RESEALED ERYTHROCYTES

INTRODUCTION:
Erythrocytes, the most abundant cells in the human body, have potential carrier capabilities for the delivery of drugs. Erythrocytes are bio compatible, biodegradable, possess very long circulation half lives and can be loaded with a variety of chemically and biologically active compounds using various chemical and physical methods. Application of erythrocytes as promising slow drug release or site-targeted delivery systems for a variety of bioactive agents from different fields of therapy has gained a remarkable degree of interest in recent years. Biopharmaceuticals are among the most widely exploited candidates for being delivered to the host body using these cellular carriers. In this review, the potential applications of erythrocytes in drug delivery have been highlighted.

ISOLATION OF ERYTHROCYTES: 

Various types of mammalian erythrocytes have been used for drug delivery, including erythrocytes of mice, cattle, pigs, dogs, sheep, goats, monkeys, chicken, rats, and rabbits. To isolate erythrocytes, blood is collected in heparinized tubes by venipuncture. Fresh whole blood is typically used for loading purposes because the encapsulation efficiency of the erythrocytes isolated from fresh blood is higher than that of the aged blood. To isolate erythrocytes, blood is collected in heparinized tubes by venipuncture.Fresh whole blood is typically used for loading purposes because the encapsulation efficiency of the erythrocytes isolated from fresh blood is higher than that of the aged blood. Fresh whole blood is the blood that is collected and immediately chilled to 4⁰c and stored for less than two days. The erythrocytes are then harvested and washed by centrifugation. The washed cells are suspended in buffer solutions at various hematocrit values as desired and are often stored in acid–citrate–dextrose buffer at  4⁰ c as long as 48 h before use. Jain and Vyas have described a well-established protocol for the isolation of erythrocytes. The loading of drugs in erythrocytes was reported separately by Ihler et al. and Zimmermann. In 1979, the term carrier erythrocytes were coined to describe drug-loaded erythrocytes.

Advantages and disadvantages of erythrocytes in drug delivery Advantages

Some of the most important advantages encouraging the use of erythrocytes in

drug delivery include:

• A remarkable degree of biocompatibility, particularly when the autologous cells are used for drug loading.
• Complete biodegradability and the lack of toxic product(s) resulting from the carrier   biodegradation.
• Avoidance of any undesired immune responses against the encapsulated drug.
• Considerable protection of the organism against the toxic effects of the encapsulated drug, e.g. antineoplasms.
• Remarkably longer life-span of the carrier erythrocytes in circulation in comparison to the 
• synthetic carriers. In the optimum condition of the loading procedure, the life-span of the 
• resulting carrier cells may be comparable to that of the normal erythrocytes.
• An easily controllable life-span within a wide range from minutes to months.
•  Desirable size range and the considerably uniform size and shape.
•  Protection of the loaded compound from inactivation by the endogenous factors.
•  Possibility of targeted drug delivery to the RES organs.
•  Relatively inert intracellular environment.
•  Availability of knowledge, techniques, and facilities for handling, transfusion, and working
• with erythrocytes. 
• Possibility of ideal zero-order kinetics of drug release.
•  Wide variety of compounds with the capability of being entrapped within the erythrocytes.
•  Possibility of loading a relatively high amount of drug in a small volume of erythrocytes,
• which, in turn, assures the dose sufficiency in clinical as well as animal studies using a limited
• volume of erythrocyte samples.
• Modification of the pharmacokinetic and pharmacodynamic parameters of the drug.
• Remarkable decrease in concentration fluctuations in steady state in comparison to the
•  conventional methods of drug administration, which is a common advantage for most of the novel drug delivery systems.
• Considerable increase in drug dosing intervals with drug concentration in the safe and effective level for a relatively long time.
• Possibility of decreasing drug side effects.

Drawbacks:

The use of erythrocytes as carrier systems also presents some disadvantages, which can be

summarized as follows:

• The major problem encountered in the use of biodegradable materials or natural cells as drug carriers is that they are removed in vivo by the RES as result of modification that occurred during loading procedure in cells. This, although expands the capability to drug targeting to RES, seriously limits their life-span as long-circulating drug carriers in circulation and, in some cases, may pose toxicological problems.
• Te rapid leakage of certain encapsulated substances from the loaded erythrocytes.
• Several molecules may alter the physiology of the erythrocyte.
• Given that they are carriers of biological origin, encapsulated erythrocytes may present some inherent variations in their loading and characteristics compared to other carrier systems.
• The storage of the loaded erythrocytes is a further problem provided that there are viable cells and need to survive in circulation for a long time upon re-entry to the host body.
• Conditioning carrier cells in isotonic buffers containing all essential nutrients, as well as in low temperatures, the addition of nucleosides or chelators, lyophilization with glycerol or gel immobilization have all been exploited to overcome this problem. 
• Possible contamination due to the origin of the blood, the equipment used and the loading environment. Rigorous controls are required accordingly for the collection and handling of the erythrocytes.

