorly soluble and the aqueous solubility, solubility in low pH is reported to

be less than 1mg/ml and is having pH dependent solubility.The drug is rapidly absorbed

following oral administration, with a bioavailability of about 23%,peak plasma concentrations of

valsartan occur 2 to 4 h after an oral dose.Therefore,it is necessary to enhance the aqueous

solubility,solubility in low pH and dissolution rate of valsartan to obtain faster onset of

action.The approach used in this study is to formulate the poorly soluble model drug, Valsartan

as drug loaded solid lipid nano dispersion which is converted into a solid form by vaccum drying

.The solid state characteristics of the dried product shall be investigated with DSC studies. The

dissolution studies of the solid dosage form of valsartan SLN’s will be carried out in

discriminating dissolution conditions and shall be compared with the commercially available

Valsartan capsule dosage form (DIOVAN).

OBJECTIVE

4. DRUG

PROFILE

4.1. API Information

Valsartan (Merck Index, rxlist.com, drugs.com, medline plus)

Valsartan (marketed as Diovan® by Novartis Company) is a nonpeptide, orally active

and specific angiotensin II antagonist acting on the AT1 receptor subtype.

Category: Angiotensin receptor blocker.

Structure:

Fig: 6 Structure of Valsartan

Chemical name: N-(1-oxopentyl)-N-[[2´-(1H-tetrazol-5-yl) [1, 1´-biphenyl]-4-yl] methyl]-L-

valine.

Empirical formula: C24H29N5O3

Molecular weight: 435.5

DRUG PROFILE

Aqueous solubility: 2.34e-02 g/l

LogP: 5.8

Melting point: 116-117oC

State: Amorphous Solid

Description:

Valsartan is a white to practically white fine powder. It is soluble in ethanol and methanol

and slightly soluble in water.

.Mechanism of Action:

Angiotensin II is formed from angiotensin I in a reaction catalyzed by angiotensin-

converting enzyme (ACE, kininase II). Angiotensin II is the principal pressor agent of the renin-

angiotensin system, with effects that include vasoconstriction, stimulation of synthesis and

release of aldosterone, cardiac stimulation, and renal reabsorption of sodium. Valsartan blocks

the vasoconstrictor and aldosteronesecreting effects of angiotensin II by selectively blocking the

binding of angiotensin II to the AT1 receptor in many tissues, such as vascular smooth muscle

and the adrenal gland. Its action is therefore independent of the pathways for angiotensin II

synthesis. Table No.3: Characteristics of Valsartan

Systematic (IUPAC) name(2S)-3-methyl-2-[N-({4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]phenyl}methyl)pentanamido]butanoic acid

CAS number 137862-53-4Formula C24H29N5 O3

Mol. mass 435.5188 g/molBioavailability 23% with high variabilityProtein binding 94 - 97% bound to serum proteinsVolume of distribution (Vd) 17 L (low tissue distribution)Metabolism Hepatic 4-hydroxylation

Half-lifeThe initial phase t1/2 α is < 1 hour while the terminal phase t1/2 β is 5-9 hours

Excretion feces Legal status ℞ Prescription only

DRUG PROFILE

Routes Oral

Pharmacodynamics:

Valsartan is a specific and selective type-1 angiotensin II receptor (AT1) antagonist

which blocks the blood pressure increasing effects angiotensin II via the renin-angiotensin-

aldosterone system (RAAS). During sympathetic stimulation or when renal blood pressure or

blood flow is reduced; renin is released from granular cells of the juxtaglomerular apparatus in

the kidneys. Renin cleaves circulating angiotensinogen to angiotensin I, which is cleaved by

angiotensin converting enzyme (ACE) to angiotensin II. Angiotensin II increases blood pressure

by increasing total peripheral resistance, increasing sodium and water reabsorption in the kidneys

via aldosterone secretion, and altering cardiovascular structure. Angiotensin II binds to two

receptors: AT1 and type-2 angiotensin II receptor (AT2). AT1 mediates the vasoconstrictive and

aldosterone-secreting effects of angiotensin II. Angiotensin receptor blockers (ARBs) are non-

peptide competitive inhibitors of AT1. ARBs block the ability of angiotensin II to stimulate

pressor and cell proliferative effects. The overall effect of ARBs is a decrease in blood pressure.

Pharmacokinetics:

Absorption — Valsartan peak plasma concentration is reached 2 to 4 hours after dosing.

Valsartan shows bi-exponential decay kinetics following intravenous administration, with an

average elimination half-life of about 6 hours. Absolute bioavailability for Diovan is about 25%

(range 10%-35%). The bioavailability of the suspension is 1.6 times greater than with the tablet.

With the tablet, food decreases the exposure (as measured by AUC) to valsartan by about 40%

and peak plasma concentration (Cmax) by about 50%. AUC and Cmax values of valsartan

increase approximately linearly with increasing dose over the clinical dosing range. Valsartan

does not accumulate appreciably in plasma following repeated administration.

Distribution — the steady state volume of distribution of valsartan after intravenous

administration is small (17 L), indicating that valsartan does not distribute into tissues

extensively. Valsartan is highly bound to serum proteins (95%), mainly serum albumin.

DRUG PROFILE

Metabolism — Valsartan is excreted largely as unchanged drug (80%) and is minimally

metabolized in humans. The primary circulating metabolite, 4-OH-valsartan, is

pharmacologically inactive and produced CYP2C9. 4-OH-valsartan accounts for approximately

9% of the circulating dose of valsartan. Although valsartan is metabolized by CYP2C9, CYP-

mediated drug-drug interactions between valsartan and other drugs are unlikely.

Excretion — 83% of absorbed valsartan is excreted in feces and 13% is excreted in urine,

primarily as unchanged drug

Indications:

Treatment of hypertension

Treatment of heart failure (NYHA class II-IV);

Reduction of cardiovascular mortality in clinically stable patients with left ventricular

failure or left ventricular dysfunction following myocardial infarction

Table No.4: Recommended dose of Valsartan

Recommended Dosing:

Indication Starting Dose Dose Range Target MaintenanceDose*

Adult Hypertension 80 or 160 mg once daily 80-320 mg once daily ---

Pediatric Hypertension

1.3 mg/kg once daily (up to 40 mg total)

1.3-2.7 mg/kg once daily (up to 40-160 mg total)

Heart Failure 40 mg twice daily 40-160 mg twice daily 160 mg twice daily Post-Myocardial Infarction 20 mg twice daily 20-160 mg twice daily 160 mg twice daily

* As tolerated by patient No initial dosage adjustment is required for elderly patients, for patients

with mild or moderate renal impairment, or for patients with mild or moderate liver

insufficiency. Care should be exercised with dosing of Diovan in patients with hepatic or severe

renal impairment. Diovan may be administered with or without food. In heart failure patients,

consideration should be given to reducing the dose of concomitant diuretics. Following

myocardial infarction, consideration should be given to a dosage reduction if symptomatic

hypotension or renal dysfunction occurs.

DRUG PROFILE

Use in specific populations:

Nursing Mothers: Nursing or drug should be discontinued.

Pediatrics : Efficacy and safety data support use in 6-16 year old patients.

