PULSINCAP RA BIBEK SINGH MAHAT

Development and Evaluation of Colon Targeted Multi-Particulate Drug Delivery

Systems (MPDDS) of Diclofenac Sodium by Pulsincap® Drug Delivery System

Literature Review

Submitted To

Department of Pharmacy,

Kathmandu University

Submitted By

Bibek Singh Mahat, Roll No.: 07

M. Pharm. (Industrial Pharmacy Group)

Kathmandu University

Literature Review of Modified Pulsincap® Drug Delivery System 2011

Page No: 2 of 51 M. PHARM. (2009) ROLL NO. : 07 Email:‐ [email protected]

TABLE OF CONTENTS

1. Multiparticulate Drug Delivery System (MPDDS) 3 1.1 Reasons for formulating a drug as a multiparticulate system 3 1.2 Drug safety vs. MPDDS 3 1.3 Multiparticulate Preparation Methods 4 1.4 Purpose of Designing MPDDS 4 1.5 Mechanism of Drug‐release from MPDDS 4 1.6 Novel Drug Delivery System based on MPDDS 5

2. Colon Targeted Drug Delivery Systems (CDDS) 12

2.1 Advantages of CDDS over Conventional Drug Delivery 12 2.2 Criteria for Selection of Drug for CDDS 14 2.3 Approaches used for Site Specific Drug Delivery to Colon 15 2.4 Approaches used for the evaluation of Drug Delivery to Colon 24

3. Pulsatile Technology (PDS) 25

3.1 Diseases targeted for pulsatile technology 26 3.2 Methodologies for PDDS 26 3.3 Time controlled Pulsatile release on Capsular system 27 3.4 Marketed Technologies 28

4. Chronobiology and Chronotherapy of Arthritic Diseases 29

4.1 Rheumatoid Arthritis 29 4.2 Biological rhythms in experimental inflammation 31

4.3 Cortisol and melatonin regulated circadian cytokine production 32

4.4 Cortisol and melatonin effects on circadian rhythms in rheumatoid arthritis 33

4.5 Conclusions of circadian symptoms in patients with RA 36 5. Hard Gelatin Capsules and CrossLinking Technology 37

5.1 Gelatin and Hard Gelatin Capsules 37 5.2 Reaction between the Proteins and Formaldehyde 38

6. Designing of the Device 42 7. Materials and Method 45 8. Work Description and Time Schedule 48 9. References 49



1. Multiparticulate Drug Delivery System (MPDDS)

Pharmaceutical invention and research are increasingly focusing on delivery systems which enhance desirable therapeutic objectives while minimizing side effects. Recent trends indicate that multiparticulate drug delivery systems (MPDDS) are especially suitable for achieving controlled or delayed release oral formulations.

Multi-particulate drug delivery systems are mainly oral dosage forms consisting of a multiplicity of small discrete units, each exhibiting some desired characteristics. In these systems, the dosage of the drug substances is divided on a plurality of subunit, typically consisting of thousands of spherical particles with diameter of 0.05-2.00mm. To deliver the recommended total dose, these subunits are filled into a sachet or encapsulated or compressed into a tablet. 1

1.1 Reasons for formulating a drug as a multiparticulate system:- 1

a) To facilitate disintegration in the stomach, b) To provide a convenient, fast disintegrating tablet that dissolves in water before swallowing

which can aid compliance in older patients and children. c) Multiparticulate systems show better reproducible pharmacokinetic behavior than

conventional (monolithic) formulations. d) After disintegration within a few minutes, the individual subunit particles pass rapidly

through the GI tract. e) If these subunits have diameters of less than 2mm, they are able to leave the stomach

continuously, even if the pylorus is closed. f) These results in lower intra and inter individual variability in plasma levels and

bioavailability.

1.2 Drug safety vs. MPDDS:- 1

Drug safety may also be increased by using multiparticulate dosage forms, particularly for modified release systems. If the film coat of a single-unit (monolithic) enteric coated tablet is damaged, the complete dose will be released into the stomach where it may cause pain or ulceration or reduced efficacy, depending on the reason for choosing the protection of the enteric coating. If there is damage to the film coating of a monolithic tablet with a sustained release formulation, this can lead to “dose dumping” and result in dramatic side effects. In multiparticulate formulation, the release characteristics are incorporated into every single subunit and any damage only affects the release behavior of the subunit involved, which represents a small part of the total dose, reducing the likelihood of safety problems.



1.3 Multiparticulates Preparation Methods:-

Multiparticulates may be prepared by several methods. Different methods require different processing conditions and produce multiparticulates of distinct qualities. Some of these methods may be broadly classified as:- Pelletization, Granulation,Spray drying,Spray congealing, Drug particles may be entrapped within the multiparticulates , Drug particles may be layered around them. These multiparticulates may be modified in many ways to achieve the desired drug release profile. One approach to the modification of drug release profile in multiparticulates is to coat them.

1.4 Purpose of Designing MPDDS:- 1

To develop a reliable formulation that has all the advantages of a single unit formulations and also devoid of the danger of alteration in drug release profile and formulation behavior due to, Unit to unit variation, Change in gastro –luminal pH and Enzyme population.

Multiparticulate systems perform better in vivo than single unit system, as they spread out through the length of the intestine cause less irritation, enjoy a slower transit through the colon and give a more reproducible drug release. Incorporating an existing medicine into a novel drug delivery system (NDDS) can significantly improve its performance in terms of efficacy, safety and improved patient compliance. In the form of a Multiparticulate NDDS, an existing drug molecule can get new life, thereby increasing its market value and competitiveness and even extending patent life.

1.5 Mechanism of Drug-release from MPDDS:-

The mechanism of drug release can occur in the following ways:

a) Diffusion: - On contact with aqueous fluids in the gastrointestinal tract (GIT), water diffuses into the interior of the particle. Drug dissolution occurs and the drug solutions diffuse across the release coat to the exterior.

b) Erosion: - Some coatings can be designed to erode gradually with time, thereby releasing the drug contained within the particle.

c) Osmosis: - In allowing water to enter under the right circumstances, an osmotic pressure can be built up within the interior of the particle. The drug is forced out of the particle into the exterior through the coating.



1.6 Novel Drug Delivery System based on MPDDS:-

a) Intestinal Protective Drug Absorption System (IPDAS)

Intestinal protective drug absorption system (IPDAS) is a multiparticulate tablet technology that has been developed to enhance the gastric tolerability of potentially irritant or ulcerogenic drugs such as the NSAIDs. It consists of high density controlled release beads that are compressed into a tablet form. The beads may be manufactured by techniques such as extrusion-spheronization. Controlled release mechanism can be achieved with the use of different polymer systems to coat the resultant beads.

Once an IPDAS tablet is ingested, it rapidly disintegrates and disperses beads containing the drug in the stomach which subsequently pass into the duodenum and along the gastrointestinal tract in a controlled and gradual manner, independent of the feeding state.Release of active ingredient from the multiparticulates occurs through a process of diffusion either through the polymeric membrane and /or the micro matrix of the polymer/active.

Figure 1: IPDAS technology

Naprelan®, which is marketed in the United States, employs IPDAS technology. This innovative formulation of naproxen sodium is a unique controlled release formulation indicated both for acute and chronic pain. The desired pharmacodynamic activity of a once-daily dosage form of naproxen requires: rapidly available naproxen for a prompt onset of analgesic activity, as well as a prolonged phase of absorption to provide 24-hour analgesic/anti-inflammatory activity.



b) Spheroidal Oral Drug Absorption Systems (SODAS)

SODAS® (Spheroidal Oral Drug Absorption System) is “Elan Drug Technologies” multiparticulate drug delivery system. It is a multiparticulate technology that enables the production of customized dosage forms and responds directly to individual drug candidate needs. This technology is based on the production of uniform spherical beads of 1-2 mm in diameter containing drug plus excipients and coated with product specific controlled release polymers. Benefits offered by the SODAS® technology include:

i. Controlled absorption with resultant reduction in peak to trough ratios

ii. Targeted release of the drug to specific areas within the gastrointestinal tract

iii. Absorption independent of the feeding state

iv. Suitability for use with one or more active drug candidate

v. Facility to produce combination dosage forms

vi. “Sprinkle dosing” by administrating the capsule contents with soft food

vii. Once or twice daily dose resembling multiple daily dose profiles

SODAS® can provide a number of tailored drug release profiles, including:-

i. Immediate release of drug followed by sustained release to give rise to a fast onset of action, which is maintained for 24 hours.

ii. Alternatively the opposite scenario can be achieved where drug release is delayed for a number of hours.

iii. An additional option is pulsatile release, where a once daily dosage form can resemble multiple daily doses by releasing drug in discrete bursts throughout the day.

Figure 2: SODAS technology



Products marketed using the SODAS® Technology:-

i) Diltiazem Once and Twice Daily

Diltiazem has a short half and was originally administered three or four times daily. The twice daily product was first launched in the US in 1986, followed by the once daily product in 1991. Elan Drug Technologies’ diltiazem is now marketed in many international markets including the U.S., Europe and Asia.

ii) Verapamil Once Daily

Verapamil, a calcium channel blocker, indicated for the treatment of hypertension was originally administered three times daily. Once daily formulation was unique in that it represented the first verapamil controlled release formulation in which the extent or rate of absorption was not affected by food intake. In addition, the capsule could be opened and the beads sprinkled on soft food for those patients unable to swallow traditional dosage forms.This verapamil is now marketed in many international markets including the U.S. and Europe.

c) Programmable Oral Drug Absorption System (PRODAS®)

Programmable Oral Drug Absorption System (PRODAS® Technology) is a multiparticulate technology, which is unique in that it combines the benefits of tableting technology within a capsule. The PRODAS® delivery system is presented as a number of mini-tablets combined in a hard gelatin capsule, to target the profile of a candidate drug. It is possible to incorporate different mini-tablets, each one formulated individually and designed to release drug at different sites so that higher dose loading is possible within the gastrointestinal tract.