METHODS OF DRUG LOADING:

Several methods can be used to load drugs or other bioactive compounds in erythrocytes, including physical (e.g., electrical pulse method) osmosis-based systems, and chemical methods (e.g., chemical perturbation of the erythrocytes membrane).the following are types of drug loading: Hypotonic hemolysis, hypotonic dilution, hypotonic preswelling, isotonic osmotic lysis, Chemical perturbation of the membrane. Electro-insertion or electron capsulation, Entrapment by endocytosis, loading by electric cell fusion, loading by lipid fusion.

OSMOTIC SHOCK:

For 0.5 study, erythrocyte suspension (1 ml, 10%) was diluted & centrifuge at 3000 rpm for 15 minute. The supernatant was estimated for % Hb release spectrophotometrically.

TURBULENCE SHOCK:

It is the measure of simulating distribution of loaded cells during injection. In this drug loaded cells are passed through a 23 gauge hypodermic at a flow rate of 10 ml/min which is comparable to the flow rate of blood. It is followed by collecting of an aliquot and centrifugation sample is estimated. Drug loaded erythrocytes appears to be less resistant to turbulence, probably indicating destruction of cells upon shaking.

ERYTHROCYTE SEDIMENTATION RATE (ESR):

It is an estimate of the suspension stability of RBC in plasma and is related to the number and size of the red cells and to relative concentration of plasma protein, especially fibrinogen and α,β globulins. This test is performed by determining the rate of sedimentation of blood cells in a standard tube. Normal blood ESR is 0 to 15 mm/hr. higher rate is indication of active but     obscure disease processes.

Use of red cell loader:

Magnani et al. developed a novel method for entrapment of non diffusible drugs into erythrocytes. They developed a piece of equipment called a “red cell loader”. With as little as 50 mL of a blood sample, different biologically active compounds were entrapped into erythrocytes within a period of 2 h at room temperature under blood banking conditions. The process is based on two sequential hypotonic dilutions of washed erythrocytes followed by concentration with a hemofilter and an isotonic resealing of the cells. There was 30% drug loading with 35–50% cell recovery. The processed erythrocytes had normal survival in vivo. The same cells could be used for targeting by improving their recognition by tissue macrophages.

Hypotonic dilution:

Hypotonic dilution was the first method investigated for the encapsulation of chemicals into erythrocytes and is the simplest and fastest. In this method, a volume of packed erythrocytes is diluted with 2–20 volumes of aqueous solution of a drug. The solution tonicity is then restored by adding a hypertonic buffer. The resultant mixture is then centrifuged, the supernatant is discarded, and the pellet is washed with isotonic buffer solution. The major drawbacks of this method include low entrapment efficiency and a considerable loss of hemoglobin and other cell components. This reduces the circulation half life of the loaded cells. These cells are readily phagocytosed by RES macrophages and hence can be used for targeting RES organs. Hypotonic dilution is used for loading enzymes such as galactosidase and glucosidase, asparginase and arginase, as well as bronchodilators such as salbutamol.

Chemical perturbation of the membrane:

This method is based on the increase in membrane permeability of erythrocytes when the cells are exposed to certain chemicals. In 1973, Deuticke et al. showed that the permeability of erythrocytic membrane increases upon exposure to polyene antibiotic such as amphotericin B. In 1980, this method was used successfully by Kitao and Hattori to entrap the antineoplastic drug daunomycin in human and mouse erythrocytes. Lin et al. used halothane for the same purpose. However, these methods induce irreversible destructive changes in the cell membrane and hence are not very popular.

Entrapment by endocytosis:

This methodwas reported by Schrier et al. in 1975. Endocytosis involves the additionof one volume of washed packed erythrocytesto nine volumes of buffer containing2.5 mM ATP, 2.5 mM MgCl2, and1mM CaCl2, followed by incubation for2 min at room temperature. The porescreated by this method are resealed byusing 154 mM of NaCl and incubationat 37⁰cfor 2 min. The entrapment ofmaterial occurs by endocytosis. The vesiclemembrane separates endocytosed materialfrom cytoplasm thus protecting itfrom the erythrocytes and vice-versa. Thevarious candidates entrapped by thismethod include primaquine and related8–amino–quinolines, vinblastine, chlorpromazineand related phenothiazines,hydrocortisone, propranolol and tetracaine.

Loading by electric cell fusion:

This method involves the initial loading of drug molecules into erythrocyte ghosts followed by adhesion of these cells to target cells. The fusion is accentuated by the application of an electric pulse, which causes the release of an entrapped molecule. An example of this method is loading a cell-specific monoclonal antibody into an erythrocyte ghost. An antibody against a specific surface protein of target cells can be chemically cross-linked to drug-loaded cells that would direct these cells to desired cells.

 Loading by lipid fusion:

Lipid vesicles containing a drug can be directly fused to human erythrocytes, which lead to an exchange with a lipid-entrapped drug. [35] This technique was used for entrapping inositol mono phosphate to improve the oxygen carrying capacity of cells. However, the entrapment efficiency of this method is very low (1%).

