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2. REVIEW OF LITERATURE

The vagina has been used for a long time as a route for drug delivery. The administration

of drugs in the vagina is believed to be as old as pharmacotherapy, first written documents

dating from 19th century BC. Until the 1920s the vagina was considered to be an organ

incapable of absorbing drugs systemically and delivery systems were indicated for

contraception or local infection. The human vagina appears to be an underutilized route of

drug delivery, that is why few delivery systems, which are available, are usually used for

contraception or to treat vaginal infection. A new age of research on novel vaginal

delivery systems is not only focused on local effect but also actively pursued for systemic

delivery of hormones, proteins and peptides. Here a concise report of literature survey on

different aspects of research project has been presented like, some notable research

outcomes in VDDS, a patent search on VDDS, marketed floating products, noticeable

advancement in solid lipid nanoparticles research, advancement in complexation

technologies.

2.1 LITERATURE SEARCH ON FORMULATIONS OF VDDS

Kast et al., 2002, designed a novel bioadhesive vaginal drug delivery system for

clotrimazole. Chitosan was modified by the introduction of thioglycolic acid by utilizing a

carbodiimide to link the carboxylic acid moieties of TGA covalently to the primary amino

groups of chitosan. The ratio was varied, resulting in chitosan-TGA-conjugates. New

chitosan-TGA conjugates are very promising vehicles for the vaginal application of

clotrimazole in treatment of mycotic infections. Water uptake, the disintegration

behaviour, the bioadhesive properties utilizing the rotating cylinder method, as well as the

release of clotrimazole from tablets based on these derivatives were studied. Amount of

water absorbed by the polymer correlated with the amount of covalently attached thiol

groups. Chitosan-TGA conjugates showed a disintegration time of > 120 min. Different

disintegration behaviour of tablets containing also 16.67% clotrimazole strongly improved

cohesive properties. Explained by the pH shift caused by clotrimazole, this is a weak base

and therefore responsible for the alteration of the pH within the matrix system.

Accordingly, the swelling of the tablets is limited and the formation of disulfide bonds is

Review of Literature Chapter 2

PhD Thesis 15 Jamia Hamdard

accelerated. No correlation between swelling behaviour and bioadhesion was observed.

Mucoadhesion was correlated with the amount of immobilized thiol group.

Yun Chang et al., 2002, reportedly developed clotrimazole containing mucoadhesive

thermosensitive gels was developed using polymers like Poloxamer 188, Poloxamer 407

and polycarbophil. Gels were prepared by using the cold method. The mucoadhesiveness

of each formulation was determined using a TA-XT2 Texture Analyzer. Oscillatory

rheometry was performed using a Carri-Med CSL2-100 rheometer (TA Instruments, USA)

with a cone and plate. The viability of cervical epithelial ME-180 cells was investigated

over different concentrations of Clotrimazole given in phosphate buffer or thermosensitive

gels. MTG vehicles did not influence the viability of the cells while Clotrimazole reduced

the viability of the cells in a dose-dependent manner. Clotrimazole did not alter the

morphology of vaginal tissue. After optimization two formulations composed of

P407/P188/PC (15/15/ 0.2 or 15/20/ 0.2) were further studied. Of the two gels, the

formulation with 15% of P188 gelled at higher temperature and revealed lower elastic

modulus. At 10 days post-dose, the CFUs of C. albicans was more than 104 -fold

decreased in gel-treated groups.

Haineault et al., 2003, reported thermo reversible contraceptive Gel containing Sodium

Lauryl Sulfate as API was developed. Block polymers polyoxypropylene and

polyoxyethylene were as gelling agent. Cold method was used to develop the gel. Polymer

(30% w/w), SLS (if present; 2%, w/w) in citrate buffer (0.05, 0.1, 0.2, or 0.4 M at pH 3.5)

and had phase transition temperatures of 25°C. It inhibited sheep testicular hyaluronidase

in a conc. dependent manner, prevented in vi'vo (rabbit model) egg fertilization, acid

buffering property with increasing molarity of citrate buffer. There were no compatibility

issues with latex condom.

Baloglu et al., 2003, investigated In Vitro investigation (bioadhesive properties and

swelling states) of vaginal tablets were carried out. Tablets prepared from the mixtures of

Carbopol 934, pectin, polyvinylpyrolidon, guar gum, ethylene maleic anhydride resins

were studied. Bioadhesive strength of tablet was done with a tensile-tester apparatus

(ZWICK D- 7900). Swelling properties of the same tablets were examined in distilled

water, lactate (pH 5) and phosphate (pH 7) buffer solutions, respectively. The most



favourable formulation resulted from a mixture of Carbopol 934 and Pectin (2:1). It

demonstrates that swelling state, bioadhesive properties and the effect on the medium pH

has significant importance for bioadhesive vaginal formulations. The general idea that

increased swelling contributes to stronger bioadhesive bonds is not quite correct.

Francois et al., 2003, Mucoadhesive, cyclodextrin-based vaginal cream formulation of Itz

was designed. Clinical investigations indicated that application of 5 g of a 2% cream was

very well tolerated and Itraconazole was not systemically absorbed. Aqueous phase was

prepared by solubilizing Itraconazole with Hcl in the presence of propylene glycol and

then adding an aqueous solution of HP-P-CD. After pH adjustment, the Itraconazole/HP-

P-CD solution was added to the oil phase (paraffin oil, trihydroxystearate, and cetyl

dimethicon copolyol) and the desired cream containing 1%, 2%, and 2.5% drug obtained

by homogenization. Deconvolution of the curve suggests stability constants of the 8929

M-1 (K 1 :1) and 27 M-1 (K 1 :2).

V

Zeljka et al., 2005, Acyclovir liposome loaded hydrogel was designed for genital herpes.

Liposomes prepared by the polyol dilution method. Phospholipid compositions used were

egg phosphatidylcholin(PC)/egg phosphatidylglycerol (PG) 9:1, egg phosphatidylcholine

(PC) and egg phosphatidycholine (PC)/stearylamine (SA) 9: 3. All liposome preparations

were characterized for particle size (320, 564 and 385 nm respectively), polydispersity

(0.185, 0.786 and 0.321 respectively), encapsulation efficiency (26.84, 8.09 and 23.78 %

respectively). Gel made from Carbopol 974P which increased the stability of the

liposome. After 4 days of storage, mean diameter increased but size distribution remained

nearly constant.

Chopra et al., 2007, reported the development and optimization of polyherbal gels using

three different carbopols (Carbopol 934P, Carbopol 974P and Polycarbophil) by a 3-

factor, 3-level Box-Behnken statistical design. Independent variables studied were the

polymer concentration, honey concentration and aerosil concentration. Dependent

variables studied were the elastic modulus, gel index, and maximum detachment force. A

Combination of polyherb selected was Trigonella foenum-graecum, Azadirachta indica,

Cichorium intybus and Curcuma longa. Carbopol 974P gels were more consistent and

provided better regulation of G0 and detachment force over a stability period of 6 months



than Carbopol 934P and Noveon AA1 gels. Aerosil 200 was also found to contribute

significantly to the improvement of consistency and rheological properties. Linearity

observed between the actual and predicted values of the response variables suggested the

prognostic ability of the RSM design.

