Exploring Drug Delivery Systems Biology Essay

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There is a swift progress in the NDDS, so as to overpower the restrictions of conventional drug delivery. Some drugs have an optimum concentration range and in the scope of this optimum range maximum benefit is derived. Some drugs can be can be toxic or produce no therapeutic benefit at all if there concentration is above or below this range. On the other hand, the very slow advancement in the efficiency of the treatment of austere diseases has suggested a growing reqirement for a multidisciplinary approach to the delivery of therapeutics to the targets in the tissues.

From this, new ideas on restraining the pharmacokinetics, pharmacodynamics, non-specific toxicity, immunogenicity, misrecognition, and efficiency of drugs were generated. This new strategy often called the drug delivery systems (DDS).The basis of DDS is the interdisciplinary approaches that involve polymer science, pharmaceutics, bioconjugate chemistry and molecular biology.

To reduce the drug degradation and loss to prevent harmful side-effects and to increase drug bioavailability ,to increase the fraction of the drug accumulated in the required zone ,many drug delivery and drug targeting systems are currently under development. Among drug carriers one can name soluble polymers, micro particles made of insoluble or biodegradable natural and synthetic polymers, liposomes, niosomes and micelles. The carriers can be caused to be slowly degradable, stimuli-reactive for e.g. it can be made pH- or temperature-sensitive and even targeted for e.g., by conjugating them with specific antibodies against certain characteristic components of the area of interest. Targeting is the ability to guide the drug-loaded system to the area of interest. Two major mechanisms can be made prominent for addressing the desired sites for drug release:

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passive

active targeting.

An example of passive targeting is the preferential accumulation of chemotherapeutic agents in the solid tumours as an outcome of the intensified vascular permeability of tumour tissues in comparison with the healthy tissue. A strategy that could allow active targeting requires the surface fictionalization of drug carriers along with ligands that are selectively acknowledged by receptors on the surface of the cells of interest. Since ligand-receptor interactions can be highly selective and hence this interaction allows a more exact targeting of the area of interest.

For developing successful formulations, controlled drug release and following biodegradation are important and potential release mechanism involves:

desorption of surface-bound /adsorbed drugs;

Diffusion through the carrier matrix.

diffusion (in the case of nanocapsules) through the carrier wall;

carrier matrix erosion;

a combined erosion /diffusion process.

The mode of delivery can be the differentiation between a drug's success and failure, as the choice of a drug is often affected by the way the medicine is administered. Sustained (or continuous) release of a drug includes the involvement of polymers that release the drug at a controlled rate due to diffusion out of polymer or either the by degradation of the polymer over time. Pulsatile release is often the preferred method of drug delivery .As pulsatile release closely copy the way by which the body naturally produces hormones such as insulin, so this release is preferred. It is accomplished by using drug-carrying polymers that react to specific stimuli (e.g., exposure to light, changes in pH or temperature).

1.2 ADVANTAGE OF NOVEL DRUG DELIVERY SYSTEM:

Reduction in the number and frequency of doses required to maintain the desired therapeutic response.

Reduce the total amount of drug administered over the period of drug treatment.

Reduced blood level oscillation characteristic of multiple dosing of conventional dosage forms.

Reduction in the incidence and severity of both local and systemic side effects in association to the high peak plasma drug concentration.

Protections from the first pass metabolism, gastro intestinal tract degradation and maximizing availability with minimum dose.

Targeting the drug molecule towards the tissue or organ leads to the reduction of the toxicity to the normal tissues and improved patient compliance.

Increased efficiency of the drug and Site specific delivery.

Reduced toxicity / side effects.

Shorter hospitalisation and increased convenience.

Workable treatments for previously incurable diseases.

Potential for prophylactic application.

Lower health care costs both short & long term.

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Preferable patient compliance.

COLLOIDAL DRUG CARRIERS

Colloidal drug carrier systems such as micellar solutions, vesicle and liquid crystal dispersions as well as the nanoparticle dispersions consisting of small particles of 10-400 nm diameter are very promising drug delivery systems (fig 1). When these formulations are developed, the goal is to obtain the systems with optimized drug loading and release properties, long shelf-life and reduced toxicity. The incorporated drug takes part in the microstructure of the system, and may even affect it due to the molecular interactions, especially if the drug has amphiphilic and/or mesogenic properties.

Micelles formed by the self-assembly of amphiphilic block copolymers (5-50 nm) in aqueous solutions are of great advantage in the case of drug delivery applications. The drugs can be physically trapped into the core of block copolymer micelles and transported at concentrations that can go farther their intrinsic water- solubility.

Liposomes are the form of vesicles that are contained of many, few or just one phospholipids bilayers. Encapsulation of polar drug molecules is possible by the polar character of the liposomal core. Amphiphilic and lipophilic molecules are solubilised in the scope of the phospholipids bilayer in accordance to their affinity towards the phospholipids. Participation of nonionic surfactants in place of phospholipids in the bilayer formation leads to the formation of noisomes.