RELEASE CHARACTERISTICS OF LOADED DRUGS:

There are mainly three ways for a drug to efflux out from the erythrocyte carriers: phagocytosis, diffusion through the membrane of the cells and using a specific transport system. RBCs are normally removed from circulation by the process of phagocytosis. The degree of cross linking determines whether liver or spleen will preferentially remove the cells. Carrier erythrocytes following heat treatment or antibody cross-linking are quickly removed from the circulation by phagocytic cells located mainly in liver and spleen. The rate of diffusion depends upon the rate at which a particular molecule penetrates through a lipid by layer. It is greatest for a molecule with high lipid solubility.

DELIVERY STRATEGIES

As mentioned earlier, there are two major strategies in the delivery of drugs using erythrocytes as carriers which include intravenous slow drug release strategy and target gene delivery.

Intravenous slow drug release strategy:

The normal life-span of an erythrocyte in systemic circulation is about 120 days. As mentioned as an advantage, in the optimum conditions of the loading procedure, the life-span of the resulting carrier cells may be comparable to that of the normal erythrocytes. [36] Erythrocytes have been used as circulating intravenous slow-release carriers for the delivery of antineoplasms, antiparasitics, antiretroviral agents, vitamins, steroids, antibiotics and cardiovascular drugs among others.

A series of mechanisms have been proposed for drug release in circulation from carrier erythrocytes, including passive diffusion out of the loaded cells into circulation, specialized membrane-associated carriers, phagocytosis of the carrier cells by the macrophages of RES and, then, depletion of the drug into circulation, accumulation of the drug in RES upon lysis of the carrier and slow release from this system into circulation, accumulation of the carrier erythrocytes in lymphatic nodes following subcutaneous injection of the cells and drug release upon hemolysis in this sites, and, finally, hemolysis in the injection sites.

Targeted drug delivery:

RES or non-RES ‘targeting’ is another important strategy using erythrocytes as carriers.

RES targeting:

It is a well-known fact that, in physiologic conditions, as a result of the gradual inactivation of the metabolic pathways of the erythrocyte by aging, the cell membrane loses its natural integrity, flexibility and chemical composition. These changes, in turn, finally result in the destruction of these cells upon passage through the spleen. The other effective site for the destruction of the aged or abnormal erythrocytes is the macrophages of the RES including peritoneal macrophages, hepatic Kupffer cells and alveolar macrophages of the lung, peripheral blood monocytes, and vascular endothelial cells. We know that aging and a series of other factors (e.g., stress during non-gentle loading methods) make the erythrocytes recognizable by the phagocyting macrophages via changing the chemical composition of the erythrocyte membrane, i.e., the phospholipids component. Therefore, a considerable fraction of carrier erythrocytes that have undergone some degrees of structural changes during the loading procedure will be trapped by the RES organs, mainly the liver and spleen, within a short time period after re-injection.

A series of approaches have been evaluated to improve RES targeting using carrier erythrocytes. In one of these approaches, the drug-loaded erythrocytes have been exposed to membrane stabilizing agents. This may increase the targeting index of the erythrocytes to RES via decreasing the deformability of these cells.

Non-RES targeting:

Recently, carrier erythrocytes have been used to target organs outside the RES. The various approaches include:

• Co-encapsulation of paramagnetic particles or photosensitive agents in erythrocytes alongwith the drug to be targeted; 
• Application of ultrasound waves; 
• Site-specific antibody attachment to erythrocyte membrane.Chiarantini et al. have reported in vitro targeting of erythrocytes to cytotoxic T-cells by coupling them to Thy-1.2 monoclonalantibody. Price et al. reported the delivery of colloidal particles and erythrocytes to tissuethrough micro vessel ruptures created by targeted micro bubble destruction with ultrasound. 
• In another study, the differential response of photosensitized young and old erythrocytes to photodynamic activation has been studied by Rollan.

 APPLICATIONS OF RESEALED ERYTHROCYTES

Resealed erythrocytes have several possible applications in various fields of human and veterinary medicine. Such cells could be used as circulating carriers to disseminate a drug within a prolonged period of time in circulation or in target-specific organs, including the liver, spleen, and lymph nodes. A majority of the drug delivery studies using drug-loaded erythrocytes are in the preclinical phase. In a few clinical studies, successful results were obtained.

Slow drug release:

Erythrocytes have been used as circulating depots for the sustained delivery of antineoplastics, antiparasitics, veterinary antiamoebics, vitamins, steroids, antibiotics and cardiovascular drugs.

The various mechanisms proposed for drug release include

•  Passive diffusion 
• Specialized membrane associated carrier transport 
• Phagocytosis of resealed cells by macrophages of RES, subsequent accumulation of drug into the macrophage interior, followed by slow release. 
• Accumulation of erythrocytes in lymph nodes upon subcutaneous administration followed byhemolysis to release the drug.

 Routes of administration include intravenous, which is the most common, followed by subcutaneous, intraperitoneal, intranasal, and oral. Studies regarding the improved efficacy of various drugs given in this form in animal models have been reported. Examples include an enhancement in anti-inflammatory effect of corticosteroids in experimentally inflamed rats, increase in half life of isoniazid and levothyroxine.