Figure 3: PRODAS technology

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f) Stabilized Pellet Delivery System:-

The active drug is incorporated in multiparticulate dosage forms such as DIFFUCAPS or Eurand MINITABS, which are then subsequently coated with pH dependent/independent polymeric membranes that will deliver the drug to the desired site. These are then filled into hard gelatin capsules. This technology is designed specifically for unstable drugs and incorporates a pellet core of drug and protective polymer outer layer(s).

g) Pelletised tablet:-

Pelletised tablet (Peltab®) system utilizes polymer-coated drug pellets or drug crystals, which are compressed into tablets. In order to provide a controlled release, a water insoluble polymer is used to coat discrete drug pellets or crystals, which then can resist the action of fluids in the GIT. This technology incorporates a strong polymer coating enabling the coated pellets to be compressed into tablets without significant breakage.

h) Multi-particle Drug Dispersing Shuttle:-

Multiparticle drug dispersing shuttle (Multipart®) consists of a tablet carrier for the delivery of controlled release beads or pellets through the GIT which preserves the integrity and release properties of the beads. The distribution of the beads is triggered by the disintegration of the tablet carrier in the stomach. Drug release from the beads is triggered by super disintegration of the tablets. It can be pH-activated or pH-independent and can occur by disintegration or osmosis. The beads can be formulated to produce first or zero order release.

i) Eurand’s Orbexa technology:-

Eurand’s Orbexa technology produces beads of a controlled size and density using granulation spheronization, and extrusion techniques. These beads provide high drug concentrations and can be coated with functional polymer membranes for additional release rate control. Orbexa beads can be filled into capsules or single-dose sachets.

Figure 6: ORBEXA® technology



Advantages of Orbexa technology:-

i. High-speed process is well suited for sensitive molecules like proteins.

ii. Suitable for high drug loading.

j) AdvaTab® technology:-

AdvaTab orally disintegrating tablets (ODT) are designed to meet FDA guidance regarding ODTs, including a disintegration time of 15 to 30 seconds without the need to chew or drink liquid. The tablets are created using polymer-coated drug particles that are uniformly dispersed in an ultra-fine, low-water content, rapidly disintegrating matrix with superior organoleptic properties.

Figure 7: AdvaTab® technology



2. Colon Targeted Drug Delivery Systems

Targeted drug delivery into the colon is highly desirable for local treatment of a variety of bowel diseases such as ulcerative colitis, Crohn’s disease, amebiosis, colonic cancer, local treatment of colonic pathologies, and systemic delivery of protein and peptide drugs. The colon specific drug delivery system (CDDS) should be capable of protecting the drug en route to the colon i.e. drug release and absorption should not occur in the stomach as well as the small intestine, and neither the bioactive agent should be degraded in either of the dissolution sites but only released and absorbed once the system reaches the colon. 2 The colon is believed to be a suitable absorption site for peptides and protein drugs for the following reasons: - 2 a) Less diversity, and intensity of digestive enzymes,

b) Comparative proteolytic activity of colon mucosa is much less than that observed in the small

intestine, thus CDDS protects peptide drugs from hydrolysis, and enzymatic degradation in duodenum and jejunum, and eventually releases the drug into ileum or colon which leads to greater systemic bioavailability.

c) And finally, because the colon has a long residence time which is up to 5 days and is highly responsive to absorption enhancers.

Oral route is the most convenient and preferred route but other routes for CDDS may be used. Rectal administration offers the shortest route for targeting drugs to the colon. However, reaching the proximal part of colon via rectal administration is difficult. Rectal administration can also be uncomfortable for patients and compliance may be less than optimal. 2 Because of the high water absorption capacity of the colon, the colonic contents are considerably viscous and their mixing is not efficient, thus availability of most drugs to the absorptive membrane is low. The human colon has over 400 distinct species of bacteria as resident flora, a possible population of up to 1010 bacteria per gram of colonic contents. Among the reactions carried out by these gut flora are azoreduction and enzymatic cleavage i.e. glycosides. These metabolic processes may be responsible for the metabolism of many drugs and may also be applied to colon-targeted delivery of peptide based macromolecules such as insulin by oral administration. 2 2.1 Advantages of CDDS over Conventional Drug Delivery :- 2,3 Chronic colitis, namely ulcerative colitis, and Crohn’s disease are currently treated with glucocorticoids, and other anti-inflammatory agents. Administration of glucocorticoids namely dexamethasone and methyl prednisolone by oral and intravenous routes produce systemic side effects including adenosuppression, immunosuppression, cushinoid symptoms, and bone resorption. Thus selective delivery of drugs to the colon could not only lower the required dose but also reduce the systemic side effects caused by high doses.



Figure 8: Comparison of conventional and Colon Targeted dosage forms

Target sites

Disease conditions

Drug and active agents

Topical action

Inflammatory Bowel Diseases,

Irritable bowel disease and Crohn’s disease. Chronic pancreatitis

Hydrocortisone, Budenoside, Prednisolone, Sulfaselazine,

Olsalazine, Mesalazine, Balsalazide.

Local action

Pancreatactomy and cystic fibrosis,

Colorectal cancer To prevent gastric irritation

Digestive enzyme

supplements 5-Flourouracil.

Systemic

action

To prevent first pass metabolism

of orally ingested drugs Oral delivery of peptides Oral delivery of vaccines

NSAIDS Steroids Insulin

Typhoid

Table 1: Colon targeting diseases, drugs and sites



2.2 Criteria for Selection of Drug for CDDS4,5,6:- The best Candidates for CDDS are drugs which show poor absorption from the stomach or intestine including peptides. The drugs used in the treatment of IBD, ulcerative colitis, diarrhea, and colon cancer are ideal candidates for local colon delivery. Drug Carrier is another factor which influences CDDS. The selection of carrier for particular drugs depends on the physiochemical nature of the drug as well as the disease for which the system is to be used. Factors such as chemical nature, stability and partition coefficient of the drug and type of absorption enhancer chosen influence the carrier selection. Moreover, the choice of drug carrier depends on the functional groups of the drug molecule. For example, aniline or nitro groups on a drug may be used to link it to another benzene group through an azo bond.

Criteria Pharmacological class

Non-peptide drugs Peptide drugs

Drugs used for local

effects in colon against GIT diseases

Anti-inflammatory

drugs

Oxyprenolol, Metoprolol,

Nifedipine

Amylin,

Antisense oligonucleotide

Drugs poorly absorbed from upper GIT

Antihypertensive and

antianginal drugs

Ibuprofen, Isosorbides,

Theophylline

Cyclosporine, Desmopressin

Drugs for colon cancer

Antineoplastic drugs

Pseudoephedrine

Epoetin,

Glucagon

Drugs that degrade in

stomach and small intestine

Peptides and proteins

Bromophenaramine,

5-Flourouracil, Doxorubicin

Gonadoreline,

Insulin, Interferons

Drugs that undergo extensive first pass

metabolism

Nitroglycerin and corticosteroids

Bleomycin, Nicotine

Protirelin,sermorelin,

Saloatonin

Drugs for targeting Antiarthritic and Antiasthamatic drugs

Prednisolone, hydrocortisone,

5-Amino-salicylic acid

Somatropin, Urotoilitin

Table 2: Criteria for selection of drugs for CDDS



2.3 Approaches used for Site Specific Drug Delivery to Colon (CDDS):- a) Primary Approaches for CDDS: i) pH Sensitive Polymer Coated Drug Delivery to the Colon8:- In the stomach, pH ranges between 1 and 2 during fasting but increases after eating. The pH is about 6.5 in the proximal small intestine and about 7.5 in the distal small intestine. From the ileum to the colon, pH declines significantly. It is about 6.4 in the cecum. However, pH values as low as 5.7 have been measured in the ascending colon in healthy volunteers. The pH in the transverse colon is 6.6 and 7.0 in the descending colon. Use of pH dependent polymers is based on these differences in pH levels. The polymers described as pH dependent in colon specific drug delivery are insoluble at low pH levels but become increasingly soluble as pH rises. Although a pH dependent polymer can protect a formulation in the stomach, and proximal small intestine, it may start to dissolve in the lower small intestine, and the site-specificity of formulations can be poor. The decline in pH from the end of the small intestine to the colon can also result in problems, lengthy lag times at the ileo-cecal junction or rapid transit through the ascending colon which can also result in poor site-specificity of enteric-coated single-unit formulations. ii) Delayed (Time Controlled Release System) Release Drug Delivery to Colon8:- Time controlled release system (TCRS) such as sustained or delayed release dosage forms are also very promising drug release systems. However, due to potentially large variations of gastric emptying time of dosage forms in humans, in these approaches, colon arrival time of dosage forms cannot be accurately predicted, resulting in poor colonical availability. The dosage forms may also be applicable as colon targeting dosage forms by prolonging the lag time of about 5 to 6 h. However, the disadvantages of this system are: a. Gastric emptying time varies markedly between subjects or in a manner dependent on type

and amount of food intake. b. Gastrointestinal movement, especially peristalsis or contraction in the stomach would result

in change in gastrointestinal transit of the drug. c. Accelerated transit through different regions of the colon has been observed in patients with

the IBD, the carcinoid syndrome and diarrhea, and the ulcerative colitis. Therefore, time dependent systems are not ideal to deliver drugs to the colon specifically for the treatment of colon related diseases. Appropriate integration of pH sensitive and time release functions into a single dosage form may improve the site specificity of drug delivery to the colon. Since the transit time of dosage forms in the small intestine is less variable i.e. about 3±1 hr. The time-release function (or timer function) should work more efficiently in the small intestine as compared the stomach. In the small intestine drug carrier will be delivered to the target side, and drug release will begin at a predetermined time point after gastric emptying. On the other hand, in the stomach, the drug release should be suppressed by a pH sensing function (acid resistance) in the dosage form, which would reduce variation in gastric residence time.