TARGETED DRUG DELIVERY SYSTEM (TDDS)

Drug targeting is a phenomenon in which the distribution of drug in the body is in such a manner that the major part of the drug interacts solely with the target tissue at a cellular or sub cellular level. The objective of the drug targeting is to aim at a desired pharmacological response at a selected site without objectionable interactions at other sites. This is especially important in the cancer chemotherapy and enzyme replacement treatment. Drug targeting is the delivery of drugs to the receptors or any other specificied part of the body to which one desires to deliver the drug.

The targeted or site specific delivery of drugs is in fact a very attractive goal as it provides one of the most possible ways to develop the therapeutic index of the drugs.

Earlier work done between late 1960s and the mid 1980s emphasized the requirement for drug carrier systems chiefly to change the pharmacokinetics of the already proven drugs whose efficiency might be improved by altering the rates for metabolism in liver. These approaches generally were not concentrated to achieve site specific or targeted delivery such as getting a cytotoxic drug to cancerous tissue while reserving other normal, though equally sensitive tissue. A number of technological advancements have since been made in the area of parenteral drug delivery leading to the progress of the sophisticated systems. This system allows drug targeting and the well maintained or controlled release of parenteral medicines.

At present drug targeting is accomplished by one or two approaches:

The first approach involves chemical modification of the parent compound to a derivative which is activated only at the target site.

The second approach uses carriers such as liposomes, niosomes, microspheres, nano particles, antibodies, cellular carriers (erythrocytes and lymphocytes) and macromolecules to direct drug to its site of action.

Recent promotion have led to the progress of many novel drug delivery system that could cause fundamental change in the method of medication and make available a number of therapeutic benefits.

The goal of any drug delivery system is to supply a therapeutic amount of drug to the proper site in the body to achieve without delay, and then maintain the required drug constant. The ideal drug delivery system delivers drug at a rate commanded by the requirement of the body over the period of treatment and it channels the active entity solely to the area of action.

Paul Ehrlich in 1906 began the era of development for targeted delivery when he pictured in mind a drug delivery mechanism that would target drugs directly to diseased cells. Numbers of carriers were used to carry drug at the target organ / tissue which include immunoglobulin, serum proteins, synthetic polymers, lipid vesicles (liposomes), microspheres, erythrocytes, reverse micelles, niosomes, pharmacosomes etc. Among the various carriers, few drug carriers reached the stage of clinical trials where phospholipids vesicle exhibit strong potential for effective drug delivery at the site of action. These carriers (liposomes) are biologically inert in nature, lacking in any antigenic, pyrogenic or allergic reactions .The components of carriers can be used as the component of biological membrane. Drugs incorporated in liposomes are not activated under physiological conditions and do not cause unfavourable side effects as well.

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There are various techniques by which drug can be targeted include (Brahmankar et al., 2001)

Nanoparticles.

Niosomes.

Resealed erythrocytes.

Microspheres.

Monoclonal antibodies.

Liposomes.

1.4.1. MERITS OF TDDS

Targeting of the drug molecule towards the tissue or organ reduces the toxicity to the normal tissues.

Increased bioavailability.

Improved treatment of chronic illness where symptoms break through occurs when the plasma level of the drug falls below the MEC.

The drug is protected from first pass metabolism and GI degradation.

Improved patient compliance can be achieved due to decrease in amount and frequency of doses administered.

Biocompatibility can be well achieved.

Maintenance of therapeutic action of the drug overnight.

Systemic and local side effects are successfully reduced due to the reduction in the total amount of the dru

1.4.2. LIMITATIONS OF TDDS

TDDS such as liposomes, resealed erythrocytes and platelets suffer serious stability problems.

Though monoclonal antibodies show very high degree of site specificity the selection and isolation procedures are too tough.

If the particle size of TDDS is high then they may be rapidly cleared by RES.

Magnetically controlled TDDS shows high specificity to superficially located organs and tissues but cannot be targeted to deep seated organs.

Monoclonal antibodies may sometimes can cause unwanted antigen - antibody reaction which leads to serious consequences.

Microspheres of particle size more than 50µg can lead to problem of thrombo­embolism in general circulation.

Once drug is administered it cannot be removed if an undesirable action is precipitated or if the drug is no longer needed.

Most of such systems are administered by subcutaneous or intraperitoneal route.

The vehicles polymer employed should be sterile, hydrogen free, non irritating, biocompatible and biodegradable into non toxic compounds within an appropriate time preferably close to duration of action.

Drugs having biological half life of 1hr or less are difficult to formulate as controlled release formulation. The high rates of elimination of such drugs from the body need an extremely large maintenance dose which provides 8 - 12 hrs of continuous therapy.

Niosomes -

Niosomes or non-ionic surfactant vesicles are microscopic lamellar structures formed on admixture of non-ionic surfactant of the alkyl, dialkyl polyglycerol ether class and cholesterol with the subsequent hydration in the aqueous media (3).