Geriatrics : overall difference in efficacy or safety vs. younger patients is not found,

but greater sensitivity of some older individuals cannot be ruled out.

Contraindications:

None Warnings and precautions:

Avoid fetal or neonatal exposure

Observe for signs and symptoms of hypotension

Use with caution in patients with impaired hepatic or renal function

Adverse reactions:

Hypertension: Most common adverse reactions are headache, dizziness, viral infection, fatigue

and abdominal pain.

Heart Failure: Most common adverse reactions are dizziness, hypotension, diarrhea, arthralgia,

back pain, fatigue and hyperkalemia.

Post-Myocardial Infarction: Most common adverse reactions which caused patients to

discontinue therapy are hypotension, cough and increased blood creatinine.

Drug Interactions:

Potassium sparing diuretics, potassium supplements or salt substitutes may lead to

increases in serum potassium, and in heart failure patients increases in serum creatinine.

NSAID use may lead to increased risk of renal impairment and loss of antihypertensive

effect.

DRUG PROFILE

Marketed formulation:

Diovan (Novartis Company) (FDA)

Diovan is available as capsules for oral administration, containing either 80 mg or 160 mg of

valsartan. The inactive ingredients of the capsules are cellulose compounds, crospovidone,

gelatin, iron oxides, magnesium stearate, povidone, sodium lauryl sulfate, and titanium dioxide.

The present drug was approved by the FDA (NDA) on December 23, 1996, as an

antihypertensive drug. The brand name of this drug is DIOVAN manufactured by NOVARTIS

Company. It is new molecular entity (NME) standard review drug with capsule dosage forms of

80mg, 160mg strengths (discontinued). Tablet dosage form of this drug was approved (NDA) on

July 18, 2001 as chemical type new formulation under review classification standard review drug

with 40mg,80mg,160mg,320mg. (ANDA)Generic drugs: marketing status none (tentative

approval) valsartan tablet oral 320mg by IVAX PHARMS approved on June 10, 2008, tablets

multiple strengths by RANBAXY approved on October 25, 2007.

DRUG PROFILE

4.2. Marketed product specifications

DiovanFig: 7 Diovan (160 mg capsule)

Generic name Valsartan capsules

Brand name DIOVAN

Market India

Manufactured and Marketed by Novartis pharmaceuticals corp.

Strength 160 mg

Dosage form Immediate release tablet

Colour Gray / Pink

Shape Capsule shaped

Imprint CG GOG

Weight 265 mg (avg of 10 caps)

Storage conditionsBelow 25º C, protected from moisture and

humidity.

Patents United States,Canada

Table No.5: Specifications of marketed product (DIOVAN)

DRUG PROFILE

5. PLAN OF

5. PLAN OF WORK

5.1. Solubility studies

5.2. Development of analytical method by UV/Visible spectrophotometer/HPLC

5.3. Formulation Development

Selection of Excipients

a. Screening of Lipids

b. Screening of Solvents

c. Screening of Surfactants

Evaluation of SLN’s

Determination of particle size and zeta potential.

Determination of drug entrapment and drug loading.

In-Vitro drug release study

Optimization of formula

Conversion of lipid dispersion (liquid) into a solid form.

Solid state charecreterization of optimized by DSC analysis.

Stability analysis.

5.4. Development of analytical methods

Dissolution methhod

Method development for particle size analysis and zeta potential by laser diffraction

technique (Malvern zeta sizer)

Method development for differential scanning calorimetry(DSC) analysis

PLAN OF WORK

6. Experimental Studies

6.1. List of materials

Table No.6: List of materials and their suppliersS.No. Name of the material Category Supplier

1 ValsartanSelective Estrogen Receptor Modulator (SERM)

Dr.Reddy’s Laboratories Ltd.

2 Compritol Phospholipid Avanti polar lipids, USA

3 Glyceryl monostearate Phospholipid Avanti polar lipids, USA

4 Tristearate Phospholipid Avanti polar lipids, USA

9 Tween-80 Surfactant Merck, Mumbai

10 Polaxamer 188 AdsorbentEvonik Degussa GmBH, Germany

11 Cremophore EL Adsorbent SD Fine chem, Mumbai

12 Dichloro methane Solvent Fischer scientifics, USA

13 Ethanol Solvent Standard Reagents

14 Potassium Chloride Analytical reagent Rankem, New Delhi

15 Hydrochloric acid Analytical reagent SD Fine Chem, Mumbai

16 Potassium hydrogen Pthalate Analytical reagent Merck, Mumbai

17Monobasic Potassium phosphate

Analytical reagent Fischer scientifics, USA

18 Sodium Hydroxide Analytical reagent Merck, Mumbai

19 Triethyl amine Analytical reagent Merck, Mumbai

20 Methanol Analytical reagent Rankem, New Delhi

21 Ortho phosphoric acid Analytical reagent Rankem, New Delhi

EXPERIMENTAL STUDIES

22 Capsules Hard Gelatin capsules Associated capsules Ltd

6.1.1. Classification of materials

Fig: 8 Classificationsof materials

Valsartan is used as drug, which is Angiotensin II blocker.

Lipids:

Lipids such as Compritol 888, Glyceryl monostearate, Glyceryl tristearate, Dynasan 118 are used to entrap the drug (Lipid matrix)

Surfactants

1. Surfactants in SLN’s are used to disperse the molten lipid in to aqueous phase and then to stabilisze the lipid/aqueous interface by covering nanoparticles surfaces after cooling.

2. Tween 80 and Polaxamer 188 are used as surfactants.

Solvents:

Materials

Drug SurfactantsLipids Solvents

Reagents

1. DCM, Ethanol, Acetone, Chloroform are used to dissolve the drug and lipid for the formation of emulsion in Solvent emulsification-Diffusion technique.

2. Methanol is used to prepare the HPLC mobile phase

Reagents:

1. Potassium Chloride,Hydrochloric acid,Potassium hydrogen Pthalate,Monobasic Potassium Phosphate,Sodium Hydroxide are used to prepare 1.2pH,3 pH,4.5 pH,6.8 pH,7.4 pH, 8 pHbuffers.

2. Triethyl amine and Ortho phosphoric acid are used to prepare triethylamine buffer (Triethylamine: Methanol =45:55), which is used as HPLC mobile phase.