Figure 9: Human GIT with lag time and pH change range

Enteric coated time-release press coated (ETP) tablets, are composed of three components, a drug containing core tablet (rapid release function), the press coated swellable hydrophobic polymer layer (Hydroxy propyl cellulose layer (HPC), time release function) and an enteric coating layer (acid resistance function). The tablet does not release the drug in the stomach due to the acid resistance of the outer enteric coating layer. After gastric emptying, the enteric coating layer rapidly dissolves and the intestinal fluid begins to slowly erode the press coated polymer (HPC) layer. When the erosion front reaches the core tablet, rapid drug release occurs since the erosion process takes a long time as there is no drug release period (lag phase) after gastric emptying. The duration of lag phase is controlled either by the weight or composition of the polymer (HPC) layer.

Figure 10: Design of enteric coated timed-release press coated tablet (ETP Tablet)



iii) Microbially Triggered Drug Delivery to Colon8:- The microflora of the colon is in the range of 1011 -1012 CFU/ mL, consisting mainly of anaerobic bacteria, e.g. bacteroides, bifidobacteria, eubacteria, clostridia, enterococci, enterobacteria and ruminococcus etc. This vast microflora fulfills its energy needs by fermenting various types of substrates that have been left undigested in the small intestine, e.g. di- and tri-saccharides, polysaccharides etc. For this fermentation, the microflora produces a vast number of enzymes like glucoronidase, xylosidase, arabinosidase, galactosidase, nitroreductase, azareducatase, deaminase, and urea dehydroxylase. Because of the presence of the biodegradable enzymes only in the colon, the use of biodegradable polymers for colon-specific drug delivery seems to be a more site-specific approach as compared to other approaches. These polymers shield the drug from the environments of stomach and small intestine, and are able to deliver the drug to the colon. On reaching the colon, they undergo assimilation by micro-organism, or degradation by enzyme or break down of the polymer back bone leading to a subsequent reduction in their molecular weight and thereby loss of mechanical strength. They are then unable to hold the drug entity any longer. iv) Prodrug Approach for Drug Delivery to Colon8:- “Prodrug” is a pharmacologically inactive derivative of a parent drug molecule that requires spontaneous or enzymatic transformation in vivo to release the active drug. For colonic delivery, the prodrug is designed to undergo minimal hydrolysis in the upper tracts of GIT, and undergo enzymatic hydrolysis in the colon there by releasing the active drug moiety from the drug carrier. Metabolism of azo compounds by intestinal bacteria is one of the most extensively studied bacterial metabolic processes. A number of other linkages susceptible to bacterial hydrolysis especially in the colon have been prepared where the drug is attached to hydrophobic moieties like amino acids, glucoronic acids, glucose, glactose, cellulose etc. Limitations of the prodrug approach are that, it is not a very versatile approach as its formulation depends upon the functional group available on the drug moiety for chemical linkage. Furthermore, prodrugs are new chemical entities, and need a lot of evaluation before being used as carriers. v) Azo-Polymeric Prodrugs8:- Newer approaches are aimed at the use of polymers as drug carriers for drug delivery to the colon. Both synthetic as well as naturally occurring polymers have been used for this purpose. Sub synthetic polymers have been used to form polymeric prodrug with azo linkage between the polymer and drug moiety. These have been evaluated for CDDS. Various azo polymers have also been evaluated as coating materials over drug cores. These have been found to be similarly susceptible to cleavage by the azo-reducatase in the large bowel. Coating of peptide capsules with polymers cross linked with azo-aromatic group have been found to protect the drug from digestion in the stomach and small intestine.



vi) Polysaccharide Based Delivery Systems8:- The use of naturally occurring polysaccharides is attracting a lot of attention for drug targeting the colon since these polymers of monosaccharides are found in abundance, have wide availability are inexpensive and are available in a verity of a structures with varied properties. They can be easily modified chemically, biochemically, and are highly stable, safe, nontoxic, hydrophilic and gel forming and in addition, are biodegradable. These include naturally occurring polysaccharides obtained from plant (guar gum, inulin), animal (chitosan, chondrotin sulphate), algal (alginates) or microbial (dextran) origin. The polysaccrides can be broken down by the colonic microflora to simple saccharides.24 Therefore, they fall into the category of “generally regarded as safe” (GRAS). b) Newly Developed Approaches for CDDS i) Pressure Controlled Drug-Delivery Systems8:- As a result of peristalsis, higher pressures are encountered in the colon than in the small intestine. Takaya et al. developed pressure controlled colon-delivery capsules prepared using ethylcellulose, which is insoluble in water. In such systems, drug release occurs following the disintegration of a water-insoluble polymer capsule because of pressure in the lumen of the colon. The thickness of the ethyl cellulose membrane is the most important factor for the disintegration of the formulation. The system also appeared to depend on capsule size and density. Because of re-absorption of water from the colon, the viscosity of luminal content is higher in the colon than in the small intestine. It has therefore been concluded that drug dissolution in the colon could present a problem in relation to colon-specific oral drug delivery systems. In pressure controlled ethyl cellulose single unit capsules the drug is in a liquid. Lag times of three to five hours in relation to drug absorption were noted when pressure-controlled capsules were administered to humans. ii) Novel Colon Targeted Delivery System (CODESTM) 8:- CODESTM is a unique CDDS technology that was designed to avoid the inherent problems associated with pH or time dependent systems. CODESTM is a combined approach of pH dependent and microbially triggered CDDS. It has been developed by utilizing a unique mechanism involving lactulose, which acts as a trigger for site specific drug release in the colon. The system consists of a traditional tablet core containing lactulose, which is over coated with and acid soluble material, Eudragit E, and then subsequently overcoated with an enteric material, Eudragit L. The premise of the technology is that the enteric coating protects the tablet while it is located in the stomach and then dissolves quickly following gastric emptying. The acid soluble material coating then protects the preparation as it passes through the alkaline pH of the small intestine. Once the tablet arrives in the colon, the bacteria enzymetically degrade the polysaccharide (lactulose) into organic acid. This lowers the pH surrounding the system sufficient to affect the dissolution of the acid soluble coating and subsequent drug release.



Figure 11: Schematics of the conceptual design of CODES™

iii) Osmotic Controlled Drug Delivery (ORDS-CT) 8:- The OROS-CT can be used to target the drug locally to the colon for the treatment of disease or to achieve systemic absorption that is otherwise unattainable. The OROS-CT system can be a single osmotic unit or may incorporate as many as 5-6 push-pull units, each 4 mm in diameter, encapsulated within a hard gelatin capsule. Each bilayer push pull unit contains an osmotic push layer and a drug layer, both surrounded by a semipermeable membrane. An orifice is drilled through the membrane next to the drug layer.

Figure 12: Cross-Section of the OROS-CT colon targeted drug delivery system



Immediately after the OROS-CT is swallowed, the gelatin capsule containing the push-pull units dissolves. Because of its drug-impermeable enteric coating, each push-pull unit is prevented from absorbing water in the acidic aqueous environment of the stomach, and hence no drug is delivered. As the unit enters the small intestine, the coating dissolves in this higher pH environment (pH >7), water enters the unit, causing the osmotic push compartment to swell, and concomitantly creates a flowable gel in the drug compartment. Swelling of the osmotic push compartment forces drug gel out of the orifice at a rate precisely controlled by the rate of water transport through the semipermeable membrane. For treating ulcerative colitis, each push pull unit is designed with a 3-4 h post gastric delay to prevent drug delivery in the small intestine. Drug release begins when the unit reaches the colon. OROS-CT units can maintain a constant release rate for up to 24 hours in the colon or can deliver drug over a period as short as four hours. c) Systems with Capsular Structures Several single unit pulsatile dosage forms with a capsular design have been developed. Most consist of an insoluble capsular body, which contains the drug, and a plug, which is removed after a predetermined lag time because of swelling, erosion or dissolution. Linkwitz et al. described the delivery of agents from osmotic systems based on an expandable orifice technology. The system is in the form of a capsule from which the drug is delivered by the capsule’s osmotic infusion of moisture from the body. The delivery orifice opens intermittently to achieve a pulsatile delivery effect. The orifice forms in the capsule wall, which is constructed of an elastic material, preferably elastomer (e.g. styrene-butadiene copolymer), which stretches under a pressure differential caused by the pressure rise inside the capsule as the osmotic infusion progresses. 9 The orifice is small enough that, when the elastic wall is relaxed, the flow rate of drug through the orifice is substantially zero; however, when the elastic wall is stretched, because of the pressure differential across the wall exceeding a threshold, the orifice expands sufficiently to allow the release of the drug at a physiologically required rate. This osmotically driven delivery device can be used as an implant in the anal-rectal passageway, in the cervical canal, as an artificial gland, in the vagina, as a ruminal bolus and so on. Niwa et al. prepared a novel capsule made from ethyl cellulose for the time-controlled release of drugs in the colon. Initially, the capsule was prepared using a gelatin capsule with ethyl cellulose, followed by dissolution of the gelatin in water. The thickness of the ethyl cellulose capsule body was varied and the effect of the wall thickness on the release of the drugs in the capsules was investigated. The ethyl cellulose capsules contained a large number of mechanically made micropores (400 µm) at the bottom. A swellable layer, consisting of low-substituted hydroxypropyl cellulose (L-HPC), was also located in the bottom of the capsule body. Above the swellable layer was the drug reservoir, which contained a mixture of the model drug, fluorescein and a bulking agent, such as lactose or starch. 9