Alam et al., 2007, introduced acid-buffering Bioadhesive Vaginal tablet for mixed vaginal

infections. Clotrimazole, metronidazole and Lactobacillus acidophilus spores were used to

challenge the infection. Excipients used - a mixture of citric acid and sodium bicarbonate

as effervescent; Sodium monocitrate as a buffering agent and Ac-Di-sol as a super

disintegrant. Tablets developed by direct compression exhibited encouraging results (Ex

vivo retention for > 24 hrs, > 80% drug release in 5 hrs, pH (4.40 ± 0.092, etc), when

Polycarbophil and Sodium CMC were used as bioadhesive materials.

Talwar et al. 2008, A polyherbal (0.36% w/v curcumin, 2.5% w/v of extracts of Amla,

1% w/v saponins from Sapindus mukorossi,2.5 % w/v purified Aloe Vera and rose water)

cream was developed. It was successfully tested against resistant and clinical isolates of

Neisseria gonorrhoeae, Candida glabrata, Candida albicans and Candida tropicalis. Also

displayed a high virucidal action against human immunodeficiency virus HIV-1NL4.3 in

CEM-GFP reporter T and P4 (Hela-CD4-LTR-betaGal) cell lines with a 50% effective

concentration (EC50) of 1:20000 dilution and nearly complete (98-99% ) inhibition at

1:1000 dilution. It was also found to be totally safe in pre-clinical toxicology carried out

on rabbit vagina.

Perioli et al., 2009, introduced Vaginal Tablet, introducing FG 90 Chitosan and its

combination with other polymers. Metronidazole tablets were prepared using bioadhesive

polymers chitosan (FG90C), polyvinyl pyrrolidone (PVPK90) and polycarbophil

(PCPAA1), blended in different ratios to treat Bacterial vaginosis. The different

combinations resulted in variable results in terms of hardness, hydration, friability,

mucoadhesion, in vitro drug release and antibacterial activity. FG 90C emerged as a good

alternative for mucoadhesive polymer.

Dobaria et al., 2009, developed Itz loaded bioadhesive film developed by solvent

evaporation and using 32 factorial designs. Overall desirability of the formulation was



0.92. The optimized formulation containing hydroxypropyl cellulose (100 mg) and

polyethylene glycol 400 (50 mg), exhibited tensile strength 9.64 N/m2, % elongation at

break 67.56% and 17.75 % drug remained at vaginal mucosa after 8 h.

Bachhav and Patravale, 2009, Designed microemulsion based vaginal gel of

fluconazole. The small-scale clinical studies indicated that the FLZ-MBG shows faster

onset of action than Candid V® gel although no difference was observed in the clinical

efficacy. Microemulsion composed of Capryol 90 (14% , w/w), Cremophore EL (43.5%,

w/w) and water (42.5%, w/w) was selected as optimized. Globule size of 24 nm and

polydispersity index of 0.98. Carbopol® ETD 2020 could successfully gel the

microemulsion without disturbing the structure. pH of the gel was observed to be 4.53.

The retention time evaluated in vitro bioadhesion studies and in vitro anti fungal studies

by the gel was significantly higher (P > 0.05) as compared to Candid- V® gel. It was

found to be well tolerated in preclinical studies.

Albertini et al., 2009, Polymer-lipid based mucoadhesive microspheres prepared by

spray-congealing for the vaginal delivery of econazole nitrate. Seven different

formulations were prepared. Lipid-hydrophilic matrix (Gelucire® 53/10) was used as

carrier and several mucoadhesive polymers such as chitosan, sodium

carboxymethylcellulose and poloxamers (Lutrol® F68 and F127) were added. Drug

content varied between 21.03 to 35.25%. Non-aggregated microspheres with high yields

(>90%, w/w) and with prevalent size in the range 100-355 ^m were obtained. Both

poloxamers significantly (p < 0.01) improved the solubility and in vitro bioavailability of

the low solubility drug and the mucoadhesive strength. Spray-congealing technology

proved to be a better option for preparation of microsphere for the given set of parameters.

Ensign et al., 2012, In this study mucus-penetrating Nanoparticles (MPPs) were used for

vaginal drug delivery to protect against Herpes Simplex virus. Vaginal distribution,

retention, and safety of nanoparticles were also evaluated. MPPs provide uniform

distribution over the vaginal epithelium, whereas conventional nanoparticles (CPs) that are

mucoadhesive are aggregated by mouse vaginal mucus, leading to poor

distribution. MPPs transported swiftly through mucus deep into vaginal folds (rugae)

within minutes. By penetrating into the deepest mucus layers, more MPPs were retained in

the vaginal tract after 6 hours compared to CPs. After 24 hours, when delivered in a




conventional vaginal gel, patches of a model drug remained on the vaginal epithelium,

whereas the epithelium was coated with drug delivered by MPPs.

Malcolm et al., 2013, Investigated the pharmacokinetics of Maraviroc, a CCR5-targeted

HIV-1 entry inhibitor, in rhesus macaques following vaginal administration of various

maraviroc-loaded aqueous hydroxyethylcellulose gels, and to correlate the PK data with

efficacy in a single high-dose vaginal HIV-162P3 challenge model. Plasma concentrations

were assessed over 72 h. A complete protection was achieved with a 3.3% w/w maraviroc

gel. Maraviroc concentrations in vaginal fluid (range 104-1 0 7 ng/mL), vaginal tissue (100

1200 ng/g) and plasma (<102 ng/mL) were highly dependent on maraviroc gel loading,

irrespective of the form of the maraviroc component within the gel (solubilized versus

dispersed). Fluid and plasma concentrations were generally highest at 0.5 or 2 h after gel

application, before declining steadily through to 72 h. Maraviroc concentrations in the

various biological compartments correlated strongly with the extent of protection against

vaginal SHIV-162P3 challenge.

2.2 PATENT SEARCH ON VAGINAL DRUG DELIVERY

Following are some important patents over VDDS.

Table 3: Patent search on vaginal drug delivery

Patent No. Inventor Claim/Title

4,551,148 Riley et al., 1985Vaginal delivery systems and their methods of

preparation and use5,266,329 Riley, TC.,1993 Vaginal delivery system

5,814,330Putteman and Snoeckx,

1998Mucoadhesive emulsion containing cyclodextrin

5,958,461 Larsen, B., 1999 Vaginal pharmaceutical compositions

US2004/0180965A1Borgman and Juul,

2005Gel composition and method for treatment of

vaginal infectionsUS2004/0192642A1 Yang et al., 2007 Prevention of urogenital infections

US2005/02551557A1 Sen N., 2005Sustained release, mucoadhesive vaginal

pharmaceutical compositions

US2006/0122095Delvin and Schnaare,

2003Mucus formulation for mucosal surfaces and uses

thereof

US2006/0172007A1 Ahmad et al., 2005Compositions and methods for reducing vaginal

pHUS2006/0240111A1 Fernandez et al., 2004 Semisolid mucoadhesive formulations

US2008/0152713A1 Singh et al., 2001 Herbal contraceptive formulations

4286587 Wong, Patrick S., 1981 Vaginal drug delivery system made from polymer

US 09/316,640Bardin, C Wayne, et al. (2000)

Intravaginal rings with insertable drug-containing core



2.3 MARKETED FORMULATIONS OF VDDS

A few examples of marketed formulation used globally for various indications of VDDS.