6.2. List of instruments

Table No.7: List of Instruments and Apparatus along with their ManufacturersS.No. Name of the Equipment Model Manufacturer

1 Semi micro analytical balance LE 225D Sartorius, India

..2 Top loading balance CP 622 Sartorius, India

3 Rota shaker SW 23 Julabo, North America

4 Heating mantle -Shital scientific industries, Mumbai

5 pH meter Orion 420 A+ Thermo orion,

6 Overhead Stirrer RZR 2051control Heidolph, Germany

7 Magnetic stirrer MR-3001,HeiTec Heidolph, Germany

8 Sonicator - Bandelin sonorex

12 Dissolution apparatus Disso 2000 Labindia

13Alliance HPLC, dual Y absorbance detector

2695 Waters, USA

14 HPLC Column4.6 ×150mm, RP18, 5µm

Xterra

15 Nano-ZS (Zeta sizer) ZEN3600 Malvern

16Differential Scanning Calorimeter

DSC Q-1000 V9.8AA 124

TA instruments, USA

17 X-Ray Diffractometer XPert Pro AA 198 PANalytical BV, Netherland

18Scanning Electron Microscope

S 3000 N Hitachi

19 Vacuum Evaporator HUSGB Hitachi

20 Ultra centrifuge - Heraeus

21 Hot air oven - Cintex

22 Milli Q Water purifier - Millipore (India) Pvt Ltd

23 Storage bottles - Schott Duran, North America

24 Glass ware - Merck, Rankem, Borosil

25 Pipettes - Vensil

26Syringe filters (0.45 µ- 47,25,13 mm)

NX047100, ZWGSFN 13045

Pall Life sciences,India

Zodiac Life sciences, India

27 Syringes - Dispo van

6.3. Methods

6.3.1. Solubility studies

The solubility studies for the drug were carried out using the Rotary shaking method.

These studies included the rotary shaking at 200 rpm and addition of excess drug till saturation

was observed for 48 Hrs. Then the samples were filtered and required dilutions were made to the

sample and was analyzed using HPLC.

6.3.1.1. Solubility in purified water:

Solubility of the drug in purified water was investigated using Rotary Shaking method at

two temperatures i.e. Room temperature (25º C). Excess drug was added to 20 ml of water in

stoppered conical flasks and were agitated continuously in a Rota shaker for 48 Hrs at 200 rpm

and respective temperatures, till saturation was observed. Then, the samples were filtered using

0.45 µ Nylon (47 mm) syringe filters. From this, samples equivalent to standard concentrations

were prepared by diluting with mobile phase and were analyzed using HPLC at 225 nm.

6.3.1.2. Solubility across pH:

Solubility of the drug across different buffers was studied. The pH ranged from 1.2 to 8.0

(1.2, 3.0, 4.5, 6.8, 7.4 and8.0). All the buffers were prepared according to USP NF. Excess drug

was added to 20 ml of water in stoppered conical flasks and were agitated continuously in a Rota

shaker for 48 Hrs at 200 rpm and Room temperature (25° C), till saturation was observed. Then,

EXPERIMENTAL

the samples were filtered using 0.45 µ Nylon (47 mm) syringe filters. From this, samples

equivalent to standard concentrations were prepared by diluting with mobile phase and were

analyzed using HPLC at 225 nm.

6.3.2. Analytical Method

6.3.2.1. Determination of max

Stock solution: Accurately weighed 100 mg of API was dissolved in little amount of methanol

and then volume was made up to 100 ml using 0.1N HCl buffer (1mg/ml)

Scanning : From the stock solution, 15-25 µg /ml solutions were prepared by pipetting 5ml to a

series of 200 ml volumetric flasks and the volume was made up to 200 ml with phosphate buffer

pH 6.8. These solutions were scanned in UV range between 200-400 nm.

6.3.2.2. HPLC Method:

The analytical methodology for estimation of drug content in the solubility samples was

adapted from in-house analytical method for estimation of valsartan. The details of the method

were given below.

Preparation of mobile phase: The mobile phase consisted of 55% Methanol and 45% of

triethylamine buffer with pH adjusted to 3.0 with o-phosphoric acid. The mobile phase was

prepared daily and degassed by sonication under reduced pressure and filtered before use.

Diluent: Methanol

Preparation of standard solution: 50 mg of pure Valsartan was added to 100 ml volumetric

flask and add about 70 ml of diluent and sonicate for 5 minutes or completely dissolved and

dilute to volume using methanol and mix well. Then 5 ml of the above solution is in to a 50 ml

volumetric flask and dilute to volume with diluents and mix well. After filtration with 0.45 µ

PVDF (25 mm) syringe filter.

Preparation of test solution: Transfer an accurately weighed portion of sample equivalent to

about 100 mg of valsartan in to 200 ml volumetric flask. Add about 100 ml of diluent and

mechanically shake for 30 minutes. Further add about 50 ml of diluents and sonicate for 30

minutes or completely dissolved and dilute to volume using methanol and mix well. After

filtration with 0.45 µ PVDF (25 mm) syringe filter.

The solubility of the drug was calculated using the formulas:

Where,

Q = Percent of drug dissolved (% w/v)

A =Standard Area

B = Test Area

C = Standard concentration (µg/mL)

Wt. = Total weight of drug added

V1 = volume of test sample taken for dilution

V2 = diluted volume of test sample

V3 = volume of diluted sample (V2) taken for dilution

V4 = diluted volume of sample (V3)

m = Amount/quantity (mg) of drug dissolved

S = Solubility (mg/mL)

6.3.3. Method development for particle size analysis by laser diffraction technique:

Laser diffraction is now one of the widely used techniques for particle size analysis. it

offers flexibility,wide dynamic range of speed of operation and yield significant advantages

compared to other methods of particle size analysis such as sieving,microscopy and

electrozone Sensing (coulter counter) Particle size analysis using wet dispersion is widely used

for obtaining Reproducible results using laser diffraction .wet analysis provides a method of

dispersion for sample across a wide particle Size range from submicron pigments to sand and

sediments. Malvern Mastersizer is used to measure the particle size distribution of drug

nanosuspension, is based on the laser diffraction Technique.

6.3.3.1. Principle and working of malvern mastersizer

The interaction of a particle and light incident upon it gives rise to four different but

inherently related scattering phenomena, namely, diffraction, refraction, reflection and

absorption of the incident beam. The magnitude of each phenomenon will vary depending upon

the nature and size of the particle and the beam. Size analysis by interpretation of the scattered

light Patterns formed due to diffraction of the incident light is of primary interest .Diffraction of

light occurs at the surface of the particle and can be thought of as the bending of light waves by

the surface of The particle .diffraction arises due to slight differences in the path length of the

light waves created upon interaction with the particle surface. These differences in the path

EXPERIMENTAL

length cause constructive and destructive interference between the sinusoidal Light waves

leading to characteristic diffraction patterns. The diffracted waves are scattered in different

diections. The Direction of scatter depends on the size and shape of the particle. Large, spherical

particles scatter mostly in the forward Direction. As the particle size gets smaller, the scattering

occurs over a broader range of angles.In practice; scattering is significantly more complex and is

influenced by the nature of polarization of the incident light, Optical properties of the particle

and surface roughness of the particle.

6.3.3.2. Light diffraction instruments are based on three basic assumptions;

a) The particles scattering the light are spherical in nature,

b) There is little to no interaction between the light scattered from different particles ( i.e.,

no multiple scattering phenomena),and

c) The scattering pattern at the detectors is the sum of the individual scattering patterns

generated by each particle interacting with the incident beam in the sample volume.

Diviations from these assumptions will introduce some degree of error due to the

inability of the mathematical algorithms for the deconvolution and inversion procedures to

account for the deviations. The assumpation of spherical particle shape is particularly

important as most algorithms in commercial instruments use the mathematical solution for

Mie, Fraunhofer and Rayleigh scattering from spherical particles.