The capsule was then capped and sealed with a concentrated ethyl cellulose solution. After administration of the drug containing capsule, water molecules penetrated the capsule through the micropores in the bottom of the capsule body. Hydration and swelling of the L-HPC induced an increase in the internal osmotic pressure, which resulted in the “explosion” of the capsule and a burst-like drug release was observed. The lag time of the drug release could be altered by altering the thickness of the capsule.

Figure 13: Capsular Drug Delivery Systems The Pulsincap® system consists of a water-insoluble capsule body, which is filled with the drug formulation. The capsule is closed at the open end with a swellable hydrogel plug. The dimensions and the position of the plug can control the lag time prior to drug release. In order to ensure rapid release of the drug, effervescent agents or disintegrants can be included in the drug formulation, in particular with water-insoluble drugs. 9

Figure 14: The Pulsincap® Systems



This system is coated with an enteric layer, which dissolves upon reaching the higher pH region of the small intestine. This system comprises insoluble capsules and plugs. The plugs consist either of swellable materials, which are coated with insoluble but permeable polymers (e.g. polymethacrylates), or of erodible substances, which are compressed (e.g. HPMC, polyvinyl alcohol, polyethylene oxide) or prepared by congealing melted polymers (saturated polyglycolated glycerides or glyceryl monooleate). The erosion of the plug can also be controlled enzymatically; a pectin plug can be degraded by incorporating pectinolytic enzymes directly into the plug. Insoluble Body Enteric Outer Layer Drug Release

Soluble Cap Cap dissolves Plug Erosion

Figure 15: The working principle of Pulsincap® Systems Another group of researchers developed a “Chronopharmaceutical capsule” using an ethyl-cellulose-coated gelatin capsule as the insoluble shell and high swelling L-HPC excipients, which was found to be a more reliable expulsion system than effervescent agents. Pulsatile systems based on multiparticulates for oral administration are described by Percel. The delivery system can be a capsule or tablet composed of a large number of pellets consisting of two or more particle populations. Each pellet has a core that contains the therapeutic drug and a water-soluble osmotic agent (e.g. NaCl). A waterpermeable, water-insoluble polymer film encloses each core. A hydrophobic water-insoluble agent that alters permeability (e.g. wax, fatty acid or salts of fatty acids) is incorporated into the polymer film. The rate at which water passes from the film coating through to the core differs for each pellet population in the dosage form. The osmotic agent dissolves in water, which causes the pellets to swell and thereby regulates the rate of diffusion of the drug from the dosage form. The effect of each pellet releasing its drug into the environment sequentially provides a series of pulsatile administrations of the drug from a single dosage form. The coating thickness may also vary among pellet populations. US Patent 20010046964 describes a capsule capable of delivering therapeutic agents into the body in a time-controlled or position-controlled pulsatile release fashion; it is composed of a multitude of multicoated particulates (beads, pellets, granules etc.). Each of these beads, except an immediate-release bead, has at least two coated membrane barriers. One is composed of a mixture of a water-insoluble polymer and an enteric polymer. The composition and the thickness of the polymeric membrane barriers determine the lag time and the duration of the drug release from each of the bead populations.



US Patent 6627223B2 describes a capsule having the capability of delivering therapeutic agents into the body in a time-controlled or position-controlled pulsatile release fashion. The dosage form comprises a multitude of multicoated particulates. The time-controlled series of pulses occurs several hours after oral administration, with or without immediate release. One of the coating membranes is composed of an enteric polymer and the second membrane barrier is composed of a mixture of a water-insoluble polymer and an enteric polymer. The composition and the thickness of the polymeric membranes determine the lag time and the duration of drug release from each of the bead populations. In other preparations, an organic acid, such as fumaric acid, citric acid, succinic acid, tartaric acid or malic acid, is included and a maleic-acid-containing membrane may be provided between the first and second membrane layers to provide for the time-separated pulses. The acids in between the membranes may delay the dissolution of the enteric polymer in the inner layer, thereby increasing the lag time as well as decreasing the rate of release of the active ingredient from the coated microparticulates. The enteric coating membrane is generally incorporated in the innermost layer to have the drugs released in the lower intestine. One of the membrane layers is made of plasticized enteric polymer whereas the other may be a mixture of a water-insoluble polymer and a plasticized water-soluble/dispersible enteric polymer. US Patent 7048945B2 provides a method for manufacturing a multiparticulate dosage form having a time-controlled series of pulses occurring several hours after oral administration, with or without an immediate-release pulse upon oral administration. The dosage form consists of an active core, a first membrane of an enteric polymer and a second membrane of a mixture of water-insoluble and enteric polymers. US Patent 6531152B1 describes a delivery system for targeted delivery with burst release within the gastrointestinal tract. The delivery system contains a core and a coating. The core contains a drug in combination with a career material. The career material has the property of swelling upon contact with an aqueous medium. The core has the ability to absorb larger quantities of fluid and disintegrates faster in that fluid. The career material comprises a water-insoluble polymer (e.g. calcium pectinate, calcium alginate etc.), which swells considerably but does not form a strong gel, a disintegrant (e.g. crospovidone) and a hardness enhancer (e.g. microcrystalline cellulose). This type of delivery system allows the controlled introduction of water from the surrounding medium into the device. When an aqueous medium comes in contact with particulate matter, the particulate matter swells. The particles eventually form channels from the outer part of the device to the core containing the drug. The core imbibes fluid and then swells, breaks the coating and disintegrates, and all or most of the drug is released with a burst effect. A time-controlled explosion system is described in US Patent 4871549. In this system, a drug is coated on to the seed along with the swelling agent, and the finished pellets are then coated with water-insoluble materials. Drug release is time controlled by the breakage of the external water-insoluble membrane, which is caused by the explosive swelling effect of the swelling agent. The coating thickness of the particles is increased to delay release of the drug. However, this system has the drawback of failing to release the drug if the swelling agent fails to rupture the water-insoluble coating. Further, it lacks the flexibility of enabling various delivery patterns because the thickness of the coating determines the release of the drug.



2.4 Approaches used for the evaluation of Drug Delivery to Colon (CDDS):- For in vitro evaluation, not any standardized evaluation technique is available for evaluation of CDDS because an ideal in vitro model should posses the in-vivo conditions of GIT such as pH, volume, stirring, bacteria, enzymes, enzyme activity, and other components of food. Generally, these conditions are influenced by the diet, physical stress, and these factors make it difficult to design a standard in-vitro model. In vitro models used for CDDS are: a) In vitro dissolution test 8 Dissolution of controlled-release formulations used for colon-specific drug delivery are usually complex, and the dissolution methods described in the USP cannot fully mimic in vivo conditions such as those relating to pH, bacterial environment and mixing forces. Dissolution tests relating to CDDS may be carried out using the conventional basket method. Parallel dissolution studies in different buffers may be undertaken to characterize the behavior of formulations at different pH levels. Dissolution tests of a colon-specific formulation in various media simulating pH conditions and times likely to be encountered at various locations in the gastrointestinal tract have been studied. The media chosen were, for example, pH 1.2 to simulate gastric fluid, pH 6.8 to simulate the jejunal region of the small intestine, and pH 7.2 to simulate the ileum segment. Enteric-coated capsules for CDDS have been investigated in a gradient dissolution study in three buffers. The capsules were tested for two hours at pH 1.2, then one hour at pH 6.8, and finally at pH 7.4. b) In vitro enzymatic tests 8 Incubate carrier drug system in fermenter containing suitable medium for bacteria (strectococcus faccium and B. Ovatus). The amount of drug released at different time intervals are determined. Drug release study is done in buffer medium containing enzymes (ezypectinase, dextranase), or rat or guinea pig or rabbit cecal contents. The amount of drug released in a particular time is determined, which is directly proportional to the rate of degradation of polymer carrier. c) In vivo evaluation 8 A number of animals such as dogs, guinea pigs, rats, and pigs are used to evaluate the delivery of drug to colon because they resemble the anatomic and physiological conditions as well as the microflora of human GIT. While choosing a model for testing a CDDS, relative model for the colonic diseases should also be considered. Guinea pigs are commonly used for experimental IBD model. The distribution of azoreductase and glucouronidase activity in the GIT of rat and rabbit is fairly comparable to that in the human. d) Drug Delivery Index (DDI) and Clinical Evaluation of CDDS 8 DDI is a calculated pharmacokinetic parameter, following single or multiple dose of oral colonic prodrugs. DDI is the relative ratio of RCE (Relative colonic tissue exposure to the drug) to RSC (Relative amount of drug in blood i.e. that is relative systemic exposal to the drug). High drug DDI value indicates better colon drug delivery. Absorption of drugs from the colon is monitored by colonoscopy and intubation. Currently, gamma scintigraphy and high frequency capsules are the most preferred techniques employed to evaluate colon drug delivery systems.