Table 4: VDDS products floating in international market

-* *- £• j 1

Therapeutic Excipients used Intended useM anufactured or

Developed byBrand nameagent

AVC Cream Sulfanilamide Lactose, propylene glycol, stearic acid, diglycol stearate, methyl paraben, propyl paraben, trolamine, lactic acid

Vulvovaginitis caused by C. albicans

King

Pharmaceuticals,USA

CanestenSolution Clotrimazole Inert solvent Fungal infection, Trichomoniasis

Bayer AG

CanestenInsert Clotrimazole Adipic acid, colloidal silicon dioxide, lactose, magnesium stearate, maize starch, Polysorbate 80, sodium bicarbonate, stearic acid

Fungal infection, Trichomoniasis

Bayer AG

ClotrimazoleCream Clotrimazole Sodium phosphate, monobasic, adipic acid, colloidal silicon dioxide, lactose, magnesium stearate, maize starch, Polysorbate 80, sodium bicarbonate stearic acid

Vaginal yeast infection

Taro Pharmaceuticals

EcostatinCream Econazole nitrate Benzoic acid, butylated hydroxy anisole, mineral oil, fragrance, palm oil, polyethylene glycol, Peg stearate

Vulvovaginalcandidiasis

Bristol-Myers Squibb, Canada

EcostatinOvules Econazole nitrate Hydrogenated vegetable oil Vulvovaginalcandidiasis

Bristol-Myers Squibb, Canada

Femstat 3Cream Butaconazole nitrate Cetyl alcohol, glyceryl stearate, polyethylene glycol 100 stearate, methyl paraben, propyl paraben, mineral oil, Polysorbate 60, propylene glycol, sorbitan monostearate, stearyl alcohol, water


Bayer Corp.

GynecureOvule Tioconazole Bees’ wax, gelatin, glycerine, glycine, hydrogenated vegetable fat, lecithin, liquid paraffin, Polysorbate, potassium sorbate, titanium dioxide, water

Vulvovaginalcandidiasis

Pfizer Consumer Healthcare

Gyne-Lotrimin Clotrimazole Insert: corn starch, crospovidone, lactose, magnesium Vaginal yeast Schering-


Review of Literature Ch apte r 2

Insert stearate, povidone; cream: benzyl alcohol, cetearyl alcohol, cetyl esters wax, octyldodecanol, Polysorbate 60, sorbitan monostearate

infection PloughCorp., NJ, USA

Gyne-Lotrimincream

Clotrimazole Benzyl alcohol, cetearyl alcohol, cetyl esters wax, octyldodecanol, Polysorbate 60, water, sorbitan monostearate


Schering- PloughCorp., NJ, USA

Monistat Ovule Miconazole nitrate Gelatin, glycerine, mineral oil, sodium ethyl paraben, titanium dioxide, white petrolatum

Vulvovaginalcandidiasis(moniliasis)

McNeil Consumer Products

Monistat Cream Miconazole nitrate Benzoic acid, butylated hydroxy anisole, mineral oil, water, Peglicol 5 oleate, Pegoxol stearate

Vulvovaginalcandidiasis(moniliasis)

Ortho Pharmaceutical Corporation, USA

Myclo-gyneCream Clotrimazole Cetyl stearyl alcohol, benzyl alcohol, Polysorbate 60, water, sorbitan monostearate, spermaceti, 2- octyldodecanol


Boehringer Engelheim, Canada Ltd

Myclo-gyneInsert Clotrimazole Adipic acid, colloidal silicon dioxide, lactose, magnesium stearate, maize starch, Polysorbate 80, sodium bicarbonate,stearic acid


Boehringer Engelheim, Canasda

Ltd.Mycelex G Tablet Clotrimazole Lactose, microcrystalline cellulose,

Hydroxypropylmethyl cellulose, silicon dioxide, magnesium stearate, corn starch, lactic acid,

crospovidone


Bayer Corp.

Mycelex 3 Cream Butoconazole nitrate Cetyl alcohol, glyceryl stearate, polyethylene glycol 400 stearate, methyl paraben, propyl paraben, mineral oil, Polysorbate 60, propylene glycol, sorbitan monostearate, stearyl alcohol, water


Bayer ConsumerCare

Mycelex 7 Cream Clotrimazole Benzyl alcohol, cetostearyl alcohol, cetyl esters wax, octyldo-decanol, Polysorbate 60, water, sorbitan monostearate


Bayer Consumer Care

TerazolSuppositories

Terconazole Butylated hydroxy anisole, palm kernel oil, coconut oil, glycerides

Vaginal candida infection

Ortho-McNeil


2.4 LITERATURE SEARCH ON SLNS

Bunjes et al., 1996, In this study the ability of tristearin, tripalmitin, trimyristin and

trilaurin to form solid lipid nanoparticles after melt-homogenization was investigated by

DSC and X-ray diffraction. Upon storage at common temperatures after preparation solid

nanoparticles are formed in tristearin and tripalmitin dispersions. In contrast to earlier

literature reports colloidal dispersions of trilaurin didn’t form solid particles under those

conditions. Depending on the mixing ratio, the time-course of polymorphic transitions

after crystallization may also be altered significantly. Trimyristin nanoparticles which can

be obtained in solid or liquid form have a larger incorporation capacity for the lipophilic

model drug menadione in the liquid than in the solid state. The kinetics of polymorphic

transitions after crystallization of triglyceride nanoparticles was slower for longer-chain

than for shorter-chain triglycerides. The structure and melting behaviour of the resulting

mixed nanoparticles were more complex than those of nanoparticles prepared from the

simple triglycerides. The melting enthalpy of the mixed nanoparticles dispersions was

usually not significantly different from that of dispersions of the simple triglycerides.

Venkateswarlu and M anjunath, 2004, SLN delivery systems of clozapine have been

developed using various triglycerides (trimyristin, tripalmitin and tristearin), soylecithin

95%, poloxamer 188 and charge modifier stearylamine. Hot homogenization of melted

lipids and aqueous phase followed by ultrasonication at temperature above the melting

point of lipid was used to prepare SLN dispersions. Analysis was done with Malvern

Zetasizer, DSC, and PXRD. Process and formulation variables have been studied and

optimized. In vitro release studies were performed in 0.1 N HCl, double-distilled water

and phosphate buffer, pH 7.4, using modified Franz diffusion cell. Stable SLN

formulations of clozapine having mean size range of 60-380 nm and zeta potential range

of -23 to + 33 mV were developed. More than 90% clozapine was entrapped in SLN. DSC

and PXRD analysis showed that clozapine is dispersed in SLN in an amorphous state. The

release pattern of drug is analyzed and found to follow Weibull and Higuchi equations.

Castelli et al., 2005, Indomethacin-loaded SLN and NLC were prepared and the

organization and distribution of the different ingredients originating each type of

nanoparticles system were studied by DSC technique. Mean particle size and percentage



of drug encapsulation were also determined. NLC lipid organization guaranteed an

increased indomethacin encapsulation in comparison with SLN. DSC static and dynamic

measurements performed on SLN and NLC showed that oil nano-compartments

incorporated into NLC solid matrix drastically influenced drug distribution inside the

nanoparticles system. Controlled release from NLC system could be explained considering

both drug partition between oil nano-compartments and solid lipid and a successive

partition between solid lipid and water.