Niosomes are formed from self-assembly of the non-ionic amphiphiles in the aqueous media out coming in the closed bilayer structures (Fig. 1).

The assembly of the amphiphiles into closed bilayers is rarely spontaneous. It also involves

some input of energy such as physical agitation or heat. An assembly in which the hydrophobic

parts of the molecule are shielded from the aqueous solvent is the result. As a result the

hydrophilic head groups enjoy the maximum contact with same. These structures are analogous

to liposomes. These structures are able to encapsulate the aqueous solutes. These structures serve

as the drug carriers.

The major advantages of Niosomes as drug carriers includes

The vesicle suspension is water-based and it offers high patient compliance in comparison with the oily dosage forms.

Niosomes possess an infrastructure consisting of hydrophilic, amphiphilic and lipophilic moieties together. As a result of this infrastructure it can accommodate drug molecules with a wide range of solubilities.

The characteristics of vesicle formulation are variable and controllable. Changing vesicle composition, size, lamellarity, tapped volume, surface charge and concentration can control the vesicle characteristics.

The vesicles may act as a depot and hence releasing the drug in a controlled manner.

They are osmotically active and stable. As a result they increase the stability of entrapped drug.

As surfactant requires no special conditions, they are easy for handling and storage.

They improve the oral bioavailability of poorly absorbed drugs and hence improve skin penetration of drugs.

They can be made to reach the area of action by the means of oral, parenteral as well as topical routes.

The surfactants are biodegradable, biocompatible as well as non-immunogenic.

They improve therapeutic performance of the drug molecules by delayed clearance from the circulation. Hence protecting the drug from biological environment and restricting effects to target cells.

Niosomal dispersion in an aqueous phase can be emulsified in a non-aqueous phase. This emulsification is done to regulate the delivery rate of drug and administer normal vesicle in external non-aqueous phase.

METHOD OF PREPARATION OF NIOSOMES

Based on the method used for the production and composition of bilayer, niosomes show a wide variation in their properties. Basis of the preparation of noisome is liposome technology. The basic process of preparation is the same i.e. hydration by aqueous phase of the lipid phase which may be either a pure surfactant or a mixture of surfactant with cholesterol. The bioactive material is dissolved in the aqueous phase/organic phase. This is the bioactive material which is entrapped. The different methods used for preparation of niosome are listed as follows:

Ether Injection method

Lipid Film Formation (Hand Shaking Method)

Sonication Method

Micro fluidization

Reverse phase evaporation

Trans membrane pH gradient (inside acidic) Drug Uptake Process (remote Loading)

Multiple membrane extrusion method

The "Bubble" Method.

Formation of niosomes from proniosomes

Method of preparation

1. Ether injection method

In this method, the niosomes are slowly introduced in a solution of surfactant dissolved in diethyl ether into warm water maintained at 60°C. The surfactant mixture in ether is injected through 14-gauge needle into an aqueous solution of material. The ether vaporizes and its vaporization leads to formation of single layered vesicles. The diameter of the vesicle range from 50 to 1000 nm. This range of the diameter depends upon the conditions being used during this method.

2. Hand shaking method (Thin film hydration technique)

The mixture of vesicles forms ingredients like surfactant and cholesterol. This mixture is a dissolved in a volatile organic solvent (diethyl ether, chloroform or methanol) in a round bottom flask. The organic solvent is removed at room temperature (20°C) using rotary evaporator. On removal of the organic solvent a thin layer of solid mixture is left deposited on the wall of the flask. Rehydration of the dried surfactant film is done with gentle agitation with aqueous phase at 0-60°C.This process forms typical multilamellar niosomes.

Raja Naresh et al /i>(15) prepared thermosensitive niosomes by evaporation of organic solvent at 60°C and a thin film of lipid was left on the wall of rotary flash evaporator resulting from the process of evaporation. The aqueous phase which contains drug was added slowly with alternate starting and stopping of the shaking of flask at room temperature. This procedure is followed by sonication.

3. Sonication

The vesicles are produced typically by the method of sonication of solution as described by Cable. In this method an aliquot of the solution of drug in buffer is added to the surfactant/cholesterol mixture in a 10-ml glass vial. The mixture is probe sonicated at 60°C for 3 minutes .This sonication is done using a sonicator with a titanium probe to yield niosomes.

4. Micro fluidization

Micro fluidization is a latest technique which is used to prepare unilamellar vesicles of definite sized distribution. Basis of this method is submerged jet principle. According to this principle the two fluidized streams interact at ultra high velocities, in micro channels which are very precisely defined and this interaction takes place within the interaction chamber. The arrangement of the impingement of the thin liquid sheet along a common front is in such a way that the energy supplied to the system does not cross the area of niosomes formation and is within it. As a result the niosomes formed have greater uniformity, small size and better reproducibility.

5. Multiple membrane extrusion method

The surfactant, cholesterol and dicetyl phosphate are mixed in chloroform and made into a thin film by the evaporating this mixture. Aqueous drug polycarbonate membranes, solution is used to hydrate the film and the extrusion product i.e. resultant suspension is placed in the series of up to 8 passages. This is a good method for controlling the size of niosome.