6.3.3.2.1. Fraunhofer Approximation

The Fraunhofer approximation (also referred to as the Fraunhofer theory )is applicable

when the diameter of the particle scattering the incident ligh Is larger than the wave length of the

incident light [ d >e.. ].Particles showing Fraunhofer scattering have a very strong forward

scattering. Intensity of Scattered light is very high. By its very nature, this model does not need

any information about the refractive index of the particle and so is extremely useful for analysis

of powders coarser than about 1 im to 2 im.

6.3.3.2.2. Mie Scattering

The Mie theory is applicable when the particle size is equal to, or smaller than, the wave

length of the incident light [d e..]. Smaller particles with Mie scattering Show decreased forward

scattering and intensity. The intensity of the light scattered decreases linearly with decrease in

particle size.

6.3.3.2.3. Rayleigh Scattering

When diameter of the particle is very small compared to the wave length of the incident

light [d<< e..], the solutions for scattering are best represented by Rayleigh scattering models.

Particles showing Rayleigh scattering have a very symmetric scattering pattern, when forward

and backward scattered light intensity is compared.these patterns have no angular information

any more; therefore particles with Rayleigh characteristics cannot be analyzed laser

Diffractometry.

6.3.3.3. Instrumentation

Light diffraction instruments comprise of a light source, tyoically a low power

(approximately 10 mw Helium – Neon, in the region of 632 nm wavelength) Laser source

optical elements to process the incident beam, a sample cell within which the sample is

introduced .sample cells have built-in ultrasonicators or agitators to keep the specimen

sample dispersed and to prevent agglomeration.sample cells also possess pumping systems to

keep the specimen circulating. Light diffraction instruments lack the ability to distinguish

between well-dispersed powders and agglomerates and thus prevention of agglomeration is a

key factor in ensuring reliability and reproducibility. Light scattered from the sample is then

focused on to a detection system, that can be a multi-element array or numerous detectors

placed at discrete locations .The detectors convert the scattered light intensity incident upon

them into electrical signals that are then processed to obtain information about the particle

size and size distribution.

Fig: 9 schematic of components in a typical laser diffraction instrument.

A typical light pattern is shown below (Fig.10).Each bar in the histogram represents the light

scattering from a particular detector.

Fig: 10 Histogram showing typical light scattering pattern

6.3.3.4. Method development for particle size determination

The wet method development steps include;

1. Representative sampling

2. Dispersant selection

3. Measurement settings

4. Pump and stirrer setting

5. Sample concentration

6. Sonication Energy

7. Solid particle Measurement

The sampling is the most important aspect of particle size analysis. Laser diffraction

is a volume –based measurement Technique and is therefore sensitive to small changes in

the amount of large particles in the sample. If sampling is controlled it should be easy to

obtain a measurement – to – measurement reproducibility within the limits defined in

ISO13320-1[2], the ISO Standard for laser diffraction measurements ( within 3% at the

d( v,0.5) and within 5% at the d( v,0.1) and d(v,0.9)). If sampling is not controlled then

measurement to measurement variations of up to 20% can be observed. The process of

wet method development for measurement of particle size distribution is as follows;

1. The particle size is measured following intitial wetting of the particles.

2. The particle size is measured during the application of ultrasound for different time

periods and at different stirrer speeds.

3. The ultrasound probe should be switched off and the particle size has to be monitored

to ensure that the dispersion is stable.

Following initial wetting of the sample in the dispersion unit the particle the particle size

may slowly decrease – this is due to the dispersion of loosely bound agglomerates under the

action of the pump and stirrer. If the obscuration reduces rapidly at this point it may suggest that

the material under test is soluble in the chosen dispersant. If this is the case, the obscuration drop

will often be associated with increase in the measured particle size, as the fines present in the

sample will be dissolved most rapidly. If this is observed a different dispersant should be used.

Following the initial stage of dispersion, ultrasound shoul be applied and the particle size

measurement followed in real – time. Rapid dispersion is normally observed at this stage as

strongly bound agglomerates are dispersed. As the time of sonication is increased the particle

size should reach a plateau where the particle size becomes stable – this represents the fully

dispersed state. If the particle size continues to reduce over time this may suggest that particle

breakup occurs during sonication. Sonication may also cause agglomeration to occur – this

would suggest that the dispersion is unstable, requiring that analyst to adjust the dispersion

conditions. Finally, the particle size should be monitored with the ultra probe switched off, once

full dispersion is achieved. If the particle size remains stable then it indicates that the dispersion

conditions are optimized. The particle size distribution of nanosuspension is represented as 10%,

50%, 90% [d (10), d (50) and d (90)]. Diameter 10%means, 10% of the partcles were below the

indicated size, diameter 50% means, 50%of the particle were below the indicated size and

diameter 90% means that 90% of the particles are below the indicated size. Water was selected

as dispersant as drug is in soluble. Different concentration of the drug (5% w/w and 10%w/w) in

polymer-stabilizer dispersions were prepared for the method development.

6.3.4. Formulation Development

When dealing with a BCS Class II or IV compound, the main formulation objective is to

increase the apparent water solubility of the API in gastro-intestinal fluid (GIF) and to maintain

it in a solubilized state until it reaches the site of absorption, which consequently means that the

API will be fully solubilized in the final dosage form.

6.3.4.1. Screening of excipients

The development of solid lipid nanoparticles begins with screening of excipients in order

to identify those that provide the best solubilization and chemical compatibility with Valsartan.

There are several types of excipients routinely used in screening. These can be classified by their

functional role in a formulation system: Water dispersible surfactant, lipid carrier and a solvent.

Fig: 11 Schematic representation of formulation development

6.3.4.1.1. Screening of lipids

Choice of the lipid cores for SLN’s preparation is dependent on many factors like their

degree of crystallinity, fatty acid chain length, and drug loading capacity in the lipids. The

loading capacity depends upon solubility of drug in lipid melt.

In the present SLN’s development, API solubility screening of lipids was carried out by visual

examination as follows

1. Lipid is allowed to melt by slowly increasing the temperature.The amount of lipid can

be used in the ratio of 1: 5 (minimum) - 1:10 (maximum) (Annette zur Muhlen,Cora

Schwarz,Wolfgang).

2. Add API to the above lipid melt which is maintained at its melting temperature. The

concentration of API used in the initial binary mixture should correspond to the target

3. The evaluation of API solubility is performed by visual observation. When the active

drug is partially solubilized, particles will be visible, when the API is completely

solubilized particles are no longer observed the composition remains visually clear.

4. The method is repeated to determine the maximum/minimum concentration of API that

is completely solubilized in the lipid excipient.

Screening of excipients

Screening of lipidsScreening of solvents Selection of surfactants

5. Depending on visual appearance, drug solubility in respective lipid is evaluated. If the

composition is clear in appearance then it is considered as ‘soluble’ and if the

composition is hazy in appearance then it is inferred as insoluble.

6.3.4.1.2. Screening of solvents:

In the preparation of SLN’s the selection of solvent is of paramount important. The

solvent used for the preparation should be partially miscible and solubilize both drug and lipid.

The miscibility of solvent is performed by visual examination of water – solvent mixture.