Targeting the liver:

Enzyme deficiency/replacement therapy:

Many metabolic disorders related to deficient or missing enzymes can be treated by injecting these enzymes. However, the problems of exogenous enzyme therapy include a shorter circulation half life of enzymes, allergic reactions, and toxic manifestations.

Treatment of hepatic tumors:

Hepatic tumors are one of the most prevalent types of cancer. Antineoplastic drugs such as methotrexate, bleomycin has been successfully delivered by erythrocytes. Agents such as daunorubicin diffuse rapidly from the cells upon loading and hence pose a problem. This problem can be overcome by covalently linking daunorubicin to the erythrocytic membrane using gluteraldehyde as a spacer. The resealed erythrocytes loaded with carboplatin show localization in liver.

Treatment of parasitic diseases:

The ability of resealed erythrocytes to selectively accumulate within RES organs make them useful tool during the delivery of antiparasitic agents. Parasitic diseases that involve harboring parasites in the RES organs can be successfully controlled by this method. Results were favorable in studies involving animal models for erythrocytes loaded with antimalarial, antileishmanial and antiamoebic drugs.

Removal of RES iron overload

Desferrioxamine-loaded erythrocytes have been used to treat excess iron accumulated because of multiple transfusions to thalassemic patients. Targeting this drug to the RES is very beneficial because the aged erythrocytes are destroyed in RES organs, which results in an accumulation of iron in these organs.

Removal of toxic agents:

Cannon et al. reported inhibition of cyanide intoxication with murine carrier erythrocytes containing bovine rhodanase and sodium thiosulfate. Antagonization of organophosphorus intoxication by resealed erythrocytes containing a recombinant phosphodiestrase also has been reported.

Delivery of antiviral agents:

Several reports have been cited in the literature about antiviral agents entrapped in resealed erythrocytes for effective delivery and targeting. Because most antiviral drugs are nucleotides or nucleoside analogs, their entrapment and exit through the membrane needs careful consideration. Nucleosides are rapidly transported across the membrane whereas nucleotides are not and thus exhibiting prolonged release profiles. The release of nucleotides requires conversion of these moieties to purine or pyrimidine bases. Resealed erythrocytes have been used to deliver deoxycytidine derivatives, recombinant herpes simplex virus type 1 (HSV-1) glycoprotein B, azidothymidine derivatives, azathioprene, acyclovir, and fludarabine phosphate.

Enzyme therapy:

Enzymes are widely used in clinical practice as replacement therapies to treat diseases associated with their deficiency (e.g., Gaucher’s disease, galactosuria), degradation of toxic compounds secondary to some kind of poisoning (cyanide, organophosphorus), and as drugs. The problems involved in the direct injection of enzymes into the body have been cited. One method to overcome these problems is the use of enzyme-loaded erythrocytes. These cells then release enzymes into circulation upon hemolysis act as a “circulating bioreactors” in which substrates enter into the cell, interact with enzymes, and generate products or accumulate enzymes in RES upon hemolysis for future catalysis.

The most important application of resealed erythrocytes in enzyme therapy is that of asparginase loading for the treatment of pediatric neoplasm. This enzyme degrades aspargine, an amino acid vital for cells. This treatment prevents remission of pediatric acute lymphocytic leukemia. There are reports of improved intensity and duration of action in animal models as well as humans. Other enzymes used for loading resealed erythrocytes include urease, galactose-1-phosphate uridyl transferase, uricase, and acetaldehyde dehydrogenase.

Improvement in oxygen delivery to tissues:

Hemoglobin is the protein responsible for the oxygen-carrying capacity of erythrocytes. Under normal conditions, 95% of hemoglobin is saturated with oxygen in the lungs, whereas under physiologic conditions in peripheral blood stream only 25% of oxygenated hemoglobin becomes deoxygenated. Thus, the major fraction of oxygen bound to hemoglobin is recirculated with venous blood to the lungs. The use of this bound fraction has been suggested for the treatment of oxygen deficiency. 2, 3-Diphosphoglycerate (2, 3-DPG) is a natural effector of hemoglobin. The binding affinity of hemoglobin for oxygen changes reversibly with changes in intracellular concentration of 2, 3-DPG. This compensates for changes in the oxygen pressure outside of the body, as the affinity of 2, 3-DPG to oxygen is much higher than that of hemoglobin.

Microinjection of macromolecules:

Biological functions of macromolecules such as DNA, RNA, and proteins are exploited for various cell biological applications. Hence, various methods are used to entrap these macromolecules into cultured cells (e.g., microinjection). A relatively simple structure and a lack of complex cellular components (e.g., nucleus) in erythrocytes make them good candidates for the entrapment of macromolecules. In microinjection, erythrocytes are used as microsyringes for injection to the host cells. The microinjection process involves culturing host eukaryotic cells in vitro. The cells are coated with fusogenic agent and then suspended with erythrocytes loaded with the compound of interest in an isotonic medium. Sendai virus (hemagglutinating virus of Japan, HVJ) or its glycoproteins or polyethylene glycol have been used as fusogenic agents. The fusogen causes fusion of co-suspended erythrocytes and eukaryotic cells. Thus, the contents of resealed erythrocytes and the compound of interest are transferred to host cell. This procedure has been used to microinject DNA fragments, proteins, nucleic acids to various eukaryotic cells.