6. Reverse Phase Evaporation Technique (REV)

Cholesterol and surfactant in the ratio 1:1 are dissolved in a mixture of ether and chloroform. An aqueous phase containing drug is added to this. The resulting two phases are sonicated at 4-5°C. After the addition of a small amount of phosphate buffered saline (PBS), the formed clear gel is sonicated further. The organic phase is removed at 40°C under low pressure. The resulting viscous niosome suspension is diluted with PBS. And then it is heated on a water bath at 60°C for 10 min so as to yield niosomes.

7. Trans membrane pH gradient (inside acidic) Drug Uptake Process (remote Loading)

Surfactant and cholesterol are dissolved in chloroform. The solvent then undergoes evaporation under reduced pressure. This results to a thin film on the wall of the round bottom flask. The film is hydrated with 300 mM citric acid (pH 4.0) by vortex mixing. The multilamellar vesicles are frozen .After freezing it is thawed 3 times and later sonicated. To this niosomal suspension, aqueous solution containing 10 mg/ml of drug is added. Then this suspension is vortexed. The pH of the sample is then raised to 7.0-7.2 with 1M disodium phosphate. This mixture is later heated at 60°C for 10 minutes to result to give niosomes.

8. The "Bubble" Method

It is novel technique for the one step preparation of liposomes and niosomes. In this method organic solvents are not used. The bubbling unit consists of round-bottomed flask with three necks positioned in water bath to control the temperature. Water-cooled reflux and thermometer is positioned in the first and second neck and nitrogen supply through the third neck. Cholesterol and surfactant are dispersed together in this buffer (pH 7.4) at 70°C. The dispersion is mixed for 15 seconds with high shear homogenizer. Then immediately afterwards "bubbled" at 70°C using nitrogen gas.

9. Formation of niosomes from proniosomes

Another method of producing niosomes is to coat a water-soluble carrier such as sorbitol with surfactant. The product of the coating process is a dry formulation. In this formulation each water-soluble particle is covered with a thin film of dry surfactant (fig c).This preparation is termed as "Proniosomes". The niosomes are recognized by the adding of aqueous phase at T > Tm .It is followed by brief agitation.

T=Temperature.

Tm = mean phase transition temperature.

Fig c: Niosome from proniosome

CHARACTERIZATION OF NIOSOMES -

a)Entrapment efficiency

After preparing the niosomal dispersion, unentrapped drug is separated by either the method of dialysis [15] entrifugation [17-18] or gel filtration [19] as described above and the drug remained entrapped in niosomes is determined by the complete vesicle disruption using 50% n-propanol or 0.1% Triton X-100.The resultant solution is analysed by the appropriate assay method for the drug. Where entrapment efficiency (EF) = (Amount entrapped total amount) x 100

b) Vesicle diameter

Niosomes, similar to liposomes, assume spherical shape. Such niosomes's diameter can be determined using either light microscopy or photon correlation microscopy or freeze fracture electron microscopy. Freeze thawing (keeping vesicles suspension at -20°C for 24 hrs and then heating to ambient temperature) of niosomes increases the vesicle diameter. This result might be attributed to fusion of vesicles during the cycle.

c) In-vitro release

A method of in-vitro release rate study includes the use of dialysis tubing. A dialysis sac is washed. This sac is soaked in distilled water. The vesicle suspension is pipetted into a bag. The bag is made up of the tubing and sealed. The bag containing the vesicles is placed in 200 ml of buffer solution in a 250 ml beaker with constant shaking at 25°C or 37°C. At various time intervals, the buffer is analyzed for the drug content by an appropriate assay method [17] .

d) Osmotic Shrinkage

Osmotic shrinkage of vesicles can be determined by monitoring reductions in vesicle diameter. This method is initiated by the addition of hypertonic salt solution to the suspension of niosomes. Niosomes prepared from pure surfactant are osmotically more sensitive in comparison to vesicles containing cholesterol.

e) Physical stability of vesicles at different temperature

Aggregation or fusion of vesicles as a function of temperature was determined as the changes in vesicle diameter by laser light scattering method. The vesicles were stored in glass vials at room temperature or kept in refrigerator (4oC) for 3 months. The changes in morphology of multilamellar vesicles (MLVs) and also the constituent separation were assessed by an optical microscope. The retention of entrapped drug was measured 72 hours after preparation. It is again measured after 1, 2 or 3 months in same formulations

f) Turbidity Measurement

The niosomes were diluted with bidistilled water to give total lipid concentration of 0.312 mM.After rapid mixing by sonication for 5 min; the turbidity was measured as the absorbance with an ultraviolet-visible diode array spectrophotometer.

APPLICATIONS

Niosomal drug delivery is potentially applicable to many of the pharmacological agents for their action against various diseases. Some of their therapeutic applications are discussed below.