Add the specified quantity of solvent selected as per IIG limits in to 100 mL of purified water.

Depending on the visual examination, solvents were graded as “miscible” where the solvent

forms clear solution, “partially miscible” where the solvent is dispersed as stable fine globules

and “immiscible” where phase separation of solvent occurs.

The quantity of partially miscible solvent is optimized by visual observation of different

percentages of (3%v/v, 5%v/v, 7.5%v/v) solvent-water mixtures as described above. Finally the

selected solvent was analyzed for its ability to solubilize both drug targeted dose and minimum

amount of lipid.

6.3.1.3. Screening of Surfactants:

The selection of water miscible surfactants was done as follows

1. Add the specified amount of surfactant selected as per IIG limits in to 100% of purified

water.

2. The water miscibility of surfactant is performed by visual observation. Depending on the

visual appearance, surfactants were graded as “miscible” when the surfactant solution

appears visually clear and “water immiscible” when the surfactant solution appears

The concentrations of surfactant and lipid were selected based on the resultant particle

size and drug entrapment efficiency when dispersed in water. Different formulations were

developed with different concentrations of surfactants. Finally formulations with optimal

surfactant concentration were studied further by increasing the concentration of lipid (1:10).

6.3.4.2. Preparation of SLN’S:

DCM and water were mutually saturated at 40+2oC for 10min in order to ensure initial

thermodynamic equlibrium of both liquids. After the equilibrium was reached add GMS to 3ml

of above water saturated solvent and stirr the mixture for 10 min until it forms clear solution.

Then add specified amount of drug to the above mixture slowly by continuous stirring. Finally

this organic solution was emulsified at 40+2oC with 50ml mixture of aqueous solution containing

different surfactants, using magnetic stirrer at 900rpm for 10 min. The SLN’s were precipitated

by adding cold water (50ml) maintained at 0 oC and stirred continuously until all the organic

solvent (DCM) was evaporated.

Figure: 12 Schematic Procedure for the preparation of solid lipid nanoparticles.

DCM and water are saturated for 10min

Add GMS and stirr for 10 min

Above organic solution is emulsified with 50ml mixture of aqueous surfactant solution, stir at 900rpm for 10 min

Add drug and stirr for 5 min

Heated at 40+ 2oC

6.3.4.3. Evaluation of SLN’s:

Particle size analysis and Z-potential analysis:

Particle size and zeta potential of SLNs in the dispersion was determined by photon

correlation spectroscopy (PCS) using Malvern zeta sizer at a fixed angle of 90° at 25 °C using

water as dispersant. Before measurement, SLN dispersions were diluted 50- fold with the

original dispersion preparation medium for size determination and zeta potential measurement.

All the measurements were performed in triplicate.

Determination of drug load and entrapment efficiency:

Ten milliliter SLN dispersion was centrifuged for 15 min at 8000 rpm−1. After centrifugation

supernant is separated.The drug content in the supernatant was measured by HPLC. The HPLC

Add cold water (50ml) at 0 oC to the initial

emulsion and stir continuously until organic

solvent is evaporated

after continual stirring for 60min.

emulsio

The dispersion is centrifuged and the sediment was vaccum dried

system (WATERS, USA), with a UV–VIS detector and Xterra column (4.6 ×150mm, RP18,

5µm) were utilized for drug separation, using methanol: Triethyl amine buffer (55:45) as mobile

phase. The flow rate and UV wavelength were 1.0 mL min−1 and 225 nm, respectively. The

equations for the drug content and loading efficiency are as follows

Entrapment efficiency (%) = WS/Wtotal ×100%

Load content (%) = WS/Wlipid ×100%

Ws = amount of VAL in the SLNs;

Wtotal =amount of VAL used informulation:

Wlipid =weight of the vehicle.

Solid state characterization:

These studies are important in estimating the crystallinity, entrapment nature, surface

morphology, particle size etc. of the developed formulation. Following studies were carried out

to characterize the SLNs:

Differential Scanning Calorimetry (DSC)

Preparation of samples:

API: The pure API was taken alone.

Placebo: The placebo was prepared with the excipients, similar to that of the formulation

excluding the pure drug.

Formulation: The formulation containing Valsartan SLNs.

Differential scanning calorimetry (DSC):

This gives us the information about the thermal properties of the sample like crystalline

or amorphous nature, entrapment of drug with lipids and transition temperature of lipids. The

thermal properties were investigated using DSC Q – 1000, Differential scanning calorimeter with

a refrigerated cooling system.

Process:

Weighed samples of 3 – 5 mg were taken in an open aluminum DSC pans and then sealed

and crimped. These samples were scanned at a ramp of 10º C / min over a range of 10 – 300 º C.

The samples included API, Placebo and SLN Formulation.

In Vitro drug release:

Selection of Discriminating Dissolution medium:

The selection of a discriminating medium was done based on the solubility of the drug in

purified water and across pH buffers. Considering the drug’s solubility, the medium with lowest

solubility of drug was selected for dissolution testing. Based on the drug’s solubility in the

discriminating medium, the conditions viz., rpm, temperature, volume of medium, sink / non-

sink conditions, time points for sample collection, etc were selected.

Dissolution:

The dissolution was carried out in discriminating conditions as mentioned above. In this the

capsule formulation representing unit dose (40 mg) of the drug was taken and dropped in the

dissolution bowl, which contained 900 mL of 1.2pH buffer and maintained at 37 ± 0.5º C. At pre

determined time intervals of 15, 30, 45 min and 60 minutes. Aliquots of sample (10 mL) were

withdrawn and were filtered through 0.45µ PVDF (13 mm) syringe filter. Then 1 mL of this

filtered sample was diluted to 10 mL using diluent (1.2pH buffer) and were analyzed using

HPLC. The cumulative percent drug dissolved at various intervals was calculated and plotted

against time.

Preparation of standard solution: 50 mg of pure Valsartan API was added to 1000 ml volumetric

flask and add 900 ml of methanol and final volume was made with water. Now it is kept in an

ultrasonic bath for 10 min and left sealed to stand overnight. After filtration with 0.45 µ PVDF

(25 mm) syringe filter.

6.3.4.4. Stability analysis: (Chrysantha Freitas, Rainer H.Muller)

Solid lipid nanoparticles are basically stable for up to 3 years; however some systems

show particle growth & drug leakage (reference). The GMS formulation stabilized with Tween-

80 was investigated for its stability as a function of storage temperature, light exposure. In

general introduction of energy to the system (temperature, light) led to particle growth and drug

leakage. This process was accompanied by a decrease in zeta potential from approximately -

25mv to -15mv.the SLN was filled in to glass vials and stored at varying conditions. Storage was

performed at different temperatures (8 ºC, 25 ºC and 50 ºC) and light exposures (dark, artificial

illumination). For each storage condition, the SLN formulation was placed in 5ml white and

brown glass vials (glass quality I). After 2 weeks cumulative % drug release of the formulation

sample was established.

7. RESULTS

AND DISCUSSIONS

7.1. Solubility studies

7.1.1. Solubility in purified water:

RESULTS AND DISCUSSIONS

The solubility of Valsartan was investigated in purified water at 25º C using the Rotary

shaking method and the results were summarized in Table No.8.