3. Pulsatile Technology:

The oral controlled-release system shows a typical pattern of drug release in which the drug concentration is maintained in the therapeutic window for a prolonged period of time, thereby ensuring sustained therapeutic action. However, there are certain conditions for which such a release pattern is not suitable. These conditions demand release of drug after a lag time. In other words, it is required that the drug should not be released at all during the initial phase of dosage form administration. Such a release pattern is known as pulsatile release.10

Recent studies have revealed that diseases have a predictable cyclic rhythm and that the timing of medication regimens can improve the outcome of a desired effect. This condition demands release of drug as a "pulse" after a time lag and such system has to be designed in a way that complete and rapid drug release should follow the lag time. Such systems are known as pulsatile drug delivery systems (PDDS), time-controlled systems, or sigmoidal release systems (Fig 16). 10

Figure 16: Schematic representation of different drug delivery systems where (a) = sigmoidal release after lag time, (b) = delayed release after lag time, (c) = sustained release after lag time, (d) = extended release without lag time.

PDDS have been developed in close connection with emerging chronotherapeutic views. In this respect, it is well established that the symptoms of many pathologies, as well as the pharmacokinetic and pharmacodynamic profiles of most drugs, are subject to circadian variation patterns.



As far as widespread chronic pathologies with night or early morning symptoms are concerned, such as cardiovascular disease (CVD), bronchial asthma and rheumatoid arthritis, remarkable efficacy, tolerability and compliance benefits could arise from modified release medications. After bedtime administration, would allow the onset of therapeutic drug concentrations to coincide with the time at which disease manifestations are more likely to occur. Performance of pulsatile delivery fulfils such goals.

3.1 Diseases targeted for pulsatile technology:-

Diseases presently targeted for chronopharmaceutical formulations are those for which there are enough scientific backgrounds to justify PDDS- compared to the conventional drug administration approach. They include: hypercholesterolemia, asthma, cancer, duodenal ulcer, arthritis, diabetes, neurological disorders, cardiovascular diseases (e.g. hypertension and acute myocardial infarction) and colonic delivery. 10

3.1.1 Arthritis

The chronobiology, chronopharmacology and chronotherapeutics of pain have been extensively reviewed. For instance, there is a circadian rhythm in the plasma concentration of C - reactive protein and interleukin-6 of patients with rheumatoid arthritis. Patients with osteoarthritis tend to have less pain in the morning and more at night; while those with rheumatoid arthritis, have pain that usually peaks in the morning and decreases throughout the day. Chronotherapy for all forms of arthritis using NSAIDs should be timed to ensure that the highest blood levels of the drug coincide with peak pain.

3.2 Methodologies for PDDS:-

Methodologies for the PDDS can be broadly classified into four classes; 10 I. Time controlled pulsatile release A.Single unit system B. Multi-particulate system II. Stimuli induced A.Thermo-Responsive Pulsatile release B.Chemical stimuli induced Pulsatile systems III. External stimuli pulsatile release A.Electro responsive pulsatile release B. Magnetically induced pulsatile release IV. Pulsatile release systems for vaccine and hormone products



3.3 Time controlled Pulsatile release on Capsular system:- Different single-unit capsular PDDS have been developed. A general -design of such systems consists of an insoluble capsule body housing a drug and a plug. The plug is removed after a predetermined time lag due to swelling, erosion, or dissolution. 10

The Pulsincap® system is an example of such a system that is made up of a water-insoluble capsule body filled with drug formulation. The body is closed at the open end with a swellable hydrogel plug. Upon contact with dissolution medium or gastro-intestinal fluids, the plug swells, pushing itself out of the capsule after a time lag. This is followed by a spontaneous release of the drug (Fig 17). 10

The time lag can be controlled by manipulating the dimension and the position of the plug. For water insoluble drugs, a spontaneous release can be ensured by inclusion of effervescent agents or disintegrants. The plug material consists of insoluble but permeable and swellable polymers (e.g.:polymethacrylates), erodible compressed polymers (e.g: hydroxypropylmethyl cellulose, polyvinyl alcohol, polyethylene oxide), congealed melted polymers (e.g: saturated polyglycolated glycerides, glycerylmonoole and enzymatically controlled erodible polymer e.g:pectin).

These formulations are well tolerated in animals and healthy volunteers, and there have been no reports of gastro-intestinal irritation. However, there was a potential problem of variable gastric residence time, which was overcome by enteric coating the system to allow its dissolution only in the higher pH region of small intestine.

Figure 17: Time controlled Pulsatile release on Capsular system



3.4 Marketed Technologies:-

CODAS® (Chronotherapeutic Oral Drug Absorption System) is one of Elan Drug Technologies, multiparticulate drug delivery systems. The CODAS® technology is designed to allow a 4-5 h delay for onset following administration of drug. This delay in release is introduced by the level of release-a controlling polymer applied to the drug-loaded beads. Applying the CODAS® technology to Verapamil Hydrochloride, Verelan® PM complimented the circadian pattern of hypertension and helped to minimize the risk of early morning cardiovascular events.11

Penwest Pharmaceuticals and Co., USA, considered to be top runner in drug delivery technologies with patented products such as TIMERx® , Geminex® and SyncroDose TM The TIMERx oral drug delivery system achieves a variety of release profiles (First order, Zero order, BurstCR, etc.) for a wide range of drugs, accommodating even the most difficult actives.

TIMERx enabled Penwest to meet the significant challenges of today's pharmaceutical marketplace head-on with 32 US issued patents and 178 patents worldwide. Alza Corporation uses OROS (Osmotic Release Oral Systems) drug delivery platform with marketed products such as Covera-HS® and Procardia XL® . Eurand Pharmaceuticals DIFFUCAPS® technology is a multiparticulate system that provides optimal release profiles for either single drugs or for a combination of drugs. 11

Table 3: The marketed technologies of Pulsatile delivery systems



4. Chronobiology and Chronotherapy of Arthritic Diseases

Chronobiology is the science concerned with the biological mechanism of the diseases according to a time structure and chronopharmacology is the science concerned with the variations in the pharmacological actions of various drugs over a period of time of the day, and based upon this, chronotherapeutics is the discipline concerned with the delivery of drugs according to inherent activities of a disease over a certain period of time. 12 Circadian rhythms are self-sustaining, endogenous oscillations that occur with a periodicity of about 24 hours. Normally, circadian rhythms are synchronized according to internal biologic clocks related to the sleep-wake cycle1. Most people sleep at night and rise in the morning. In night-shift workers (who typically sleep during the day), most circadian rhythms are shifted to match their sleep-wake cycle. 12 The goal of chronotherapeutics is to match the timing of treatment with the intrinsic timing of illness. Theoretically, optimum therapy is more likely to result when the right amount of drug is delivered to the correct target organ at the most appropriate time. 14

Figure 18: Circadian Rhythms of Diseases (Peak Time of Event/Variable) 13

Arthritic diseases such as rheumatoid arthritis, osteoarthritis, ankylosing spondylitis and gout exhibit profound circadian rhythms in the manifestation and intensity of symptoms. 14

4.1 Rheumatoid Arthritis

Rheumatoid arthritis is a chronic inflammatory autoimmune disorder. The cardinal signs of rheumatoid arthritis are stiffness, swelling and pain of one or more joints of the body characteristically most severe in the morning. Rheumatoid arthritis shows a marked circadian variation in its symptoms.