Saraf et al., 2006, double emulsion-solvent evaporation (w/o/w) method was selected for

the preparation of hepatitis B surface antigen (HBsAg) loaded lipid micro particles.

Intranasal route was considered for mucosal administration and hence to prepare the

delivery system biocompatible and least irritable, soyalecithin (phospholipid) was taken

instead of polymer because phosphatidylcholine is the major component of endogenous

lung surfactant. The studies performed in present work included antigen characterization,

development of lipid micro particles, stability studies of the prepared lipid micro particle

formulations, percent mucoadhesion, ex vivo cellular uptake studies and in vivo studies.

The general order obtained from in vivo studies for mucosal immune response (IgA)

followed the sequence: LMST-HBsAg (IN) > LM-HBsAg (IN) > alum-HBsAg (IN) >

LMST-HBsAg (IM) > alum-HBsAg (IM)>LM-HBsAg (IM) > plain HBsAg (IN) > plain

HBsAg (IM).

Zhang et al., 2006, to design and characterize lectin-modified solid lipid nanoparticles

(SLNs) containing insulin and to evaluate the potential of the lectin-modified colloidal

carriers for oral administration of peptide and protein drugs, SLNs were prepared by three

different methods. For comparison, some insulin-loaded SLNs were modified with wheat

germ agglutinin-N-glutaryl-phosphatidylethanolamine (WGA-N-glut-PE). The particle

size, zeta potential and entrapment efficiency of insulin-loaded SLNs were determined.

These results demonstrated that SLNs and WGA-modified SLNs promoted the oral

absorption of insulin. Insulin-loaded SLNs prepared by an appropriate modification of the

double dispersion method yielded the highest drug entrapment efficiency, which was more

than 60%. In vivo experiments were carried out using insulin-loaded SLNs and WGA-

modified SLNs prepared by this method. SLNs and WGA-modified SLNs protected



insulin against degradation by digestive enzymes in vitro. The stabilizing effect of WGA-

modified SLNs was greater than that observed in SLNs. After oral administration of

insulin-loaded SLNs or WGA-modified SLNs to rats, the relative pharmacological

bioavailability were 4.46% and 6.08%, and the relative bioavailability were 4.99% and

7.11%, respectively, in comparison to subcutaneous injection of insulin.

M uller et al., 2006, developed an optimized oral formulation for cyclosporine A. 2% of

this drug has been formulated in solid lipid nanoparticles (SLN™, mean size 157 nm) and

as nanocrystals (mean size 962 nm). The encapsulation rate of SLN was found to be

96.1%. Nanocrystals are composed of 100% of drug. For the assessment of the

pharmacokinetic parameters the developed formulations have been administered via oral

route to three young pigs. Comparison studies with a commercial Sandimmun

Neoral/Optoral® used as reference have been performed. In this study it has been proved

that using SLN™ as a drug carrier for oral administration of cyclosporine A a low

variation in bioavailability of the drug and simultaneously avoiding the plasma peak

typical of the first Sandimmun® formulation can be achieved. The blood profiles observed

after oral administration of the commercial microemulsion Sandimmun® revealed a fast

absorption of drug leading to the observation of a plasma peak above 1000 ng/ml within

the first 2 h. For drug nanocrystals most of the blood concentrations were in the range

between 30 and 70 ng/ml over a period of 14 h. These values were very low, showing

huge differences between the measuring time points and between the tested animals. On

the contrary, administration of cyclosporine-loaded SLN led to a mean plasma profile with

almost similarly low variations in comparison to the reference microemulsion, however

with no initial blood peak as observed with the Sandimmun Neoral/Optoral®. Comparing

the area under the curves (AUC) obtained with the tested animals it could be stated that

the SLN™ formulation avoids side effects by lacking blood concentrations higher than

1000 ng/ml.

Liu et al., 2007, constructed isotretinoin-loaded SLN (IT-SLN) formulation with skin

targeting for topical delivery of isotretinoin. Precirol ATO 5 was selected as the lipid of

SLN. Tween 80 and soybean lecithin were used as the surfactants to stabilize SLN. The

hot homogenization method was performed to prepare the drug-loaded SLN. The



penetration of isotretinoin from the IT-SLN formulations through skins and into skins

were evaluated in vitro using Franz diffusion cells fitted with rat skins. These results

indicate that the studied IT-SLN formulation with skin targeting may be a promising

carrier for topical delivery of isotretinoin. PCS showed that the SLN formulations had low

average size between 30 and 50 nm. TEM studies showed that the IT-SLN formulation

had a spherical shape. All the formulations had high entrapment efficiency ranging from

80% to 100%. The in vitro permeation data showed that all the IT-SLN formulations can

avoid the systemic uptake of isotretinoin in skins; however the control tincture had a

permeation rate of 0.76±0.30 ^g cm-2 h-1 through skins. The IT-SLN consisting of 3.0%

Precirol ATO 5, 4.0% soybean lecithin and 4.5% Tween 80 could significantly increased

the accumulative uptake of isotretinoin in skin and showed a significantly enhanced skin

targeting effect. The studied IT-SLN showed a good stability.

del Pozo-RodrA-guez et al., 2007, Different formulations based on SLN-DNA

complexes were formulated in order to evaluate the influence of the formulation

components on the “in vitro” transfection capacity. SLNs composed by the solid lipid

Precirol® ATO 5, the cationic lipid DOTAP and the surfactant Tween 80, and SLN-DNA

complexes prepared at different DOTAP/DNA ratios were characterized by studying their

size, surface charge, DNA protection capacity, transfection and cell viability in HEK293

cultured cells. The incorporation of Tween 80 allowed for the reduction of the cationic

lipid concentration. The formulations prepared at DOTAP/DNA ratios 7/1, 5/1 and 4/1

provided almost the same transfection levels (around 15% transfected cells), without

significant differences between them (p > 0.05). Other assayed formulations presented

lower transfection. Transfection activity was dependent on the DOTAP/DNA ratio since it

influences the DNA condensation into the SLNs. DNA condensation is a crucial factor

which conditions the transfection capacity of SLNs, because it influences DNA delivery

from nanoparticles, gene protection from external agents and DNA topology.

Almeida and Souto, 2007, In this report, Solid lipid nanoparticles, lipid microparticles

and lipospheres have been sought as alternative carriers for therapeutic peptides, proteins

and antigens. The research work developed in the area confirms that under optimized

conditions they can be produced to incorporate hydrophobic or hydrophilic proteins and


PhD Thesis T1 Jamia Hamdard

seem to fulfil the requirements for an optimum particulate carrier system. Proteins and

antigens intended for therapeutic purposes may be incorporated or adsorbed onto SLN,

and further administered by parenteral routes or by alternative routes such as oral, nasal

and pulmonary. Formulation in SLN confers improved protein stability, avoids proteolytic

degradation, as well as sustained release of the incorporated molecules. Important peptides

such as cyclosporine A, insulin, calcitonin and somatostatin have been incorporated into

solid lipid particles and are currently under investigation. Several local or systemic

therapeutic applications may be foreseen, such as immunization with protein antigens,

infectious disease treatment, chronic diseases and cancer therapy.