Advantages of this method include quantitative injection of materials into cells, simultaneous introduction of several materials into a large number of cells, minimal damage to the cell, avoidance of degradation effects of lysosomal enzymes, and simplicity of the technique. Disadvantages include a need for a larger size of fused cells, thus making them amenable to RES clearance, adverse effects of fusogens, and unpredictable effects on cell resulting from the co- introduction of various components. Hence, this method is limited to mainly cell biological applications rather than drug delivery.

Other applications of resealed erythrocytes include

•  surface modification with antibodies 
• surface modification with gluteraldehyde 
• surface modification with carbohydrates such as sialic acid 
• entrapment of paramagnetic particles along with the drug 
• Entrapment of photosensitive material 
• antibody attachment to erythrocyte membrane to get specificity of action

 NOVEL APPROACHES:

Erythrosomes:These are specially engineered vesicular systems that are chemically cross-linked to human erythrocytes’ support upon which a lipid bilayer is coated. This process is achieved by modifying a reverse-phase evaporation technique. These vesicles have been proposed as useful encapsulation systems for macromolecular drugs.

Nanoerythrosomes:These are prepared by extrusion of erythrocyte ghosts to produce small vesicles with an average diameter of 100 nm. Daunorubicin was covalently conjugated to nanoerythrosomes using gluteraldehyde spacer. This complex was more active than free daunorubicin alone.

CONCLUSION:
During the past decade, numerous applications have been proposed for the use of resealed erythrocytes as carrier for drugs, enzyme replacement therapy etc. The use of resealed erythrocytes looks promising for a safe and sure delivery of various drugs for passive and active targeting. However, the concept needs further optimization to become a routine drug delivery system. The same concept also can be extended to the delivery of biopharmaceuticals and much remains to be explored regarding the potential of resealed erythrocytes. For the present, it is concluded that erythrocyte carriers are “golden eggs in novel drug delivery systems” considering their tremendous potential.Most of the studies in this area are in the in vitro phase and the ongoing projects worldwide remain to step into preclinical and, then, clinical studies to prove the capabilities of this promising delivery system.

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Controlled Released Systems

    Over past 30 year as the expanse and complication involved in marketing new drug entities have increased, with concomitant recognition of the therapeutic advantages of controlled drug delivery, greater attention has been focused on development of sustained or controlled release drug delivery systems. There are several reasons for the attractiveness of these dosage forms. It is generally recognized that for many disease states, a substantial number of therapeutically effective compounds already exist.

The effectiveness of these drugs, however, is often limited by side effects or the necessity to administer the compound in a clinical setting, the goal in designing sustained or controlled delivery system is to reduce the frequency of dosing or to increase effectiveness of the drug by localization at the site of action, reducing the dose required, or providing uniform drug delivery. Sustained release constitutes any dosage form that provides medication over and extended time. Controlled release, however, denotes that the system is able to provide some actual therapeutic control, whether this is of a temporal nature, spatial nature or both.

This correctly suggests that there are sustain release system that can not be considered controlled release system. In general, the goal of a sustained release dosage from is to maintain therapeutic blood or tissue levels of drug for an extended period this is usually accomplished by attempting to obtain zero-order release from the dosage form; zero-order release constitutes drug release from the dosage form. Sustained release systems generally do not attain this type of release and provides drug is a slow first order fashion. In recent year sustained release dosage forms continue to draw attention in the search for improved patient compliance and decreased incidence of adverse drug reactions. Sustained release technology is relatively cow field and as a consequence, research in the field has been extremely fertile and has produced many discoveries. New and more sophisticated controlled release, sustained release delivery systems are constantly being developed and tested.

Sustained release, sustained action, prolonged action controlled release, extended action, timed release, depot and repository dosage forms are terms used to identify drug delivery system that are designed to achieve or prolonged therapeutic effect by continuously releasing medication over an extended period of time after administration of a single dose.²

Fig.1: Drug level verses time profile showing differences between zero order, controlled release, slow first order sustained release and release from conventional tablet.²

Systems that are designed as prolonged release can also be considered as attempts at achieving sustained-release delivery. Repeat action tablets are an alternative method of sustained release in which multiple doses of drug are contained within a dosage form, and each dosage is related to a periodic interval. Delayed release systems, in contrast may not be sustaining, science often function of these dosage forms is to maintain the drug within the dosage form for some time before release. Commonly the release rate of drug is not altered and does not result in sustained delivery once drug release has begun.