Targeting of bioactive agents

To reticulo-endothelial system (RES)

The cells of RES preferentially take up the vesicles. The uptake of niosomes by the cells is by circulating serum factors known as opsonin.This factor which mark them for clearance. Such localized drug accumulation has nevertheless been exploited in the treatment of animal tumors known to metastasize to the liver, spleen and in parasitic infestation of liver.

To organs other than RES

It has been suggested that carrier system can be directed to specific areas in the body by use of antibodies. Immunoglobulins seem to bind quite readily to the lipid surface and hence offering a convenient means for targeting of drug carrier. Many cells possess the intrinsic ability to recognize and bind particular carbohydrate determinants. This can be exploited to direct carriers system to particular cells.

2) Neoplasia

The anthracyclic antibiotic named doxorubicin has broad spectrum anti tumor activity.This antibiotic shows an irreversible cardio toxic effect. This effect is dose dependent. The niosomal delivery of this drug to mice bearing S-180 tumor lead to the growth in their life span. This delivery reduced the rate of proliferation of sarcoma. The entrapment of the niosome increased the half-life of the drug and extends duration of its circulation, hence lead to alteration in its metabolism. The administration of the methotrexate intravenously entrapped in the niosomes to S-180 tumor bearing mice lead to the total regression of tumor ,higher plasma level and slower elimination.

3) Leishmaniasis

In the treatment of diseases where the infecting organism dwells in the organ of reticulo-endothelial system, niosomes can be used for targeting the drug. A disease in which the parasite enters and causes harm to cells of liver and spleen is Leishmaniasis. The commonly prescribed drugs are antimonials, which have relation with arsenic. The antimonials at high concentration will affect and damage heart, liver and kidney.

4) Delivery of peptide drugs

When oral delivery of 9-desglycinamide, 8-arginine vasopressin is entrapped into the niosomes in an in-vitro intestinal loop model, it was reported that the peptide's stability increased consequently.

5) Immunological application of niosomes

The nature of the immune response aroused by the antigens has been studied using the niosomes. In terms of the immunological selectivity, low toxic level and stability the niosomes act as potent adjuvant.

6) Niosomes as carriers for Hemoglobin.

Niosomes can act as a carrier for hemoglobin. The suspension of niosomes points a visible spectrum super imposable to the free hemoglobin. The vesicles are permeable to oxygen and dissociation curve for hemoglobin can be moderated in the similar manner as to non-encapsulated hemoglobin.

7) Transdermal delivery of drugs by niosomes

The slow penetration of the drug via skin is the major disadvantage of the transdermal route of delivery. A growth in penetration rate has been accomplished by the transdermal delivery of drug included as a part in niosomes. Niosomes on hairless mouse are the various formulations based on the topical delivery of erythromycin. As per the studies and the confocal microscopy, it was found that non-ionic vesicles could be devised to target the pilosebaceous glands.

8) Other Applications

Sustained Release

The liver plays the role of depot for methotrexate, after niosomes the role of depot for metotexate is played by the liver cells. The released action of niosomes is applicable to the drugs with low therapeutic index and having low water solubility since these conditions for the drug can be maintained in the circulation through the encapsulation of niosome.

Localized Drug Action

The delivery of drug through niosomes is one of the approaches to accomplish the localized drug action, since the size and low penetrability of the niosomes through the epithelium and connective tissue keeps the drug confined at the area of administration.

The localized drug action results in the improvement of the effectiveness of the potency of the drug and at the corresponding time it decreases its systemic toxic effects e.g. Antimonials encapsulated within niosomes are received up by the mononuclear cells which results in the localization of the drug, increase in potency and therefore reduce in dose as well as toxicity.

PRONIOSOMES - A STABLE PRECURSOR FOR NIOSOMES

Proniosomes offer a variety vesicle drug delivery concept with latent ability for delivery of drugs (Gupta et al., 2007).These dry niosomes is dry free flowing; granular product upon addition of water. The dry niosomes disperses or dissolves to form a suspension of multilamellar niosomes.

Preparation of proniosomes

There are many components present in proniosomes with non -ionic surfactants and cholesterol, among them lecithin is the main ingredient. The required characteristics of the selected carrier that could be used in the preparation of proniosomes were formally speeched by Payne et al. (2008).These include: safety and non-toxicity, free flowability,poor solubility in the loaded mixture solution and good water solubility for easy hydration (Abd-Elbary et al.,2008). In the proniosome preparation, different carriers and non ionic surfactants and membrane stabilizers are used. As shown in table 1. Three different methods were reported for the preparations of proniosomes .They are:

Slurry method: Maltodextrin powder 10 g as carrier is added to a 250-mL round-bottom flask and the whole volume of the surfactant solution (14.5 mL) was mixed directly to the flask to form slurry. If the surfactant solution volume is less, then some extra amount of organic solvent can be mixed to get slurry. The flask was attached to the rotary evaporator and vacuum was applied until the powder appears to be dry and having free flow. The flask was removed from the evaporator and kept under vacuum overnight. Proniosome powder was stored in the sealed containers at 4°C. The time needed to produce proniosomes is not dependent on the ratio of surfactant solution to carrier material and appear to be scalable (Blazek-Welsh and Rhodes, 2001a and b; Solanki et al., 2007 and Perrett et al., 1991).