Suresh et al., 2007, studied the bioavailability enhancement of lovastatin by

administering lovastatin solid lipid nanoparticles (SLN) duodenally to rats. Lovastatin

SLN were developed using triglycerides by hot homogenization followed by ultra-

sonication. Particle size and zeta potential were measured by photon correlation

spectroscopy. The entrapment of the drug in the SLN and lipid modification was

characterized. Bioavailability studies were conducted in male Wistar rats after

intraduodenal administration of lovastatin suspension and SLN. Stable lovastatin SLN

having a mean size range of 60 to 119 nm and a zeta potential range of -1 6 to -21 mV

were developed. More than 99% of the lovastatin was entrapped in the SLN. Lovastatin

was dispersed in an amorphous state, and triglycerides were in p1 form in the SLN. In

vitro stability studies showed the slow release and stability of lovastatin SLN. The relative

bioavailability of lovastatin and lovastatin hydroxy acid of SLN were increased by ~173%

and 324%, respectively, compared with the reference lovastatin suspension.

Liu et al., 2007, SLNs loaded with insulin-mixed micelles (Ins-MMs) were prepared by a

novel reverse micelle-double emulsion method, in which sodium cholate (SC) and

soybean phosphatidylcholine (SPC) were employed to improve the liposolubility of

insulin, and the mixture of stearic acid and palmitic acid were employed to prepare insulin

loaded solid lipid nanoparticles (Ins-MM-SLNs). Some of the formulation parameters

were optimized to obtain high quality nanoparticles. The drug release behavior was

studied by in situ and externally sink method and the release pattern of drug was found to

follow Weibull and Higuchi equations. Results of stability evaluation showed a relatively



long-term stability after storage at 4 °C for 6 months. The particle size and zeta potential

measured by PCS were 114.7±4.68 nm and-51.36±2.04mV, respectively. Nanospheres

observed by TEM and SEM showed extremely spherical shape. The entrapment efficiency

(%EE) and drug loading capacity (%DL) were 97.78±0.37% and 18.92±0.07%,

respectively. DSC of Ins-MM-SLNs indicated no tendency of recrystallisation. The core

shell drug loading pattern of the SLNs was confirmed by fluorescence spectra and

polyacrylamide gel electrophoresis (PAGE) which also proved the integrity of insulin after

being incorporated into lipid carrier.

Radomska-Soukharev, A., 2007, it was an investigation of chemical stability of lipids

used as excipients in the production of SLN. Different lipids and amounts of surfactants

were considered. Most of the formulations were produced using identical binary surfactant

mixtures and concentrations to analyze the effect of the chemical nature of the lipids on

their stability in SLN. In some formulations, surfactants were exchanged or their

concentration was increased to assess the contribution of surfactants on stability of lipids

particles. SLN were characterized by PCS, LD, zeta potential determination and DSC.

Potential effects of lipid crystallinity and modifications were assessed. All formulations

were produced by the hot homogenization technique. A GC analysis in combination with a

method for lipid extraction from aqueous SLN dispersions was also used to investigate the

chemical stability of the lipid excipients forming the particle matrix. The production

process of SLN itself did not affect the chemical stability of lipid excipients forming the

particle matrix. The formulations where lipids consisted of triglycerides showed a

negligible decomposition of the structure during incubation at 25°C. Dynasan 118 showed

the highest chemical stability (lossb4%) within two years.

Han et al., 2008, influence of surfactants on properties of NLC was investigated. Four

types of surfactants and their mixtures were used in the absence of model drugs thereby

avoiding the interaction between surfactants and drugs were taken. The hot high-pressure

homogenization method was employed to produce NLC and the physicochemical

properties of NLC, such as particle size distribution, zeta potential and DSC analysis were

investigated. The results indicated that ionic surfactants such as sodium deoxycholate

(SDC), showed obviously low emulsification efficiency in the preparation. However, it



increased the zeta potential of nanoparticles leading to improve the physical stability of

the system. Non-ionic emulsifier, especially Poloxamer 188, offered additional stearic

stabilization effect avoiding aggregation of the nanoparticles in the colloidal system. The

formulation in the study combined four types of additives including ionic surfactant

(SDC), non-ionic emulsifier (Poloxamer 188 and Tween-80), and Lecithin to obtain

favorably stable NLC drug delivery system, which could stabilize for more than 1 year

without phase separation at 4° C.

M uller et al., 2008, SLNs were produced loaded with cyclosporine A in order to develop

an improved oral formulation. In this study, the particles were characterized with regard to

the structure of the lipid particle matrix, being a determining factor for mode of drug

incorporation and drug release. DSC and WAXS measurements were employed for the

analysis of the polymorphic modifications and mode of drug incorporation. Particles were

produced using Imwitor 900 as lipid matrix (the suspension consisted of 10% particles,

8% Imwitor 900, 2% cyclosporine A), 2.5% Tagat S, 0.5% sodium cholate and 87%

water. The processing of the bulk lipid into nanoparticles was accompanied by a

polymorphic transformation from the P to the a-modification. After production, the drug-

free SLN dispersions converted back to P-modification, while the drug-loaded SLN stayed

primarily in a -modification. After incorporation of cyclosporine A into SLN, the peptide

lost its crystalline character. Based on WAXS data, it could be concluded that

cyclosporine is molecularly dispersed in between the fatty acid chains of the liquid-

crystalline a-modification fraction of the loaded SLN.

Yuan et al., 2008, in this report water-in-oil (w/o) mini-emulsion was used to prepare

SLNs by solvent diffusion method. n-Hexane, Tween 80 and Span 80 were used as the oil

phase and surfactant combination, respectively. Clobetasol propionate was used as a

model drug. Mini-emulsion with the particle size of 27.1±7.6 nm was obtained when the

composition of water/Tween 80/Span 80/n-hexane was 1 ml/18 mg/200 mg/10 ml. The

SLN prepared by the novel method displayed smaller particles size and higher drug

entrapment efficiency than those of SLN prepared by the conventional method. The drug

entrapment efficiency decreased with increasing of charged amount of drug, and 15.9% of

drug loading was achieved as the charged amount of drug was 20%. The in vitro drug



release tests indicated that the drug release rate was faster than that of SLN prepared by

the conventional method, and the drug content in SLN did not affect the in vitro drug

release profile.

Li et al., 2008, Surface-modified SLNs were prepared from precipitation in an oil-in-

water microemulsion, using monocaprate as solid matrix, polyethylene glycol sorbitan

monooleate (Tween80) as emulsifier and poly(ethylene glycol) stearate (PEG-S) as

surface modifier. Effect of PEG-S on the phase behaviour of MC/Tween80/H2O system as

well as on the properties of surface-modified SLNs was evaluated. Three different PEG-

Ss: PEG8-S, PEG40-S and PEG100-S with varying PEG chain length were used to modify

SLN. The effect of PEG-S on pseudo-ternary phase showed that the presence of PEG-S

extended the areas of liquid crystal and microemulsion in the phase diagram. Meanwhile,

with increasing the PEG chain length, liquid crystal and microemulsion areas extended to

the water corner. TEM and ESEM showed that the modified SLNs to be spherical

particles, and the average diameters measured by DLS were between 71 nm and 140 nm.

XRD showed that both surface-modified and non-modified SLN existed in the amorphous

state.