Table No.8: Solubility studies of Valsartan in water

From the above data it was stated that Valsartan is a poorly water soluble drug.

7.1.1. 2.Solubility across pH:

The solubility of Valsartan was also investigated in various buffers differing in their pH

using the Rotary shaking method at 25º C. The solubility results of drug in various buffers were

summarized in Table No.9.

Table No.9: Solubility studies of Valsartan across pH

MediumImmediate Saturation

Solubility at 25°C (mg/mL)

Saturation

Solubility(mg/ml)(48hrs)

pH-1.2 (0.1 N HCl) 0.014 0.01823

pH-3.0 (0.001 N HCl) 0.324 0.4283

pH-4.5 (Acetate buffer) 0.7234 0.7854

pH-6.8 (Phosphate buffer) 7.5 8.5

MediumImmediate Saturation

Solubility at 25°C (mg/mL)

Saturation

Solubility(mg/ml) (48hrs)

Water 0.013 0.01793

The solubility results indicate that the solubility of Valsartan is pH dependent. At low pH, the

solubility was found less and on increasing pH the solubility was significantly increased. The pH

solubility data clearly shows that drug is highly soluble in pH-8.0 buffer and less soluble in 0.1 N

HCl (pH-1.2).

Solubility enhancement is the single most important driving force in prototype

formulation selection and optimization. As a general rule, if solubility is < 1% (w/v) or 10 mg/ml

in the aqueous or buffers, it is necessary to further evaluate solubility in pharmaceutical

excipients and vehicles.

7.2. Analytical method

7.2.1. Determination of max of API in 0.1N HCl:

The spectrum obtained in scanning of the drug was as shown in Figure 13.

Figure 13: UV spectra of API

From the above spectrum (Figure 12), the absorption maximum (max) of API was

found to be 248.0 nm and this wavelength was used for HPLC method.

7.2.2. HPLC Method:

The HPLC chromatographic conditions used for anlaysing the samples are summarized in Table

Two washing solvents were prepared using purified water and Acetonitrile (90:10 and

10:90 respectively).

7.3. Method for particle size analysis by laser diffraction technique:

The specification used for particle size analysis of the drug nanodispersion was

summarized in Table 11. Water was selected as dispersant since the drug is insoluble in aqueous

media.

Table No.10: Chromatographic conditions for anlalytical method by HPLC

HPLC SYSTEM WATERS 2695 Separations module

DETECTOR WATERS 2996 Photo Diode Array Detector

COLUMN 4.6 ×150mm, Xterra RP18, 5µm or equivalent.

COLUMN TEMPERATURE Ambient

FLOW RATE 1.0 ml/min

RUN TIME 15 min

LOAD VOLUME 10 µL

UV WAVELENGTH 225 nm

TYPE OF FLOW Isocratic

Table: 11. Parameters used for particles size analysis using Malvern Mastersizer

7.4. Dissolution method:

The conditions for discriminating dissolution were summarized in Table No.12.

The formulation samples were evaluated in the discriminating dissolution media using

the following parameters

Parameter Value

Refractive Index of water 1.33

Refractive Index of drug 1.50

Obscuration range 5-15

Pump speed 2000 rpm

Sonication Not applicable

Table No.12: Dissolution conditions for the Discriminating medium

Medium Purified (De mineralized) Water

Volume 900 mL

Replacement Volume 10 mL

Temperature 37º ±0.5ºC

Method USP I (basket)

Rpm 100

Time Points 10, 30, 45min, 60 minutes

7.5. Formulation Development

7.5.1. Screeninig of Excipients:

Screeninig of Lipids:

The solubility of drug in various lipids is summerized in Table 13. The solubility was

evaluated based on the Visual clarity of the solution after addition of drug to the lipid at its

melting temperature.

Table No.13: Solubility of drug in various Lipids

S. NoAmount of

Drug Lipids (Qty) Meltingpoint Visual Observation1 40 mg Compritol 888 (400 mg) 59.3-70.5°C Hazy

240 mg Glyceryl Monostearate

(400 mg) 68°C Clear3 40 mg Dynasan 118 (400 mg) 71-73 ºC Hazy4 40 mg Tristearin (400 mg) 68°C Hazy

The results described in Table 13, indicates that Compritol 888, Dynasan 118 and tristearin were

‘hazy’ in appearance when the drug was added to the lipids individually and Glyceryl

monostearate was ‘clear’ in appearance indicating the drug solubility in GMS. Based on these

observations Glyceryl monostearate was selected as suitable lipid carrier for developing SLN

formulation.

The concentration of Glyceryl monostearate required for formulation development was

selected by studying the solubility of targeted dose (40 mg) of drug in various amounts of lipid

and the results were summarized in Table 14.

Table No.14: Solubility of drug targeted dose in Glyceryl monostearate

Amount of lipid (mg)

SNO Lipids Meltingpoint

Visual obseravation

40 80 160 200

1 Glyceryl Monostearate 68°C Hazy Hazy

Slightly Hazy Clear

The results from Table 14 indicate that 200 mg of GMS is required for solubilizing the drug

therefore this amount was choosen for formulation development.

Screeninig of Solvents:

The results of water miscibility of various solvents were summarized in the following Table

Table No.15: Water miscibility of various solvents

SNO Solvents Qty Miscibility with 100 mL

water1 Methanol Miscible 2 Alcohol Miscible 3 Acetone Miscible 4 DCM Partially miscible5 Chloroform Immiscible

From the Table No.15 it was clear that DCM is the partially water miscible solvent and

therefore it was selected as solvent for the formulation development.

After the selection of suitable solvent, the solubility of targeted dose (40 mg) of drug in

presence of lipid (200 mg) by appling gentle heat (40° C) was studied by visual clarity and the

results were summarized in the following Table 16.

Table No.16: Solubility of drug in the drug and lipid

SNO Ingredients Solvent Visual Observation

1 Drug (40 mg) + GMS (200 mg) DCM (3 mL) Clear

From the solubility data given in the Table No.16 it was clear that dichloromethane

(DCM) can solubilise both drug and the lipid.

Screeninig of surfactants:

In solvent emulsification-diffusion technique preparation of aqueous surfactant solution is the

secondary step for preparing the SLNs. For this purpose Tween-80 and Poloxamer 188 were

selected for prototype formulation development. Various concentrations of Tween 80 and

Polaxamer 188 were evaluated by characterizing SLN’s Particle size analysis and drug

entrapment. The composition evaluated for prototype formulation development are summarized

in Table 17 Table 17 selection of surfactants with optimal concentrations

Weights (mg)SNO Ingredients VSX 1 VSX 2 VSX 3 VSX 4 VSX 5 VSX 6 VSX 7

1 Drug 40 40 40 40 40 40 40

2Glyceryl monostearate 200 200 200 200 200 200 200

Surfactants

3Polaxamer-188 30 50 70 - - - -

4 Tween-80® - - - 100 200 300 400 Solvents(ml)5 DCM 3 3 3 3 3 3 36 Purified water 50 50 50 50 50 50 507 Total weight 270 290 307 340 440 540 640

Formula explanation (in brief):

VSX-1: In this formulation 0.75% of Polaxamer 188 was used as surfactant

VSX-2: In this formulation 1.25% of Polaxamer 188 was used as surfactant

VSX-3: In this formulation 1.675% mg of Polaxamer 188 was used as surfactant

VSX-4: In this formulation 2.5% mg of Tween 80 was used as surfactant

VSX-5: In this formulation 5% mg of Tween 80 was used as surfactant.