Successful fabrication of sustained release products is usually difficult & and involves consideration of physicochemical properties of drug, pharmacokinetic behavior of drug, route of administration, disease state to be treated and, most importantly, placement of the drug in dosage form total will provide the desired temporal and spatial delivery pattern for the drug³

The slow first order release obtained by a sustained release pre parathion is generally achieved by the release of the drug from a dosage form. In some cases in some cases, this achieved by making slow the release of drug from a dosage form. In some cases, this is accomplished by a continuous release process⁴

2. Potential advantages of Controlled drug therapy 

1. Patient compliance due to reduction in the frequency of designing.
2. Employ minimum drug.
3. Minimize or eliminates local and systemic side effects.
4. Obtain less protentiation or deduction in drug activity with chronic use.
5. Minimize drug accumulation with chromic dosing.
6. Improves efficacy in treatment.
7. Cure or control confirm more promptly.
8. Improve control of condition i.e. reduce fluctuation in drug level.
9. Improve bioavailability of same drugs.
10. Make use of special effects, e.g. sustained release aspect for morning relief of arthritis by dosing before bedtime.

3. Disadvantages of controlled release dosage forms

1. They are costly.
2. Unpredictable and often poor in-vitro in-vivo correlations, dose dumping, reduced potential for dosage adjustment and increased potential first pass clearance.
3. Poor systemic availability in general.
4. Effective drug release period is influenced and limited by GI residence time.

4. Rationale of Controlled –Drug Delivery:

The basic rationale for controlled drug delivery is to alter the pharmacokinetic and pharmacodynamics of pharmacologically active moieties by using novel drug delivery systems or by modifying the molecular structure and/or physiological parameters inherent in a selected route of administration. It is desirable that the duration of drug action become more to design properly. Rate controlled dosage form, and less, or not at all, a property of the drug molecules inherent kinetic properties.

As mentioned earlier, primary objectives of controlled drug delivery are to ensure safety and to improve efficiency of drugs as well as patient compliance. This achieved by better control of plasma drug levels and frequent dosing. For conventional dosage forms, only the dose (D) and dosing interval (C) can vary and, for each drug, there exists a therapeutic window of plasma concentration, below which therapeutic effect is insufficient, and above which toxic side effects are elicited. This is often defined as the ratio of median lethal dose (LD 50) to median effective dose (ED50) ²³

5. Controlled Release Systems:

Diffusion controlled

·Reservoir

·Matrix

·Reservoir and monolithic

Dissolution controlled

·Encapsulation

·Matrix

Water penetration controlled

·Osmotically controlled

·Swelling controlled

Chemically controlled

·Erodible systems

·Drug covalently linked with polymer

Hydrogels

·Chemically controlled

·Swelling controlled

·Diffusion controlled

·Environment responsive

Ion-exchange resins

·Cationic exchange

·Anionic exchange

Diffusion controlled systems:-

The basic mechanism of drug release from these two systems is fundamentally different besides these simple systems, combination of reservoir and monolithic systems also exist in practice18.      

Diffusion systems are characterized by release rate of drug is dependant on its diffusion through inert water insoluble membrane barrier.

There are basically two types of diffusion devices.

(I)Reservoir devices  

(II)Matrix devices

Reservoir Devices :

Reservoir Devices are those in which a core of drug is surrounded by polymeric membrane. The nature of membrane determines the rate of release of drug from system.

The process of diffusion is generally described by a series of equations governed by Fick’s first law of diffusion.

J = -D (DC/ DX)…….(1)

Where J :  is the flux of drug across the membrane given in units of amount / area time.

D : is diffusion coefficient of drug in membrane in units of area / time. This is reflecting to drug molecule’s ability to diffuse through the solvent and is dependent on the factors as molecular size and charge.

dc/ dt : represents rate of change in concentration C relative to a distance X in the membrane.

The law states that amount of drug passing across a unit area, is proportional to the concentration difference across that plane.

Schematic representation of reservoir diffusion device Cm (o), and Cm (d) represent concentration of drug inside surfaces of membrane and C (o) & C(d) represents concentration in adjacent  regions.

Fig 2

If it is assumed that the drug on the both side of membrane is in equilibrium with its respective membrane surface which in equilibrium between the membrane surfaces and their bathing solutions as shown in Figure.

Therefore the concentration just inside the membrane surface can be related to the concentration in the adjacent region by following expression.

K  = Cm (o) /C(d)      at  X = o                                  (2)

K  = Cm (d) / C(d)     at  X = d                                  (3)

Where K  = partition coefficient.

If we consider K & D are constants then equation (1) becomes,

J  = D KC/d                                                           (4)

Where c is the concentration difference across the membrane and d is path length of diffusion.

The simplest system to consider is that of slab, where drug release is from only one surface as shown Figure  in this case equation (4) becomes

dMt/ dt  =    ADKC/ d                                              (5)

Figure 3 Diagrammatic representation of slab configuration of reservoir diffusion system.

  

Non permeable polymer shell

Where Mt  = Mass of drug released after time t, dMt/dt. Steady state drug release rate of time‘t’.

A :  surface area of device.

In equation (7) if variables of right side of equation remain constant, then left side of equation represents release rate of system, a true controlled release system with a zero-order release rate.