Li et al., 2008, Solid polymer-lipid hybrid nanoparticles (PLN) were prepared by using a

rationally selected composition. The solid-state properties of the components, their

molecular interactions and the morphology, particle size and internal structure of PLN

were determined by use of DSC, XRD, 13C NMR, FT IR, TEM and DLS. The distribution

of drug in PLN was examined by TEM imaging using a cationic gold tracer. Drug release

studies were conducted in various media. Drug loading as high as 36% and loading

efficiencies up to 99% were achieved in the rationally formulated PLN. Hydrogen bonding

between drug, polymer and lipid and a uniform distribution of amorphous drug within the

solid lipid matrix were evident. Sustained drug release from the PLN was mainly

controlled by ion exchange and diffusion processes. The results demonstrated that strong

molecular interactions among the drug, the polymer and the lipid in the optimized

formulation were responsible for the improved drug loading and release performance of

the PLN.



Hu et al., 2008, to overcome the disadvantages such as lower drug entrapment efficiency

of lipid nanospheres prepared by conventional solvent diffusion method, a solvent

diffusion method in drug saturated aqueous system was developed. Nimodipine was used

as a model drug to incorporate into lipid nanospheres. The monostearin solid lipid

nanoparticles produced by conventional method under different production temperature.

The highest drug loading was reached up to 4.22 wt% when 8 wt% drug was charged in

the preparation of lipid nanospheres. 24.40-30.21 wt% EE, and relatively higher EE was

achieved when the production temperature was 0°C. The EE could be enhanced by the

incorporation of liquid lipid (caprylic/capric triglycerides, CT) into SLN and the

employing of drug saturated dispersion medium. The NLC with higher CT content

indicated the highest EE as the drug saturated aqueous solution was used as dispersion

medium. The DSC results demonstrated the present method could improve the drug

encapsulation into lipid nanospheres. In vitro drug release experiments indicated the

present preparation method could delay the drug release rate from lipid nanospheres, and

the drug release rate could adjust by the CT content in lipid nanospheres.

Bhaskar et al., 2009, prepared aqueous dispersions of lipid nanoparticles - flurbiprofen

solid lipid nanoparticles and flurbiprofen nanostructured lipid carriers by hot

homogenization followed by sonication technique and then incorporated into the freshly

prepared hydrogels for transdermal delivery and characterized. Both the SLN and NLC

dispersions and gels enriched with SLN and NLC possessed a sustained drug release over

period of 24 h but the sustained effect was more pronounced with the SLN and NLC gel.

Anti-inflammatory effect in the Carrageenan-induced paw edema in rat was significantly

higher for B1 and A1 formulation than the orally administered flurbiprofen. More than

50% of the particles were below 300 nm after 90 days of storage at RT. The

pharmacokinetics of flurbiprofen in rats following application of SLN gel (A 1) and NLC

gel (B1) for 24 h were evaluated. The Cmax of the B1 formulation was 38.67±2.77 ^g/ml,

which was significantly higher than the A1 formulation (Cmax = 21.79 ± 2.96 ^g/ml). The

Cmax and AUC of the B1 formulation were 1.8 and 2.5 times higher than the A1 gel

formulation respectively. The bioavailability of flurbiprofen with reference to oral

administration was found to increase by 4.4 times when gel formulations were applied.



2.5 LITERATURE SEARCH ON INCLUSION COMPLEXATION

Miyake et al., 1999, In this report, the interaction of Itraconazole, with 2-hydroxypropyl-

P-cyclodextrin (HP-P-CD) in water and 10% v/v propylene glycol: water solution at pH

2.0 was investigated by the solubility method and ultraviolet and 1H-nuclear magnetic

resonance (NMR) spectroscopy. The solubility of Itraconazole in water significantly

increased as the concentrations of HP-P-CD was augmented, showing an Ap type phase

solubility diagram. The upward curvature closely corresponded to the simulation curve

which was calculated on the basis of the 1:2 (guest: host) complexation model. The 1:2

complex was formed even in the presence of 10% v/v propylene glycol, although the co

solvent system made the interaction with HP-P-CD weaker due to the competitive

inclusion. The ultraviolet spectroscopic studies also supported the 1:2 complex formation

of Itraconazole with HP-P-CD in 10% v/v propylene glycol: water solution at pH 2.0. The

1H-NMR spectroscopic studies suggested that the triazole and triazolone moieties of

itraconazole are involved in the 1:2 inclusion complexations.

Wen et al., 2004, Inclusion complex of P-cyclodextrin with carvedilol was prepared by

using a convenient new method of microwave irradiation. The structure of inclusion

complex was determined by fluorescence spectroscopy and NMR, NMR

measurements in solution. The solid inclusion was characterised by infrared spectroscopy,

differential scanning calorimetry (DSC) and element analysis. Phase-solubility studies

demonstrated the ability of ^ -cyclodextrins to complex with carvedilol and increase drug

solubility. These experimental results confirmed the existence of 1:2 inclusion complex of

carvedilol with P-cyclodextrin, the formation constant of complex was determined by the

fluorescence method. Molecular modelling predicted the energy-minimized structure of

the complex.

Jun et al., 2007, Inclusion complex of Simvastatin (SV) with hydroxypropyl ^ -

cyclodextrin (HP-P-CD) prepared using supercritical antisolvent (SAS) process were

investigated to improve the aqueous solubility and the dissolution rate of drug. Inclusion

complexation in aqueous solution and solid state was evaluated by the phase solubility

diagram, differential scanning calorimetry (DSC), powder X-ray diffractometry (PXRD),

Fourier-transform infrared spectroscopy (FT-IR) and scanning electron microscopy



(SEM). No endothermic and characteristic diffraction peaks corresponding to SV was

observed for the inclusion complex in DSC and PXRD. FT-IR study demonstrated the

presence of intermolecular hydrogen bonds between SV and HP-P-CD in inclusion

complex, resulting in the formation of amorphous form. Aqueous solubility and

dissolution studies indicated that the dissolution rates were remarkably increased in

inclusion complex, compared with the physical mixture and drug alone. The phase

solubility diagram with HP-P-CD was classified as AL-type at all temperatures

investigated, indicating the formation of 1:1 stoichiometric inclusion complex. The

apparent complexation constants ( ^ 1:1) calculated from phase solubility diagram were 774,

846 and 924 M-1 at 25, 37 and 45 ± 0.5 °C, respectively. Moreover, SV/HP-P-CD

inclusion complex performed better than SV in reducing total cholesterol and triglyceride

levels. This could be primarily attributed to the improved solubility and dissolution

associated with inclusion complex between drug and HP- ^ -CD. In conclusion, SAS

process could be a useful method for the preparation of the inclusion complex of drug with

HP-P-CD and its solubility, dissolution rate and hypolipidemic activity were significantly

increased by complexation between SV and HP-P-CD.

Lee et al., 2008, feasibility of a supercritical fluid process, called aerosol solvent

extraction system (ASES), for producing solid-state inclusion complexes of Itraconazole

(ITR) with 2-hydroxypropyl-P-cyclodextrin (HP-P-CD) was investigated. ITR was

complexed with HP-P-CD at temperatures of 35-55°C, pressures of 83-140 bars, CO2

densities of 0.498-0.801 g/cm3, and solution concentrations of 1-5% (w/v). The ASES-

processed ITR/HP-P-CD powders showed a significant enhancement in the ITR solubility

(up to 753.6 ^g/mL) in an aqueous medium of pH 1.2. The aqueous solubility of ITR

increased with pressure at a constant temperature, and we could obtain a relatively high

solubility of 341 ^g/mL at 140 bars and 35°C. In a solution concentration range of 1-5%

(w/v), the solubility increased with decreasing concentration, yielding 289-407 ^g/mL.