A group of British volunteers self-assessed the pain and stiffness of affected finger joints every 2 to 3 hours daily for several consecutive days. They also measured the circumference of the arthritic joints to gauge the amount of their swelling, and they performed grip strength tests to determine the effect of the arthritic condition on the hands. Ratings of the severity of joint pain swelling and stiffness were about 3 times higher between 08:00 and 11:00 than at bedtime. In contrast, hand strength was lower by as much as 30% in the morning than at night.15

Figure 19: Diagram of chronological biological processes in rheumatoid arthritis (RA) compared with those in healthy controls. 15

It is well known that some clinical signs and symptoms of rheumatoid arthritis (RA) vary within a day and between days, and the morning stiffness seen in patients with RA has become one of the diagnostic criteria of the disease. 16

Figure 20: Clinical signs and symptoms in RA depend on the time of day16



Clinical signs and symptoms of articular inflammation in patients with RA change consistently as a function of the hours of the day. Pain and joint stiffness are greater after waking up in the morning than in the afternoon or evening. Among the clinical signs of joint inflammation in patients with RA, the intensity of pain changes consistently as a function of the hours of the day. Pain is greater after waking up in the morning than in the afternoon or evening. In patients with RA circadian variations are also found in joint swelling and finger size and these symptoms are in phase with the circadian rhythm of pain. The RA rhythms differ in phase by about 12 hours from the circadian changes of left and right hand grip strength. Greater grip strength is seen when joint circumferences and the subjective ratings of stiffness and pain are least and vice versa. Therefore, clinical signs and symptoms in RA show a rhythm that seems driven by a biological clock. 16

4.2 Biological rhythms in experimental inflammation

Biological rhythms have been seen in different models of inflammation, and maximal inflammation occurred during the activity period of the animals, that is, between midnight and 8.00 am. Biological rhythms with a periodicity longer than 24 hours have also been detected, and a circaseptan rhythm (almost seven days) of paw oedema, over a period of 30 days, was observed, with peak of inflammation every 6–7 days. 16

Furthermore, circannual variations have been identified in different models of inflammation showing that maximal articular oedema was significantly larger in spring and lowest in winter. A time dependent change of blood flow at the inflammatory site may also explain the circadian variations in experimental oedema; some studies in rat models showed that the blood flow was greater in the night and lower in the morning. 16

The mechanisms of the time dependent variations of the inflammatory reaction are complex and include several systems of mediators (that is, histamine, bradikinin, prostaglandin, and mainly, pro- and anti-inflammatory cytokine production). However, the circadian changes in the metabolism or secretion of endogenous corticosteroids are certainly implicated in the time dependent changes seen during the inflammatory response. This assumption is supported by data showing that adrenalectomy abolished the circadian variation in the rate of formation of experimental oedema and that this was restored by hydrocortisone administration. 16

More recently, melatonin (MLT), another circadian hormone that is the secretory product of the pineal gland, has been found to be implicated in the time dependent inflammatory reaction, with effects opposite to those of cortisol. In several species, pinealectomy or any other experimental procedure that inhibits MLT synthesis and secretion induces a state of immunodepression, which is counteracted by MLT replacement. 16

In general, MLT displays an immunoenhancing effect. MLT can activate T lymphocytes, monocytes, NK cells, and even neutrophils, activates antibody dependent cellular cytotoxicity, and enhances antibody responses in vivo. In animal models, as well as in human and in vitro experiments, MLT enhances inflammatory cytokine and nitric oxide production. In addition, the in vitro effects exerted by glucocorticoids on the immune function seem modulated by MLT in physiological to pharmacological concentrations. 16



4.3 Cortisol and melatonin regulated circadian cytokine production

In adult primates, only visible light (400–700 nm) is received by the retina. This photic energy is then transduced and delivered to the visual cortex and, by an alternative pathway, to the suprachiasmatic nucleus, the hypothalamic region that directs circadian rhythms. Visible light exposure also modulates the pituitary and pineal glands, leading to neuroendocrine changes. MLT, norepinephrine, and acetylcholine decrease with light activation, whereas cortisol, serotonin, γ-aminobutyric acid, and dopamine levels increase. 16

Therefore, ocular light seems to be the predominant modulator and major determinant of circadian rhythm for many neurohormones, with cortisol and MLT showing an opposite response to the light. The light conditions in the early morning have a strong impact on the morning cortisol peak, whereas direct inhibitory effects of light on pineal activity may contribute to phasing of the onset and termination of MLT production in a strictly nocturnal pattern. Melatonin counteracts the effects of cortisol. Recently, a diurnal rhythmicity in healthy humans between cellular (Th1 type) or humoral (Th2 type) immune responses has been found and related to immunomodulatory actions of cortisol and MLT. 16

Figure 21: A diurnal rhythmicity in healthy humans between cellular (Th1 type) or humoral (Th2 type) immune responses has been found and related to the immunomodulatory effects exerted by cortisol and melatonin, respectively.

In particular, the production of the interferon γ (IFNγ; type 1) and interleukin 10 (IL10; type 2) in human whole blood stimulated with lipopolysaccharide or tetanus, as well as the IFNγ/IL10 ratio, exhibited a significant diurnal rhythmicity. The IFNγ/IL10 ratio peaked during the early morning and correlated negatively with plasma cortisol and positively with plasma MLT; the IFNγ/IL10 ratio decreased by >70% after the administration of oral cortisone acetate (25 mg). Therefore, these findings support the concept that plasma cortisol and possibly MLT seem to regulate diurnal variations of cytokine production.



In normal subjects,

a) MLT peaks at about 3 am, whereas cortisol peaks at about 4 am. b) Interestingly, IL1, IL6, and soluble IL2 receptors peak at 1–4 am and are low throughout the

day. c) MLT stimulates IL1 and IFNγ production by human monocytes, and serum IL2 increases

during the night concomitantly with the rise in MLT; d) MLT also seems to enhance IL2 immunomodulating effects. e) In addition, MLT increases the production of IL12 and nitric oxide by cultured human

synovial macrophages, enhances IL2, IL6, and IFNγ production by human circulating CD4+ lymphocytes, and up regulates the level of gene expression of tumour necrosis factor α (TNFα) and macrophage-colony stimulating factor.

f) On the contrary, cortisol was found to be negatively correlated with the IFNγ/IL10 ratio, and cortisone administration markedly reduced this ratio with a clear causal relationship.

g) Furthermore, similarly to IFNγ and IL1, TNFα and IL12 also exhibit distinct diurnal rhythms that peak in the early morning, and these changes are inversely related to the rhythm of plasma cortisol.

h) In conclusion, because IFNγ and IL10 might be considered markers of cellular (type 1) and humoral (type 2) immunity, respectively, these circadian studies suggest that there is a bias towards cellular immunity during the night and early morning (peak of MLT) when the IFNγ/IL10 ratio is high and, conversely, a relative bias towards humoral (type 2) immunity during the rest of the day.

4.4 Cortisol and melatonin effects on circadian rhythms in rheumatoid arthritis

The inflammatory cytokines (that is, IL6, IL1, TNFα), as soluble products of the activated immune system, stimulate in the central nervous system, the production of corticotrophin releasing hormone (CRH) in the hypothalamus: CRH release leads to pituitary production of adrenocorticotrophic hormone (ACTH), followed by glucocorticoid secretion by the adrenal cortex. These components constitute the hypothalamic-pituitary-adrenocortical axis (HPA). Recently, intact ACTH secretion, but impaired cortisol response in patients with active RA has been described and this observation was consistent with a relative adrenal glucocorticoid insufficiency. HPA axis function is a normal response to the stress of inflammation and may be mediated by central and peripheral actions of circulating cytokines.

Besides IL1 and TNFα, IL6 seems to be a major factor mediating interactions between the activated immune system and both the anterior pituitary cells (central) and the adrenal (peripheral) steroidogenesis. 16

However, recent studies in patients with RA have shown that the overall activity of the HPA axis remains inappropriately normal (or low) and is apparently insufficient to inhibit ongoing inflammation, at least in patients with early untreated arthritis. In particular, in the early morning hours, an earlier surge of plasma ACTH and cortisol was seen in patients with RA, who at the same time had significantly increased IL6 levels and a pronounced circadian variation of plasma levels in comparison with healthy subjects. 16



In addition, in the patients with RA, a positive temporal correlation was found between plasma IL6 levels and ACTH/cortisol, with raised levels of IL6 before the increases of ACTH and cortisol by one and two hours, respectively. In the same patients, a negative effect of cortisol upon IL6 was found, exerted with a delay of five hours, confirming that the HPA axis in RA is apparently insufficient to inhibit ongoing inflammation.

Recent studies have evaluated MLT levels in patients with RA, together with an analysis of circadian variations. Interestingly, MLT serum levels at 8 pm and 8 am were found to be significantly higher in patients with RA than in controls (p<0.05). The differences were greater in the older patients with RA (age >60 years) than in the younger ones. Both in patients with RA and healthy subjects, MLT levels progressively increased from 8 pm to the early hours of the morning, but reached a peak in patients with RA at 12 pm, at least two hours before those in controls. Subsequently, in patients with RA, MLT concentrations reached a plateau, lasting 2–3 hours; this effect was not evident in the controls. After 2 am, MLT levels decreased similarly both in patients with RA and healthy subjects. The results of the study confirm the existence of a nocturnal rhythm of MLT also in patients with RA. However, the peak appears earlier in the night and lasts longer in the early morning than in healthy controls. 16

Figure 22: A lower than expected cortisol secretion as seen during testing in patients with RA, should be clearly regarded as a “relative adrenal insufficiency” in the setting of a sustained inflammatory process (that is, high IL6 serum levels). 16



As previously discussed, IFNγ and IL2, as well as IL1, IL6, IL12, and TNFα production (Th1 cytokines) reach peak levels during the night and in the early morning, when MLT serum levels are highest and plasma cortisol levels are lowest.

Therefore, MLT may play a part in the induction of a more active inflammatory response during the night, at least in patients with RA, because the disease is considered to be a Th1-cytokine driven immune disease. At that time; the lower than expected levels of cortisol seen in patients with RA; are less efficient in counteracting the effects of MLT. Recently, MLT has been found at a rather high concentration in the synovial fluids of patients with RA, and binding sites for MLT have been detected in synovial macrophages. 16

Figure 23: IFNγ, IL2, as well as IL1, IL6, IL12, and TNFα production (Th1 cytokines) reach a peak during the night and early morning, when MLT serum levels are highest and plasma cortisol levels the lowest. 16 Therefore, MLT may be implicated in a more active inflammatory response during the night, and the clinical symptoms follow this rhythm in patients with RA. 16



4.5 Conclusions of circadian symptoms in patients with RA

An altered functioning of the HPA axis and of the pineal gland seems to be an important factor in the perpetuation of clinical circadian symptoms in patients with RA. The clinical symptoms show a circadian variation, with joint stiffness and pain being more prominent in the early morning. Consistently, human pro-inflammatory cytokine production exhibits a diurnal rhythmicity, with peak levels during the night and early morning when plasma cortisol level is lowest and MLT level is highest.