Coacervation phase separation method: This method is widely adopted to formulate proniosomal gel. In a clean and dry wide mouthed glass vial of 5.0 ml capacity exactly weighed amounts of surfactant, lipid and drug are taken and alcohol (0.5 ml) is added to it. After warming, all the ingredients are mixed well with a glass rod. Then the open end of the glass bottle is covered with a lid to prevent the loss of solvent from it and then it is warmed over water bath at 60-70°C for about 5 min until the surfactant mixture is dissolved completely. Then the aqueous phase (0.1% glycerol solution) is added and warmed on a water bath till a clear solution was formed which then gets converted into proniosomal gel when it is cooled (Vora et al.,1998 and Gupta et al., 2007).

Slow spray-coating method: This method includes the formulation of proniosomes by spraying surfactant in the organic solvent onto sorbitol powder and then evaporating the solvent. It is important to repeat the process until the desired surfactant loading has been achieved because the sorbitol carrier is soluble in the organic solvent. The surfactant coating on the carrier is very thin and hence hydration of this coating will allow multilamellar vesicles to form as the carrier dissolves (Bangham et al., 1965 and Yoshioka et al., 1994). The resulting niosomes are very similar to those produced by the conventional methods. The resulting niosomes's size distribution is much uniform. It is suggested that this preparation could provide a suitable method for formulating hydrophobic drugs in a lipid suspension without any trouble over instability of the suspension or susceptibility of the active ingredient to hydrolysis (Hu and David G.Rhodes, 1999). This method was reported to be tiresome since the sorbitol carrier for formulating proniosomes is soluble in the solvent used to deposit the surfactant. Sorbitol is also found to interfere with the encapsulation of certain drugs.

Anatomy of the hair follicle

Hair is the keratinized product that grows from the hair follicle, which is a tube-like structure continuous with the epidermis at its upper end. The follicles are comparatively angled in the dermis, and longer follicles extend in to the subcutaneous layer. An obliquely shaped muscle, the arrector pili, runs from the mid-region of the follicle wall to a point in the papillary dermis close to the junction of the dermis and epidermis. Above the muscle, one or more sebaceous glands, and an apocrine gland (in some parts of the body) open into the follicle. The fibre in the hair is made up of three cell layers namely an outer cuticle, the cortex (which forms the bulk of the fibre in most hair types) and a variable central medulla, all of which are derived from cells that are highly proliferative in the hair bulb at the base of the follicle. Cells in the hair bulb also give rise to the inner root sheath which surrounds the hair fibre and this sheath disintegrates before the hair emerges from the skin. The inner root sheath is again enclosed by the outer root sheath, which is a continuous structure extending right from the hair bulb to the epidermis, although the functions and microscopic structure of the outer root sheath usually vary throughout the length of the follicle.

The hair follicle has a specialized dermal component, which includes the dermal or connective tissue sheath surrounding the follicle, and the dermal papilla which invaginates the hair bulb.

The hair follicle is divided into two regions: the upper part of the hair follicle consisting of the infundibulum and isthmus and the lower part consisting of the hair bulb and suprabulbar region. The upper follicle is a relatively constant structure, whereas the lower follicle undergoes continuous episodes of regression and regeneration during the hair cycle.

On the scalp, and some other regions of the skin, hair follicles are arranged in groups of three or more follicles which are known as the follicular units (Fig. 2). Several follicles that are contained within a follicular unit may merge so that hairs emerge out through a common infundibulum.

The hair cycle (Fig. 3)

Hair follicles undergo a continuous repetitive sequence of growth and rest which is known as the hair cycle. The timing of the phases of the hair cycle and its overall duration differs between species, follicles in different regions of the skin in the same species and, in some animals, different follicle types, such as guard hairs and under hairs, in the same region of the skin.

The phase of active hair growth is known as anagen and the duration of this phase is responsible for determining the final length of the hair.

The hair cycle is constituted by three phases viz, anagen (growth phase), catagen (involutional phase) and the telogen (resting phase). The Anagen phase lasts for 3-5 years, catagen phase for 2 weeks and the telogen phase lasts for a time period of 3 months. Thus the anagen

to telogen hair ratio is usually in the order of 12:1. Hair shedding (exogen) occurs in the telogen phase and the sub-phase of telogen that follows shedding is called the latent phase.

Androgenetic alopecia

This type of Common baldness is the result of a progressive, hair loss that has a defined pattern and occurs when individuals with genetic predisposition are exposed to androgens. The lack of balding in eunuchs, pseudo hermaphrodites and individuals with insensitivity syndrome to androgens confirms the fact that androgens are a prerequisite for common baldness. As there is a difference in the pattern of hair loss in men and women, the terms to differentiate them i.e. male pattern hair loss and female pattern hair loss are commonly in use. Considering a person bald, and particularly prematurely bald, depends purely on subjective assessment. The process by which common baldness occurs is androgen-mediated conversion of susceptible terminal hairs into vellus hairs, and has been termed androgenetic alopecia (AGA).