A constant effective area of diffusion, diffusional path length, concentration difference, and diffusion coefficient are required to obtain a release rate that is constant. Reservoir diffusional systems have several advantages over conventional dosage forms. They can after zero order release of drug, kinetics of which can be controlled by changing the characteristics of the polymer to meet the particular drug and therapy conditions.

Pot showing approach to steady state for reservoir device that has been stored for an extended period (the burst effect curve) and for device that has been freshly made (the time lag curve)

Fig 4

Common methods used to develop reservoir type of devices include micro encapsulation of drug particles and press coating of tablets containing drug cores. In most cases particles coated by microencapsulation form a system where the drug is contained in the coating film as well as in the core of micro capsule. The drug release generally involves combination of dissolution and diffusion with dissolution being process that controls the release rate. If encapsulating material is selected properly will be the controlling process. Some materials such as membrane barrier coat alone or in combination, are hardened gelatin, methyl or methylcellulose, polyhydroxymethacrylate hydroxypropylmethylcellulose, polydroxymethacrylate, polyvinyl acetate & various waxes.

Matrix devices:

A matrix device, as the name implies, consists of drug dispersed homogenously throughout a polymer.

Matrix diffusion system before release (time =0) & after partial drug release (time = t)

Fig5

Time= 0                                                        Time=t                                   

In this model drug in out side layer exposed to the bathing solution is dissolved first and diffused out of the matrix.  This process continues with the interface between  bathing solution and the solid drug moving controlled, the rate of dissolution of drug particles within the matrix must be faster that the diffusion rate of dissolved drug leaving matrix.

Following assumptions are made in retrieving the mathematical models are.

i.A pseudo steady state is maintained during drug release.

ii.The diameter of drug particles is less than the average distance of drug Diffusion through the matrix.

iii.The bathing solution provides sink conditions.

iv.The diffusion coefficient of drug in the matrix remains constant.

The next equation that describes the rate of release drugs dispersed in an inert matrix system has been derived by Higuchi.

Figure 6 : Schematic representation of the physical model used for a planer slab matrix diffusion device.

The change in amount of drug released per unit area dM and change in the thickness of the zone of the matrix that has been depleted of the drug,

dM/dh = Co dh – Cs /2                                               (6)

by Fick’s first law,

dm = (DmCs/h) dt.                                                     (7)

where Dm is diffusion coefficient in matrix if equation (6)  & (7) are equated & solved for D that value of h sustituted back into the integrated form of equation (7) An equation for M is obtained.

M= [ Cs Dm (2Co – Cs) t] ½                          (8)

Similarly, a drug released from porous or granular matrix is described.

M= [ Ds Ca (є/τ)( 2Co – єCa) t           ] ½   (9)

Where    e = Porosity of matrix

              τ = tortuosity.

Ca = Solubility of drug in release medium

Ds = diffusion coefficient of drug in release medium.

In this system drug is leached from matrix through channels or pores.

For purpose of data treatment equation (8) & (9) are reduced to

M = Kt½

          (10)

Where K is constantan so, that plot amount of drug released verses square root of time should be linear if the release of drug from the matrix is diffusion controlled.  The release rate of drug from such a device is not zero order, since if decreases with time but as previously mentioned, this may be clinically equivalent to constant drugs. 5

Water Penetration Controlled Systems:

In water penetration controlled delivery systems, rate control is obtained by the penetration of water into the system. Two general types of these systems include, swelling controlled release systems and osmotically controlled delivery systems.

Swelling Controlled Systems:

Swelling controlled release systems are initially dry and when placed in the body absorb water or other body fluids and swell. Swelling increases the aqueous solvent content within the formulation as well as the polymer mesh size, enabling the drug to diffuse through the swollen network into the external environment. Figure 1-18 (A and B> illustrates swelling reservoir and matrix systems, respectively. Most of the materials used in swelling controlled release systems that will swell without dissolving, when exposed to water or other biological fluids. These hydrogels can absorb a great deal of fluid and at equilibrium, typically comprise 60-90% fluid and only 10-30% polymer In case of polymer hyfrogel containing dispersed water-soluble agent, initiall3ç the diffusion coefficient of agent in the dehydrated hydrogel is very 10% however a significant increase is noticed as the gel imbibes water. Thus the release of active agent from the system is a function of rate of uptake of water from the vicinity nd the rate of drug diffusion.²⁸

6. Factors Affecting Sustained Release Dosage Forms:-

Physicochemical properties of drug

a) Dose Size:

If an oral product has a dose size greater that 0.5gm it is a poor candidate for sustained release system, Since addition of sustaining dose and  possibly the sustaining mechanism will, in most cases generates a substantial volume product that unacceptably large.

b) Aqueous Solubility :

Most of drugs are weak acids or bases, since the unchanged form of a drug preferentially permeates across lipid membranes drugs aqueous solubility will generally be decreased by conversion to an unchanged form for drugs with low water solubility will be difficult to incorporate into sustained release mechanism. The lower limit on solubility for such product has been reported 0.1mg/ml. drugs with great water solubility are equally difficult to incorporate in to sustained release system. pH dependent solubility, particularly in the physiological pH range, would be another problem because of the variation in pH throughout the GI tract and hence variation in dissolution rate

c) Partition Coefficient:

Partition coefficient is generally defined as the fraction of drug in an oil phase to that of an adjacent aqueous phase. Accordingly compounds with relatively high partition coefficient are predominantly lipid soluble and consequently have very law aqueous solubility. Compounds with very law partition coefficients will have difficulty in penetrating membranes resulting poor bioavailability. 