In particular, Th1 type cytokines that are mainly involved in RA, significantly increase, with an earlier peak in relation to altered peaking of both cortisol and MLT. An inappropriate low secretion of cortisol is a typical feature of the inflammatory disease in patients with RA.

On the contrary, the nocturnal rhythm of MLT shows an earlier peak and longer peak duration in the early morning in patients with RA than in normal subjects. Therefore, an imbalance between anti-inflammatory effects exerted by cortisol and proinflammatory effects exerted by MLT during the night seems evident in patients with RA and suggests that this imbalance may have a crucial pathogenic role in RA, and may also drive the circadian rhythm of the clinical symptoms that is, morning stiffness and pain.



5. Hard Gelatin Capsules and CrossLinking Technology

5.1 Gelatin and Hard Gelatin Capsules Gelatin is a generic term for a mixture of purified protein fractions obtained either by partial acid hydrolysis (type A gelatin) or by partial alkaline hydrolysis (type B gelatin) of animal collagen obtained from cattle and pig bone, cattle skin (hide), pigskin, and fish skin. Gelatin may also be a mixture of both types. The protein fractions consist almost entirely of amino acids joined together by amide linkages to form linear polymers, varying in molecular weight from 20,000–200,000. 18 Gelatin is an amphoteric material and will react with both acids and bases. It is also a protein and thus exhibits chemical properties characteristic of such materials; for example, gelatin may be hydrolyzed by most proteolytic systems to yield its amino acid components. Gelatin will also react with aldehydes and aldehydic sugars, anionic and cationic polymers, electrolytes, metal ions, plasticizers, preservatives, and surfactants. It is precipitated by alcohols, chloroform, ether, mercury salts, and tannic acid. 18 Gelatin is widely used in a variety of pharmaceutical formulations, including its use as a biodegradable matrix material in an implantable delivery system, although it is most frequently used to form either hard or soft gelatin capsules. Gelatin capsules are unit-dosage forms designed mainly for oral administration. Soft capsules on the market also include those for rectal and vaginal administration. Gelatin is soluble in warm water (>30°C), and a gelatin capsule will initially swell and finally dissolve in gastric fluid to release its contents rapidly. 18 Hard capsules are manufactured in two pieces by dipping lubricated stainless steel mold pins into a 45–55ºC gelatin solution of defined viscosity, which depends on the size of the capsules and whether cap or body are to be formed. The gelatin is taken up by the pins as a result of gelation, and the resulting film thickness is governed by the viscosity of the solution. The capsule shells are passed through a stream of cool air to aid setting of the gelatin, and afterwards they are slowly dried with large volumes of humidity controlled air heated to a few degrees above ambient temperature and blown directly over the pins. The capsule halves are removed from their pins trimmed and fitted together. 18 Gelatin that is used to produce hard capsules may contain various coloring agents and antimicrobial preservatives. Surfactants may be present in small quantities in the shells being a residue of the pin lubricant. However, the use of preservatives is no longer encouraged in line with current GMP principles. Capsule shells may be treated with formaldehyde to make them insoluble in gastric fluid.18



5.2 Reaction between the Proteins and Formaldehyde

A study of the literature relating to the reaction between the proteins and formaldehyde suggests that some divergence of opinion exists. a) Harris and Birch in 1930 showed that the titration of amino acids, with hydrochloric acid in

the presence of formaldehyde, is not affected, while the titration with sodium hydroxide is markedly affected. Harris explained this phenomenon as the repression of acidic groups upon acid titration and the repression of basic groups upon alkaline titration.19

b) Tomiyama believes the anionic form of the amino acid reacts with the formaldehyde. He also

considers the protein-formaldehyde reaction in terms of the electronic theory. He pictures the formaldehyde as a dipolar molecule and since the amino or imino group of the anionic form of the amino acid has unshared electrons, the two components react to give the accompanying formula. 19

c) Levy and Silberman have shown mathematically from their studies that 2 molecules of formaldehyde combine with 1 molecule of amino acid. Bergmann et al. have isolated a triformyl compound and further shown that the triformyl derivative changes to the monoformyl upon addition of alkali. Reiner and Marton postulated the following reaction between protein and formaldehyde, 19



d) The aldehyde being held to the amino group by secondary valence. Einhour showed that the

acid amides fix formaldehyde and suggested the following reaction, 19

R-CONH2 + CH2O + R-CONH.CH2OH

e) Cherbuliez and Bergmann found that diketopiperazines react with formaldehyde, taking up 2

molecules of the aldehyde. 19

f) Levy and Silberman have taken exception to the interpretations of Tomiyama, maintaining that he made no distinction between amino and imino groups. Balson and Lawson have suggested that the number of formaldehyde groups which can be introduced corresponds to the number of hydrogen atoms attached to the nitrogen atom and have therefore proposed Reactions 1, 2, and 3. 19



g) Stiasny has suggested that formaldehyde reacts with gelatin in possibly two ways, in one, with the basic groups, changing them to neutral ones, and in the other, with the imino groups of the peptide linkage. He suggests that the first reaction proceeds through an intermediate formation of a triformyl derivative which then changes to the monoformyl. In the second reaction, Stiasny postulates a binding of the formaldehyde with the weakly basic imino groups, forming methyol compounds, 19

h) Stiasny further suggests that the free amino groups of the gelatin react rapidly, while the

peptide groups only do so gradually. He believes that the action of the formaldehyde on the basic groups is such that not only the acid and base fixation capacity is influenced but also that of the fixation of tanning materials and dyes. 19

i) It was suggested by Meyer and Kinzel that the tanning and hardening effect of formaldehyde

on proteins might be ascribed to the formation of methylene cross-links. The work of Custavson and Hadorn included experimental evidence and indicated that free amino groups were essential for the cross-linking. Proteins containing appreciable amounts of amino and also phenol or imidazole groups bound considerably more formaldehyde in a manner irreversible by acid hydrolysis than did derivatives in which the amino groups had been selectively acetylated.20

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7. Materials and Method

7.1 Materials:

S. NO. ITEM ITEM CODE QTY REQUIRED* UNIT

MAIN INGREDIENTS

1. Diclofenac Sodium

DFS

200.00

GRAMS

2. Microcrystalline Cellulose (pH 102)

MCC

200.00

GRAMS

3. PVP- K30

PVP

50.00

GRAMS

4. Isopropyl alcohol

IPA

100.00

ML

5. Hydroxypropyl Methylcellulose

HPMC

100.00

GRAMS

6. Hydroxypropyl cellulose

HPC

100.00

GRAMS

7. Sodium Alginate

SA

100.00

GRAMS

8. Ethyl Cellulose

EC

25.00

GRAMS

9. Ethanol

200.00

ML

10. Empty Hard Gelatin Capsules Size “0”

EHGC

1000.00

NOS.

11. Cellulose Acetate Phthalate

CAP

100.00

GRAMS

12. Acetone

200.00

ML

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1. Formaldehyde 15% V/V

FD

500.00

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10.00

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*Quantities required are approximate value for the Research Works to be carried out.



7.2 Methods:-

7.2.1 Preparation of cross-linked gelatin capsules:-

a) Few ml of 15% (v/v) Formaldehyde will be taken into desiccator and Potassium Permanganate will be added to it, to generate formalin vapors.

b) The wire mesh containing the empty bodies of the 0 size hard gelatin capsule will be then exposed to formaldehyde vapors.

c) The caps will be not exposed leaving them water-soluble. d) The reaction will be carried out for 12 h after which the bodies will be dried at 45OC for 30

minutes to ensure completion of reaction between gelatin and formaldehyde vapors. e) The bodies will be then dried at room temperature to facilitate removal of residual

formaldehyde. f) These capsule bodies will be capped with untreated caps and stored in a polythene bag. g) Various physical tests such as, identification attributes, visual defects, dimension changes,

solubility studies were carried out.

7.2.2 Formulation of pulsatile drug delivery system:-

Granulation:-

a) The multiparticulate mixed bed powders (Sieved by BSS#40) will be prepared by using ratio of Diclofenac Sodium: Microcrystalline Cellulose (pH 102) (1:1).

b) PVPK-30 will be used on the 1 to 1.5% ratios as a binder with Isopropyl Alcohol. c) After complete mixing on a polybag non-aqueous wet granulation method will be carried out

to form granules of active with additives. d) The powder mixture will be kneaded with IPA until a damp mass will be formed. e) The damp mass will be passed manually with low pressure through BSS# 20 to obtain

uniformly sized granules. f) The resulting granules will be dried at 600C and size segregated by BSS# 16/20 and these

spherical granules will be used in the study. Filling and Plugging:- g) The multiparticulates equivalent to 100 mg of Diclofenac Sodium will be accurately weighed

and filled into the treated bodies by hand filling. h) The capsules containing the multiparticulates will be then plugged with different amounts

(e.g. 20, 30 and 40 mg) of various polymers, i.e., guar gum, hydroxylpropylmethylcellulose and sodium alginate.

i) The joint of the capsule body and cap will be sealed with a small amount of the 25% ethyl cellulose on ethanol solution.