When the molar ratio of ITR to HP-P-CD was varied from 1:1 to 1:3, the ITR solubility

increased with HP-P-CD content, giving a value of 753.6 ^g/mL for the 1:3 ratio. For the

ASES-processed ITR/HP- P-CD powders, the percent dissolution of ITR also increased

considerably and about 90% of ITR was dissolved within 5-10 min.



Brewster et al., 2008, the report assessed the ability of hydrophilic cyclodextrins to

solubilize compounds via stabilization of supersaturated drug solutions presumably by

inhibition of nucleation and arresting crystal growth. To these points, the effects of 2-

hydroxypropyl-P-cyclodextrin (HP-P-CD) and sulfobutylether-P-cyclodextrin (SBE-P-CD)

on equilibrium solubility was assessed via phase solubility analysis as were the

interactions of these excipients on drug solubility under conditions favouring super

saturation. Phase-solubility analysis indicated that different profiles were generated as a

function of the cyclodextrin examined and the pH of the complexing medium. When

kinetic solubility measurements were completed, the cyclodextrins were found to stabilize

concentrations of Itraconazole significantly in excess of their equilibrium solubility when

supersaturated solutions were formed using the co-solvent/solvent quench approach.

These solutions were stable over 240 min falling in concentration at the 24h time point of

the experiment unlike those formed using surfactants and other polymers which

demonstrated a rapid decrease in concentration over time. These data suggest that

hydrophilic cyclodextrins might be useful formulation adjuncts in supersaturating drug

delivery systems.

M irza et al., 2011, the study has assessed the comparative abilities of Fulvic acid and

Humic acid as complexing agents. These two complexing agents have been compared on

various indices such as their abilities to cause complexation and enhance solubility,

permeability and dissolution. The present study also compared pharmacodynamic and

biochemical profiles after oral administration of complexes. With the help of various

pharmaceutical techniques such as freeze drying, physical mixture, kneading and solvent

evaporation two molar ratios (1:1 and 1:2) were selected for complexation and evaluated

for conformational analysis (molecular modelling). Complex formed was further

characterized by DSC, FT-IR, MS and XRD. Preclinical study on rodents with CBZ-HA

and CBZ-FA has yielded appreciable results in terms of their anticonvulsant and

antioxidants activities. However, CBZ-HA (1:2) demonstrated better result than any other

complex.

M irza et al., 2011, the Complexation property of Fulvic acid with Carbamazepine was

explored for the first time and different in vitro, ex vivo and pharmacodynamic parameters



were evaluated. Complexes were evaluated by DSC, FT IR, XRD, solubility analysis,

release profile study, computational method and molecular modelling. Optimized

complexes were undergone, permeation study across intestine, anticonvulsant activity

(Maximal Electro Shock) and biochemical estimations (TBARS and glutathion). These

studies confirmed the entrapment efficiency of the fulvic acid. Increased solubility

(2268.75% ), better release profile (~ 81% in 60 min), rat intestinal permeability (>3

times), biochemical and pharmacodynamic studies conferred the best to freeze dried

(ratio; 1:2) and kneading (ratio; 1:2) complexes.

M irza et al., 2010, Humic acid was used in this study to increase the dissolution,

intestinal permeation, and pharmacodynamic response of CBZ by the technique of

complexation and other related mechanism reported with humic substances. Different

complexation techniques were explored in this study for the entrapment of CBZ, which

was authenticated by molecular modelling and conformational analysis. These were

further characterized using DSC, FT- IR, and XRD. Solubility analysis and dissolution

release profile were carried out to access the in vitro parameters. Molecular modeling

approach and instrumental analysis (DSC, XRD, and FT-IR) confirmed the entrapment of

CBZ inside the complexing agent. Increased solubility (-1 7 4 2 % ), sustained release

(~78% ), better permeability (- 3 .5 times), and enhanced pharmacodynamic responses

conferred the best to 1:2 freeze dried and then 1:2 kneading complexes compared with

pure CBZ. Conclusion: Now it could be concluded that HA may be tried as a complexing

agent for antiepileptic drug and other classes of low water-soluble drug.

Aigner et al., 2012, the steps of formation of an inclusion complex produced by the co

grinding of gemfibrozil and dimethyl-P-cyclodextrin were investigated by differential

scanning calorimetry (DSC), X-ray powder diffractometry (XRPD) and Fourier transform

infrared (FTIR) spectroscopy with curve-fitting analysis. These studies demonstrated that

co-grinding is a suitable method for the complexation of gemfibrozil with dimethyl-P-

cyclodextrin. XRPD analysis revealed the amorphous state of the gemfibrozil-dimethyl-P-

cyclodextrin product. FTIR spectroscopy with curve-fitting analysis may be useful as a

semi quantitative analytical method for discriminating the molecular and amorphous states

of gemfibrozil. The endothermic peak at 59.25 °C reflecting the melting of gemfibrozil



progressively disappeared from the DSC curves of the products on increase of the duration

of c o-grinding. The crystallinity of the samples too gradually decreased, and after 35 min

of co-grinding the product was totally amorphous. Up to this co-grinding time, XRPD and

FTIR investigations indicated a linear correlation between the cyclodextrin complexation

and the co-grinding time. After co-grinding for 30 min, the ratio of complex formation did

not increase.

2.6 LITERATURE SURVEY ON HERBAL REMEDIES

In India, the Ayurvedic (indigenous) system of medicine has been in use for over 3000

years and there are more than 8000 formulations related to gynecological disorders. A

concise report on literature survey over indigenous formulations is given below (Table 5).

Even in Europe, the use of medicinal plants for symptomatic treatment of STDs dates back

at least to 1574 AC when ‘sarsaparilla’ (Smilax officinalis , family Liliaceae) was first

introduced for the treatment of syphilis. In clinical studies, sarsaparillax was observed to

be effective in about 90% cases of acute syphilis and 50% chronic cases (Mitra et al.,

1997). The most commonly used alternative therapies for yeast infection and BV is

Lactobacillus re-colonization by yogurt or capsules, boric acid douching, tea tree oil and

garlic. Even now in Europe, herbal therapies have been considered as the most popular

complementary medicine used by HIV infected individuals.

2.6.1 LITERATURE SURVEY ON TEA TREE OIL

Following are the concise result of an exhaustive literature search on tea tree oil.

Mondello et al., 2006, Oophorectomized, pseudoestrus rats under estrogen treatment were

used for experimental vaginal infection with azole (fluconazole, itraconazole) -susceptible

or -resistant strains of C. albicans. All these strains were preliminarily tested for in vitro

susceptibility to TTO, terpinen-4-ol and 1,8-cineole for their antifungal properties, using a

modification of the CLSI (formerly NCCLS) reference M27-A2 broth micro-dilution

method. In vitro minimal inhibitory concentrations (MIC90) values were 0.06% (v/v) for

terpinen-4-ol and 4% (volume/volume) for 1,8-cineole, regardless of susceptibility or

resistance of the strains to fluconazole and Itraconazole. Fungicidal concentrations of

terpinen-4-ol were equivalent to the candid-static activity. In the rat vaginal infection



model, terpinen-4-ol was as active as TTO in accelerating clearance from the vagina of all

Candida strains examined.