VSX-6: In this formulation 7.5% of Tween 80 was used as surfactant

VSX-7: In this formulation 10% of Tween 80 was used as surfactant

In the above formulations drug: lipid ratio is 1:5

7.5.2. Evaluation of SLNs

The results of partcle size and zeta potential of various formulations (Table 20) were summarized

in the Table 18.

Particle size analysis and measurement of potential

Table: 18 Z-Average size (d.nm), zeta potential and PDI of different SLN formulations (0 day)

Particle size & Z-Potential analysis

Formulae Z-Average size (d.nm) PDI Z-Potential (mv)

VSX 1 8492 + 30 1 -18.9 + 2

VSX 2 6738 + 25 1 -0.95 + 1

VSX 3 3522 + 10 0.824 -0.68 + 1

VSX 4 6300 + 20 0.923 -17.8 + 0.23

VSX 5 2939 + 5 0.7 -15.6 + 3

VSX 6 772 + 20 0.432 -14.8 + 3

VSX 7 723 + 5 0.841 -11.9 + 5

Fig: 14 Graph representation of Z- average size (d.nm) of different SLN formulations

The mean particle size of VAL-SLN prepared with different formulations, ranged from

723 + 5to 8492 + 30 nm (Table 21). Higher surfactant concentrations reduce the lipid/water

interfacial tension, resulting in a decrease in particle size subsequent increase in surface area.

The mean diameters, PDI of VSX 3 and VSX 6 were in the range of approximately 3522 + 10

nm, 0.8-0.9 and 772 + 20 nm, 0.4-0.5 respectively (Table 21.). The VSX 3 and VSX 6 SLNs had

a zeta potential 0.68 + 1,-14.8 + 3 mV respectively (Table 21). As concentration of Polaxamer

188 and Tween 80 increased, the zeta potential decreased significantly.

Fig: 15 Peak showing Z-average size (d.nm) of formulae VSX 6 in zeta sizer (0 day)

Determination of drug load and entrapment efficiency:

The results of entrapment efficiency of various formulations were summarized in the Table

Table: 19 Entrapment efficiency of different SLN formulations

Determination of drug entrapmentFormulae Entrapment efficiency (%)

VSX 1 28.17 + 5VSX 2 25.78 + 4VSX 3 23.44 + 3VSX 4 28.34 + 5VSX 5 32.78 + 7VSX 6 78 + 4VSX 7 62.5 + 2

Fig: 16 Graph representation of Entrapment efficiency of different SLN formulations

Fig: 17 Chromatogram representing standard preparation in determination of entrapment efficiency

Fig: 18 Chromatogram representing VLX 6 (blank) in determination of entrapment efficiency

Fig 19 Chromatogram representing VLX 6 in determination of entrapment efficiency

From Table 22 it was clear that VAL has high entrapment efficiency in formula VSX 6

(78 + 4 %) SLN’s, and the entrapment efficiency increases as the amounts of the surfactants

were increased. The entrapment efficiency of formulations containing Polaxamer 188 was lower

than that of formulations containing Tween 80 and (Table 22). These results may have

contributed to the lower stabilization of polaxamer 188 compared with Tween 80.

From the above discussions of Table 21 and Table 22, it was clear that VSX 6

formulation was selected as best formulation with optimal particle size (772 + 20 nm) and drug

entrapment (78 + 4 %) among all other Tween 80 and polaxamer 188 formulations, so VSX 6

was studied against increase in lipid concentration to 400 mg (1:10) as shown in the Table 20.

Table No.20: Optimization of the lipid concentration.

VSX-8: In this formulation drug: lipid is 1:10 and 7.5% of Tween 80 was used.

The results of partcle size and zeta potential analysis of VSX 8 were summarized in the

Table 21

Table: 21 Effect of lipid concentration on the Partcle size (0 day)

Particle size & Z-Potential analysis

Formulae Z-Average size (d.nm) PDI Z-Potential (mv)

VSX 6 772 + 20 0.432 -14.8 + 3

Weights (mg)SNO Ingredients VSX 6 VSX 8

1 Drug 40 40

2 Glyceryl monostearate 200 400 surfactants

3 Polaxamer-188 - 4 Tween-80® 300 300 Solvents(ml)5 DCM 3 36 Purified water 50 507 Total weight 540 740

VSX 8 3832 +10 1 -18.5 + 2

Fig: 20 Graph representation of the effect of lipid concentration on the Partcle size

The results of drug entrapment and load content of formulation VSX 8 were summarized in the

following Table 22.

Table: 22 Effect of lipid concentration on the drug entrapment (0 day)

Determination of drug entrapment

Formulae Entrapment efficiency (%)

VSX 6 78 + 4

VSX 8 80 + 5

Fig: 21 Graph representation of the effect of lipid concentration on the drug entrapment (0 day)

Table: 22 reports that as the concentration of lipid increases particle Z-Average size, PDI

and zeta potential also increases. Formula VSX 6 where drug: lipid is 1:5 was showing Z-

Average, Z-Potential and PDI in the range of 772 + 20 nm,-14.8 + 3 mv, and 0.432 to 0.5

respexctively. Formula VSX 8 where drug: lipid is 1:10 was showing Z-Average, Z-Potential

and PDI in the range of 3832 +10nm, -18.5 + 2 mv, and 0.932 to 1. From Table 25, even though

VSX 8 has having high drug entrapment (80 + 5 %) than VSX 6 (78 + 45%) , it has shown larger

particle size and PDI when compared to formulation VSX 6. Therefore VSX 6 was selected as

the final formulation for in-vitro characterization.

Drug release of VAL from SLNs

Drug release from VSX 6 was observed in pH 1.2 buffer by comparing with API

Solution and marketed formulation (DIOVAN) at time intervals of 10, 20,30,45,60

min.dissolution was carried out using Purified (De mineralized) Water 800 mL,0 mL (Non-sink

conditions),USP I (basket) and at temperature37º ±0.5ºC.

Table: 23 Cumulative % release of VAL from VSX 6, API and Marketed formulation (DIOVAN) in

dissolution media: 0.1 M HCl (0 day)

Fig: 22: Chromatogram corresponding to the standard preparation

Fig: 23 Chromatogram corresponding to the VSX 6 Placebo

Time(min) VSX 6(%) API (%)Marketed formulation

0 0 0 0

10 53.57 0.3 5

20 64.43 0.9 10

30 75.8 1.9 13

45 76.13 4.3 17

60 80.16 6.8 20

Fig: 24 Chromatogram corresponding to the VSX 6 formulation

From the above chromatograms, it was clear that, there was no interference of the blank

and placebo in the analysis of formulation. The peak represented by the test preparation’s

chromatogram completely elucidated the pure drug’s peak. Also the Retention time of the peak

and standard were almost equivalent along with a good shape and peak purity.