Aetiology: There are four separate but interrelated factors that determine whether an individual will become bald and those factors are: susceptibility to AGA, age of onset of baldness, rate of progression and the pattern of hair loss. Among men, susceptibility to AGA is the universal factor, and the other three factors explain the variations in baldness.

Hamilton [2] defined the progressive pattern of male baldness and produced the first useful scale for grading of baldness. This classification was further modified by Norwood [3], who added grades Illa, III vertex, IVa and Va (Fig. 66.14). Although the grades are imprecise measures for hair loss patterns that are seen in adult males, they are useful as diagnostic aids and in the classification of extent of hair loss in clinical investigations.

The lifetime risk of MPB can be defined by studying hair patterns in men aged 80 years and above. In Norwood's cohort, 16% had a type I hair pattern, and by definition are not bald despite many demonstrating at least some degree of bitemporal AGA. Fourteen per cent had a type 2 hair pattern, with a moderate degree of bitemporal AGA, but would not be considered bald. The remaining 70%had a type III (16%), IV (12%), V (12%), VI (13%) or VII (17%) pattern of hair loss and would be considered to be balding.

The age of onset of MPB varies widely. If early MPB is defined as stage II pattern, then 40% of men begin to develop MPB between the ages of 18 and 29 years, and a further 24% first develop MPB in their thirties, 3% in their forties, 5% in their fifties, 9% in their sixties, 2% in their seventies and 1% at or beyond the age of 80 years.

The word androgenetic encompasses the two dominant causal factors, namely genetic susceptibility and androgens.

Genetic susceptibility

A familial tendency to androgenetic alopecia is well recognized as clearly as race variation [1, 2]. Genetic factors modify the magnitude of response to circulating androgens given by the follicle. Those with a stronger predisposition begin to bald in their teens, and those with a weak predisposition may not bald until they are in their 60s or 70s. As few as than 15% of men have little or no baldness by the age of 70 [3]. Osborne, in 1916 suggested that the baldness gene acts as an autosomal dominant manner in men and an autosomal recessive fashion in women [4].Androgenetic alopecia is polygenic.

Current modelling suggests involvement of at least four genes that act combindly to modify the age of onset, pattern and rate of progression of androgenetic alopecia [1]. The other candidate gene and chromosomal areas have been examined which include SRDA1 and SRDA5 genes coding for the two variants of the 5α-reductaseenzymes [7], the insulin gene [8], the aromatase gene, the gene for the Era oestrogen receptor, the non-recombinant area of the Y chromosome, and the type II insulin-like growth factor genes.[19 ..Book...... α α α α α α α α α α α ααα

Hormonal influences

Systemic hormonal effects-

Androgenetic alopecia is induced by activation of the androgenic receptors in the follicle by dihydrotestosterone. Increase in dihydrotestosterone levels have been found in balding scalp in comparision with non-balding scalp [13] and androgen receptors have been found in hair follicle. However, the specific mechanism of the androgen effect is not known.

Intrafollicular over activity may be due to the local or systemic factors. Possible local factors are increased number of androgen receptors, functional changes of the androgen receptor, increased production of dihydrotestosterone, or reduced degradation of dihydrotestosterone. Systemic factors are increase in circulating androgens that provide increased substrate for the conversion to dihydrotestosterone, or increase in systemic production of dihydrotestosterone at distant sites such as the prostate gland.

Dihydrotestosterone binds the androgen receptor with five times the affinity of testosterone and is more potent in causing downstream activation. Two 5α-reductase isoenzymes have been characterized, based on their different pH optima namely Type 1 5 α -reductase & Type 2 5 α -reductase.

The type 2 enzymes has been found by immunohistochemistry to be in the dermal papilla, the inner layer of sheath, the sebaceous ducts and proximal inner root sheath of scalp hair follicles [17].

Local hormonal effects-

Beard dermal papilla cells secrete growth-inducing autocrine factors in response to testosterone, leading to an increase in dermal papilla size and enlargement of the hair follicle. This response is not seen in occipital scalp hair follicles [20, 22]. Insulin-like growth factor- 1 has been identified as a major component of secreted cytokines [23]. Similar investigations on dermal papilla cells from the balding scalp of the stump-tailed macaque show that testosterone inhibited the growth and proliferation of keratinocytes [24].