Reichling et al., 2006, the capability of terpinen-4-ol, the main compound of TTO, to

permeate human skin was investigated. In static Franz diffusion cells permeation

experiments with heat separated human epidermis were carried out using infinite dosing

conditions and compared to liberation experiments. No relationship between permeation

and liberation was found. The flux values of three different semisolid preparations with

5% TTO showed the rank order semisolid O/W emulsion (0.067 ^l/cm2 h) > white

petrolatum (0.051 ^l/cm2 h) > ambiphilic cream (0.022 ^l/cm2 h). In comparison to the

flux value obtained with the native TTO (0.26 ^l/cm2 h), the flux values are remarkably

reduced due to the lower amount of terpinen-4-ol. Papp values for cream (2.74 ± 0.06 • 10'7

cm/s) and native TTO (1.62 ± 0.12 X 10'7 cm/s) are comparable whereas white petrolatum

(6.36 ± 0.21X 10'7 cm/s) and semisolid O/W emulsion (8.41 ± 0.15 X 10'7 cm/s)

demonstrated higher values indicating a penetration enhancement.

Kim et al., 2009, nonionic sucrose ester microemulsions composed of sucrose laurate

(SL), propylene glycol (PG) and water were prepared with the essential oil of Melaleuca

alternifolia, commonly known as tea tree oil (TTO), as oil phase to investigate the phase

behaviour, microstructure, and antiradical activity. The pseudo-ternary phase diagrams

were constructed to elucidate the phase behaviour of the microemulsion formations at

different weight ratios of surfactant and co-surfactant (Sm = SL/PG) of 1:1, 2:1, and 3:1.

Micro structural aspects were studied by electrical conductivity and pulsed gradient spin

echo (PGSE) NMR measurements along water titration line L28 (Ro = 2:8). The results

from these combined techniques were in good agreement in regard to the microstructure

transition points. The extension of the microemulsion zone was found to be strongly

dependent on the Sm ratios. The single phase microemulsion domain, especially o/w

microemulsion region increased when Sm ratio is increased from 1:1 to 3:1 and no liquid

crystalline structure was observed for all formulations studied. The microstructural

inversion of w/o to bi-continuous micro emulsions occurred at ~30 wt. % water while the

transition from bi-continuous to o/w structure occurred at ~55 wt. % water. The physical

stability on storage temperature and time was examined by dynamic light scattering after



the centrifuge test and freeze-thaw cycles. The droplet size was kept almost the same

without any phase separation, providing less temperature-sensitivity up to 70 °C and good

stability for 3 months at room temperature.

Sudjana et al., 2012, investigated the effects of the volatile terpene-rich oil

from Melaleuca alternifolia (tea tree oil) on the formation of bio films and the adhesion

of C. albicans cells to both biotic and abiotic surfaces. Reductions in adhesion were not

due to loss of viability (at concentrations of <0.125% )or interactions between the TTO

and polystyrene. These data demonstrate that sub-inhibitory TTO reduces the adhesion

of C. albicans to both human cells and polystyrene, inhibits bio film formation and

decreases cell surface hydrophobicity. Bio film formation on polystyrene was significantly

inhibited for 70% of the isolates at the lowest test concentration of 0.016% of tea tree oil

(TTO) when quantified by XTT and 40% of isolates when measured by crystal violet

staining. Adhesion to polystyrene, quantified by crystal violet staining, was significantly

reduced for 3 isolates at 0.031%, 6 isolates at 0.062% and 0.125% and for all 7 isolates at

0.25% TTO. Similarly, adhesion to buccal epithelial and HeLa cells was also significantly

reduced in the presence of 0.016-0.062% TTO. Treatment with 0.125% TTO, but not

0.062%, decreased the cell surface hydrophobicity of C. albicans, indicating one potential

mechanism by which adhesion may be reduced.

Yeh et al., 2011, prepared alginate calcium microcapsules containing tea tree oil and

measured the release behaviour. The release rate observed at different temperatures was

almost constant with time. The data were analyzed by a theory for a model system

consisting of encapsulated coexisting liquid and vapour phases. The observed release

curves were expressed well by the theoretical equation for the release, which supports the

validity of the theory. The theory predicts a polynomial-type release which is much more

uniform than the exponential-type release appearing in conventional microcapsule systems

and regarded as roughly constant release. From the fitting of the data to the theory, the

time constant for vaporization in the core and that for permeation through the

microcapsule membrane were determined. The activation energy for permeation through

the membrane determined from the fitting parameters is much larger than that for

vaporization.



Low et al., 2013, in this study, liposome-encapsulated TTO, Ag+ and TTO plus Ag+ were

added to suspension cultures of Pseudomonas aeruginosa (Ps. aeruginosa),Staphylococcus

aureus and Candida albicans (C. albicans). Treatment of these cultures using the agents in

combination at subminimal lethal concentrations resulted in an enhanced loss of viability

compared to treatment with individual agents. The activity of alternative antimicrobial

agents such as tea tree oil (TTO) and silver ions (Ag+) with multiple target sites impedes

the development of antibacterial resistance and might be useful in improving the current

treatment strategies for various chronic wound infections. These results showed the

potential for improving antimicrobial efficacy by delivering lower effective concentrations

of alternative agents, via controlled release systems.



Review of l.iterature Ch ap ter 2

Table 5: Some ayurvedic formulations used in female genital disorders

Formulation Im portant /major plant drug English name P art(s) Im portant therapeutic usesused

Ashokghrita Asoka with 27 others Saraca asoca (Roxb.) De. Wilde Stembark

Pain in female genital tract, excessive vaginal discharge

Asokarista Asoka and 15 others Saraca asoca (Roxb.) De. Wilde Stembark

Vaginal pain, leucorrhea, menorrhagia, dysmenorrhea

Candraprabha vati Trivrt, danti, patraka and 34 others

Ipomoea turpethum R. Br., Baliospermum montanum Muell-Arg

Root,root

Menstrual pain

Darvyadi kvatha curna

Daruharidra, bhallataka and 5 others

Berberris aristate D.C., Semecarpus anacardium Linn. f.,

Stem,

fruitLeucorrhea

Goksuradi Churna Goksura Tribulus terrestris Linn. Fruit Threatened abortion

Goksuradi guggul Goksura with 9 others Tribulus terrestris Linn. Fruit Leucorrhea

Grahanimihira taila Dhanyaka and 33 others Coriandrum sativum Linn. Fruit Abortion

Kumarivatika, Kumari Pak

Kumari Aloe barbadensis Mill. Leaf pulp Dysmenorrhea, resistance in menstruation

Lodhrasava Lodhra and 30 others Symplocos racemosa Roxb. Stembark

Diseases of the uterus

Nyagrodhadi kvatha curna

Nyagrodha and 20 others Ficus benghalensis Linn. Stembark

Diseases of female genital tract

Satavari Ghrita Satavari rasa Asparagus racemosus Willd. Roottuber

Galactagogue, threatened abortion, menorrhagia

Triphala ghrta Haritaki,bibhitaka,amalaki Terminalia chebula Retz, Terminalia bellerica

Roxb.,Emblica officinalis GaertnFruit Leucorrhea



Phd Thesis 42 Jamia Hamdard

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