Typical relationship between drug activity and partition

 Coefficient K, generally known as Hansch Correlation.    

d) Pka :

The relationship between Pka of compound and absorptive environment. Presenting drug in an unchanged form is adventitious for drug permeation but solubility decrease as the drug is in unchanged form

e) Drug Stability :

Orally administered drugs can be subject to both acid base hydrolysis and enzymatic degradation. Degradation will proceed at the reduced rate for drugs in the solid state, for drugs that are unstable in stomach, systems that prolong delivery ever the entire course of transit in GI tract are beneficial. Compounds that are unstable in the small intestine may demonstrate decreased bioavailability when administered form a sustaining dosage from. This is because more drug is delivered in small intestine and hence subject to degradation

f) Molecular size and diffusivity:

The ability of drug to diffuse through membranes its so called diffusivity & diffusion coefficient is function of molecular size (or molecular weight).

Generally, values of diffusion coefficient for intermediate molecular weight drugs, through flexible polymer range from 10-8 to 10-9 cm2 / sec. with values on the order of 10-8 being most common for drugs with molecular weight greater than 500, the diffusion coefficient  in many polymers frequently are so small that they are difficult to quantify i.e. less than 16-12 cm2/sec.  Thus high molecular weight drugs and / or polymeric drugs should be expected to display very slow release kinetics in sustained release device using diffusion through polymer membrane.

g) Protein binding:

It is well known that many drugs bind to plasma proteins with a concomitant influence on the duration of drug action. Since blood proteins are for the most part re-circulated and not eliminated, drug Protein binding can serve as a depot for drug producing a prolonged release profile, especially if a high degree of drug binding occurs.

Extensive binding to plasma proteins will be evidenced by a long half life of elimination for drugs and such drugs generally most require a sustained release dosage form. However drugs that exhibit high degree of binding to plasma proteins also might bind to bio-polymers in GI tract which could have influence on sustained drug delivery.  The presence of hydrophobic moiety on drug molecule also increases the binding potential.

Biological factors:

a) Biological Half Life:

The usual goal of an oral sustained release product is to maintain therapeutic blood levels over an extended period. To action this, drug must enter in the circulation of approximately the same rate of which it is eliminated. The elimination rate is quantitatively described by half-life (t1/2). Therapeutic compounds with short half lives are excellent candidates for sustained release preparations. Since this can reduce dosing frequency. In general drugs with half-lives shorter than 3hrs are poor candidates of sustained release dosage forms of dose size will increase as well as compounds with long half lives, more than 8 hrs are also not used in sustained release forms because their effect is already sustained.

b) Absorption:

The rate, extent and uniformity of absorption of a drug are important factors when considered its formulation into a sustained release system. As the rate limiting step in drug delivery from a sustained-release system is its release from a dosage form, rather than absorption. Rapid rate of absorption of drug, relative to its release is essential if the system is to be successful.6 It we assume that transit time of drug  must in the absorptive areas of the GI tract is about 8-12 hrs. The maximum half life for absorption should be approximately 3-4 hrs. Otherwise device will pass out of potential absorption regions before drug release is complete.

c) Distribution:

The distribution of drugs into tissues can be important factor in the overall drug elimination kinetics. Since it not only lowers the concentration of circulating drug but it also can be rate limiting in its equilibrium with blood and extra vascular tissue, consequently apparent volume  of distribution assumes different values depending on time course of drug disposition. For design of sustained/ controlled release products, one must have information of disposition of drug.

d) Metabolism:

Drugs that are significantly metabolized before absorption, either in lumen or the tissue of the intestine, can show decreased bioavailability from slower-releasing dosage forms. Most intestinal wall enzymes systems are saturable. As drug is released at a slower rate to these regions less total drug is presented to the enzymatic. Process device a specific period, allowing more complete conversion of the drug to its metabolite.6

7. Compounds Those Are Unsuitable For Controlled Release

For drugs with elimination half life less than two hrs, as well as those that are administered in large doses, a controlled release dosage from may contain in prohibiting large quantity of drug, on the other hand, drugs with elimination half lives of 3 hrs or more are sufficiently sustained in the bldg from conventional doses, and controlled release is generally not necessary.

Administering drugs like warfarin, whose pharmacological effect is delayed relative to its blood profile, offers no clinical advantage, similarly, incorporating drugs like fluorouracil, and perhaps some beta lactum antibiotics and thiamine diuretics that appears to exhibit an “absorption window” may reduce absorption efficiency. Problems of first pass clearance of sustained release drugs.