Studies done for examining the distribution and expression of androgen receptors have shown varying results. Two studies showed that androgen receptors are only found in the nuclei of dermal papilla cells [20, 25]. Another study showed extensive distribution of receptors including the hair bulb [26]. Considering different anatomical sites, higher numbers of androgen receptors are seen in the pubic hair follicles and beard dermal papilla cells, with lower levels of occipital scalp follicles [27]

Scalp hair loss progresses in an orderly and reproducible pattern, and is a dependent of factors particular to each hair follicle. Laboratory studies have shown that the hair follicles are able to self-regulate their response to androgens by regulating the expression of 5α-reductase and androgen receptors [27-29]. This self-regulation is created to produce the accountable difference in androgen receptor numbers [27,30] and enzyme activity [28,31] that is observed between balding and non-balding areas. This is best seen in hair transplantation experiments: occipital hairs maintain their resistance to androgenetic alopecia when transplanted to the vertex, and scalp hairs from the vertex transplanted to the forearm miniaturise at the same pace as hairs surrounding the donor site [32].

Pathogenesis

The three important features of pathogenesis of androgenetic alopecia are alteration of hair cycle dynamics, follicular miniaturisation and inflammation.

Hair cycle dynamics

The active growth to resting phase hair count is usually in the order of 12:1. Hair shedding (exogen) occurs within the telogen phase and the sub-phase of telogen that follows exogen is called the latent phase [34]. In androgenetic alopecia, the duration of anagen is decreased as the cycle progresses, whereas resting phase duration remains constant or is increased. This leads to a reduction of the anagen to telogen ratio [1]. Balding patients often face periods of excessive hair shedding, most noticeable while combing or washing. This is due to the relative increase in the number of follicles in telogen phase. Thus with each successively foreshortened hair cycle, the length of each hair shaft is reduced. Ultimately the duration of anagen becomes so short that the growing hair does not attain sufficient length to reach up to the surface of the skin, which leaves an empty follicular pore. In androgenetic alopecia, the latent phase is prolonged, reducing hair numbers, further contributing to the balding process [34].

Hair follicle miniaturisation

Along with the changes in hair cycle dynamics, there is progressive, stepwise miniaturisation of the entire follicle (Figure 2). As the dermal papilla plays a major role in the maintenance and control of hair growth, it has the highest possibility to be the target of androgen-mediated events leading to miniaturisation and hair cycle changes [35-37].

There constant geometric relation between the dermal papilla size and the size of the hair matrix which [38] suggests that the size of the dermal papilla is the parameter that determines the size of the hair bulb and the shaft thus produced [39]. A tenfold fall in overall cell numbers is likely to cause decrease in hair follicular size [40]. The mechanism for which remains unexplained, and may be due to apoptotic cell death, reduction in proliferation of keratinocytes [41], cell displacement with loss of adhesion of cell leading to dermal papilla fibroblasts dropping off into the dermis, or migration of dermal papilla cells into the dermal sheath associated with the outer root sheath of the hair follicle [39].

Inflammation

In 40% of cases of androgenetic alopecia, a moderate perifollicular, lymphohistiocytic infiltrate, perhaps with concentric layers of perifollicular collagen deposition is present. Occasional eosinophils and mast cells can be seen.

In some cases, the cellular inflammatory changes also occur around lower follicles and occasionally involve follicular stellae. The diagnostic and prognostic significance of the degree of the inflammation is not known [44].

Management of male pattern baldness -

1.Minoxidil lotion. Minoxidil is a piperidinopyrimidine derivative and a potent vasodilator that is effective orally for severe hypertension. When applied topically in a lotion or other form, minoxidil was found to increases terminal hair density in up to 30% of individuals. Terminal hair regrowth at the margins were appeared, but complete covering of the bald areas was seen in less than 10% of responders. De Villez suggested that people who responded best to minoxidil treatement were those in whom the balding process was at an early stage i.e., with a maximum diameter of the bald area of less than 10 cm2. . The benefit is most pronounced in the first six months of therapy and thereafter is marginal.

Topical minoxidil appears to be a safe therapy with less side effects which includes local irritation and hypertrichosis of the temples, and there is very low incidence of contact dermatitis. Once the treatment is stopped, clinical regression occurs within 6 months, to the state of baldness that would have existed if treatment had not been applied. Patients should be warned very well that in order to maintain any beneficial effects, applications must continue twice daily for the rest of their lives.

Finasteride. Finasteride is a synthetic aza-steroid which is a potent and highly selective antagonist of 5α-reductase type 2. It inhibits the conversion of testosterone to dihydrotestosterone (DHT), which is a more powerful androgen than testosterone (as it has a much higher affinity for the androgen receptor). By inhibiting this enzyme, finasteride blocks the conversion of testosterone into the more powerful androgen DHT. This reduces androgenic activity in the scalp, thus treating hair loss at its hormonal source .A scalp biopsy study of patients with AGA found that after 12 months of finasteride treatment, terminal hair counts increase and vellus hair counts decrease, proving the ability of finasteride to reverse the miniaturization process and to encourage the growth of terminal hairs. An oral dosage of 1 mg/ day has found to reduce the scalp DHT by 64% and serum DHT by 68%. Clinical trial data proved that, after 1 year, patients on finasteride have a 10% increase in the mean number of terminal hairs compared with baseline counts.