Fig: 25 Comparative Cumulative % release profiles of VSX 6 formulations API and Marketed formulation

(DIOVAN) in dissolution media: 0.1 M HC (0 day)

Table: 26 Reports that the %Cumulative drug release of VSX 6 was higher than API

solution and marketed formulation (DIOVAN) in pH 1.2 media.

7.5.3. Optimization of formula

From the above results, we concluded that the surfactants (polaxamer 188 and Tween 80)

made an important contribution to the differences between the release from the two SLN

formulations, diffusion from API and marketed formulation (DIOVAN). Surfactants altered the

barrier properties of the aqueous boundary layer around drug particle, resulting in a high release

velocity of VAL in SLN dispersion. The API would not have this effect. In addition, the

concentration of VAL in SLN dispersion was close to saturation (maximal thermodynamic

activity), while in API, although the overall concentration of VAL was identical with that in SLN

dispersion, with the appearance of microcrystals the real concentration of drug dissolved in

solution would be greatly lowered, since thermodynamic activity is the driving force for

transport, so the diffusion of VAL was slow compared with that in the SLN dispersion. The

above dissolution results indicated convincing for the formulation VSX 6 on comparison with

API and DIOVAN. Hence, it was concluded that, the solid lipid nanoparticles (VSX 6) showed

greater dissolution profile, when compared to that of, API and DIOVAN and better particle

size,PDI,Zetapotential,drug Entrapment when compared to that of formulations (VSX 3,VSX 8).

Hence, VSX 6 was concluded as the optimized formula for Valsartan SLN’s and further solid

state characterization studies were coducted on the formula VSX 6.The optimized concentration

of ingredients for the lipid based formulation is mentioned below in Table No.24.

Table No: 24 Optimized Formula for Valsartan SLN’s Formulation

Weights (mg)SNO Ingredients VSX 6

1 Drug 40

2 Glyceryl monostearate 200 surfactants

3 Polaxamer-188 -4 Tween-80® 300 Solvents(ml)5 DCM 36 Purified water 507 Total weight 540

7.5.4. Solid state characterization of optimized formula:

The solid state characterization studies were carried out for API, Placebo Lipid

formulation and Granulated Lipid formulation.

Table No: 25 Sample compositions for Solid State Characterization

S.No. Sample Drug (mg) Excipients (mg)

1 API 40 0

2 Placebo 0 500

3 Formulation 40 500

Differential Scanning Calorimetry (DSC)

Figure 37 represents the thermograms of pure API, Placebo and Lipid Formulation.

The DSC curve of pure Valsatan exhibited a single endothermic peak at 98.1º C corresponding to

the melting of the drug and the sharp peak indicated its crystallinity.The DSC curve of Placebo

lipid formulation and VSX 6 formulation exhibits broad endotherms at 75.3º C, 86.68 º C

respectively, corresponding to Glyceryl monostearate and Tween 80 respectively, but the drug’s

peak was no longer observed. It could be attributed to complete entrapment of the drug in the

lipids. Solid state studies did not indicate chemical decomposition of the components (drug and

excipients), showing compatibility and formation of homogenous systems.

Fig: 26 Thermograms of API, Placebo and VSX 6 Formulation

7.5.5. Stability Studies:

A 2 weeks storage stability study was conducted on the lipid based formulation.

Dissolution runs were conducted on the stressed samples at 2 weeks to assess any changes in

release behavior of Valsartan. Accordingly, the formulations was placed in 5ml white and brown

glass vials (glass quality I) and charged for stability studies at at different temperatures (8 ºC,25

ºC and 50 ºC) and light exposures (dark, artificial illumination). At pre-determined time of 2

weeks the samples were analyzed for the percent drug content and the drug dissolution rates. The

dissolution studies were carried out in the developed 100 % release medium. Table No.26

corresponds to the dissolution conditions and results carried out for stability studies.

Table: 26 Stability results for optimized formula VSX 6

VSX 6 LIGHT DARK

Parameter Initial 2 weeks 2 weeks

Temperature 8oC 25oC 50oC 8oC 25oC 50oC Dissoultion Profile

10 min 53.57 53.53 50.6 51.8 53.53 43.45 40.3215 min 64.43 59.1 50.12 54.23 60.12 55.35 50.2430 min 75.8 60.8 55.29 57.15 67.25 56.2 51.345 min 76.13 53.13 58.25 56.19 68.23 54.1 52.660 min 80.16 65.25 60.23 57.25 70.8 58.2 51.8

The above results indicated the intermediate stability of the formulation. As the storage temperature increased, there was a slight decrease in the release of Valsartan from the formulation. A decrease in the dissolution rate of the drug in light exposure conditions when compared to dark conditions was observed in the formulation

The decrease in stability as a result of decreasing dissolution rates in the formulation indicating the effect of light and temperature on formulation.

8. SUMMARY AND CONCLUSION

The goal of any drug delivery system is to provide a therapeutic amount of drug to the proper

site in the body and also to achieve and maintain the desired plasma concentration of the drug for

a particular period of time. However, poor water solubility, incomplete release of the drug,

shorter residence times of dosage forms in the upper GIT leads to lower oral bioavailability.

Such limitations of the conventional dosage forms have paved way to an era of novel drug

delivery systems.

Valsartan is a poorly water soluble drug having an oral bioavailability of 23 to 25%.

Valsartan belongs to the category of Angitotensin II blocker, used in the treatment of

Hypertension. Thus in order to overcome these drawbacks, solid lipid nanoparticles based

formulation approach was selected.

The solubility studies across pH and water indicated the poor solubility of Valsartan in

water.

Biologically compatible Lipid excipients, stabilizers were screened in order to reduce the

particle size of drug to enhance the solubility of Valsartan in water. Based on solubility

results appropriate excipients were selected for formulation development.

Prototype formlations were prepared using the Solvent emulsification-diffusion

technique.

The prepared prototype formulations were characterized for Particle size, zeta potential,

drug entrapment and in-vitro relase characteristics.

The final optimized formulation was characterized for its physical properties Viz.,

particle size, zeta potential, DSC and drug release rate. The final SLN formulation has

shown significant increase in drug relase when comared to API and marketed product

(DIOVAN) in physiologically relevant media.

The optimized lipid based formulations was charged on stability studies for a period of

two weeks and the results indicated that temperature and light have significant effect on

the formulation stability. Therefore storing the formuation in light resistant containers

and at low temperatures and also use of stabilizers in the formulation can provide a stable

drug product.

Summary AND conclusion

To sum up a poorly aqueous-soluble drug VAL was successfully incorporated into SLNs by a

Solvent emulsification-Diffusion technique. The physicochemical characterization and short-

term physical stability were investigated. The in-vitro release tests showed that the release rate of

the SLNs is significantly higher compared with the API and marketed product. Thus the prepared

solid lipid nanoparticles were proved to be a potential technology for enhancing the

bioavailability.

Summary AND conclusion

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