Enteric Coating:- j) The sealed capsules will be completely enteric coated with 5% w/w Cellulose Acetate

Phthalate (CAP) in 8:2 (v/v) mixture of acetone and ethanol plasticized with Dibutylphthalate (0.75%), to prevent variable gastric emptying.

k) Coating will be repeated until 10 to 12% increase in weight is obtained. l) Percentage weight gain of the capsules before and after coating will be determined.

7.3 Evaluation of in-vitro release profile of Modified Pulsincap Drug Delivery System:-

a) Dissolution studies will be carried out by using USP dissolution test apparatus (Basket) method. Capsules will be placed in a basket so that the capsule should be immersed completely in dissolution media but not float.

b) In order to simulate the pH changes along the GI tract, three dissolution media with pH 1.2, 7.4 and 6.8 will be sequentially used referred to as sequential pH change method.

c) While performing experiments, the pH 1.2 medium will be first used for 2 hours (since the average gastric emptying time is 2 h), then removed and the fresh pH 7.4 phosphate buffer saline (PBS) will be added.

d) After 3 hours (average small intestinal transit time is 3 h), the medium will be removed and fresh pH 6.8 dissolution medium will be added for subsequent hours.

e) Nine hundred milliliters of the dissolution medium will be used at each time. f) Rotation speed will be maintained at 100 rpm and temperature will be maintained at 37±0.5

◦C. g) The withdrawn samples will be analyzed by UV absorption spectroscopy and the cumulative

percentage release will be calculated over the sampling times.



8. Work Description & Time Schedule



9. References

1. NS Dey, S Majumdar and MEB Rao, Multiparticulate Drug Delivery Systems for Controlled Release, (Review Article), Tropical Journal of Pharmaceutical Research, September 2008; 7 (3): 1067-1075.

2. Navnit H. Shah, Ph.D., Multi-Particulate Dosage Form for Oral Controlled Release : Development Considerations, Power Point Presentation, Hoffmann La-Roche, Inc.

3. Shaji J., Chadawar V.,Talwalkar P., Multiparticulate Drug Delivery System, The Indian Pharmacist,

June 2007.

4. Tang E. S.K., Chan L.W, Heng P.W.S, Amer J., Coating of Multiparticulates for Sustained Release, Drug Delivery 2005

5. Preparing Modified Release Multiparticulate Dosage Forms with Eudragit Polymers, Pharma Polymers, November 2002

6. Laila Fatima, Ali Asghar and Sajeev Chandran, Multiparticulate Formulationm Approach to Colon Specific Drug Delivery: Current Perspectives; Formulation Development & Pharmacokinetic Laboratory, Pharmacy Group, Birla Institute of Technology & Science, Pilani, Rajasthan, INDIA, J. Pharm Pharmaceutical Science , 9 (3): 327-338, 2006

7. Prof V. R. Sinha, Site Specific Drug Delivery to Colon : Approaches and Recent Developments, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160014

8. Anil K. Philip, Betty Philip, Colon Targeted Drug Delivery Systems: A Review on Primary and Novel Approaches, Oman Medical Journal 2010, Volume 25, Issue 2, April 2010

9. Anil K. Anal, Time-Controlled Pulsatile Delivery Systems for Bioactive Compounds, Recent Patents

on Drug Delivery & Formulation 2007, 1, 73-79, Bentham Science Publishers Ltd., New Zealand

10. Sharma GS, Srikanth MV, Uhumwangho MU, Phani Kumar KS and Ramana Murthy KV, Recent trends in pulsatile drug delivery systems - A review, International Journal of Drug Delivery 2, (2010) , 200-212

11. Shidhaye SS, Lotlikar VM, Ghule AM, Phutane PK, Kadam VJ. Pulsatile delivery systems: An

approach for chronotherapeutic diseases. Systamatic Review in Pharmacy , 2010;1:55-61. 12. Bi Bottic , Youan, Overview of Chronopharmaceutics.

13. Jha N. , Bapat S., Chronobiology and Chronotherapeutics, Department of Pharmacology, Kathmandu

Medical College, Kathmandu University Medical Journal (2004) Vol. 2, No. 4, Issue 8, 384-388



14. J. D. Patel, K. Anjela, S. H. Majmudar, Pulsatile Drug Delivery System: An user friendly dosage form, JPRHC, Volume 2, Issue 2, 204-215, April 2010

15. J W G Jacobs, J W J Bijlsma, Modified release prednisone in patients with rheumatoid arthritis, University Medical Center Utrecht, Utrecht, The Netherlands, Rheum Dis 2010;69:1257-1259 doi:10.1136/ard.2010.132738

16. M Cutolo, B Seriolo, C Craviotto, C Pizzorni, A Sulli, Circadian rhythms in Rheumatoid Arthritis, Author Affiliations: Research Laboratory and Division of Rheumatology, Department of Internal Medicine, University of Genova, Viale Benedetto XV,6 16132 Genova, Italy.

17. SS Shidhaye, VM Lotlikar, AM Ghule, PK Phutane, VJ Kadam, Pulsatile delivery systems: An approach for chronotherapeutic diseases, Department of Pharmaceutics, Vivekanand Education Society's College of Pharmacy, Behind Collector Colony, Chembur (E), Mumbai - 400 074, India

18. Handbook of Pharmaceutical Excipients, 5th Edition, Edited by: Raymond C Rowe, BPharm, PhD, DSc, FRPharmS, CChem, FRSC, CPhys, MInstP, 2006, 295-296.

19. Edwin R. Theis, The Protein and Formaldehyde Reaction, Biochemistry Division, Department of

Chemistry, Lehigh University, Bethlehem, March 8, 1944.

20. Heinz Frankel Conrat, The Reaction of Formaldehyde with Protein, Demonstration of Intermolecular Crosslinking by means of Osmotic Pressure measurement, Sep 7, 1948

21. John A. Kiernan, Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: What they are and

what they do. Microscopy Today, 2000- Issue 1, pp. 8-12

22. Sven Stegemann, Capsugel Borenem, Hard Gelatin Capsules, Today and Tomorrow, 2nd edition 2002, Capsugel library.

23. Shivakumar H.N, Suresh S, Desai B.G., Design and evaluation of controlled onset extended release multiparticulate systems for chronotherapeutic delivery of ketoprofen, Indian journal of Pharmaceutical Science 2006.

24. Dr. M.S.Srinath, Vijaya D.C., Development and Comparative In-Vitro evaluation of Sustained release bilayered matrix tablet and sustained release capsule of model antihypertensive drug, Rajiv Gandhi University of Health Sciences, Karnataka, Department of Pharmaceutics, Government College of Pharmacy,

25. Cheng G, An F, Zou MJ, Sun J, Hao XH, He YX. Time- and pH dependent colon-specific drug

delivery for orally administered Diclofenac Sodiumand 5-aminosalicylic acid, World J Gastroenterol 2004; 10(12): 1769-1774, http://www.wjgnet.com/1007-9327/10/1769.asp

26. M. Najmuddin, Vishal Ashok Patel, Azgar Ali, S. Shelar, K Tousif, Development and evaluation of

pulsatile drug delivery system of flurbiprofen, Research Journal of Pharmaceutical, Biological and



Chemical Sciences, Department of Pharmaceutics, Luqman College of Pharmacy, Gulbarga-585102, Karnataka, India

27. J W G Jacobs, J W J Bijlsma, Modified release prednisolone in patients with rheumatoid arthritis,

Department of Rheumatology and Clinical Immunology, F02.127, University Medical Center Utrecht, PO Box 85500, 3508 GA Utrecht, The Netherlands; [email protected] , June 2010

28. Ina Krogel, Roland Bodmeier, Pulsatile Drug Release from an Insoluble Capsule Body Controlled by an Erodible Plug, December 5, 1997. Pharmaceutical Reserch , Volume 15 , No. 3, 1998.

29. U.Y. Nayak , G. V. Shavi , Y. Nayak , R. K. Averinen , S. Mutalik , S. Meka Reddy, P. Das Gupta and N. Udupa, Chronotherapeutic drug delivery for early morning surge in blood pressure: A programmable delivery system, Journal of Controlled Release 136 (2009) 125–131.

30. Mukesh C. Gohel, Manhapra Sumitra G, Modulation of active pharmaceutical material release from a novel ‘tablet in capsule system’ containing an effervescent blend, Journal of Controlled Release 79 (2002) 157–164.

31. T. Bussemer, R. Bodmeier, Formulation parameters affecting the performance of coated gelatin capsules with pulsatile release profiles, International Journal of Pharmaceutics 267 (2003) 59–68.

32. V.S. Mastiholimath , P.M. Dandagi, S. Samata Jain, A.P. Gadad, A.R. Kulkarni, Time and pH dependent colon specific, pulsatile delivery of theophylline for nocturnal asthma , International Journal of Pharmaceutics 328 (2007) 49–56.

33. Howard N.E. Stevens, Clive G. Wilson, Peter G. Welling, Massoud Bakhshaee , Julie S. Binns, Alan C. Perkins, Malcolm Frier, Elaine P. Blackshaw, Margaret W. Frame, Don J. Nichols, Michael J. Humphrey, Steve R. Wicks, Evaluation of Pulsincap™ to provide regional delivery of dofetilide to the human GI tract, International Journal of Pharmaceutics 236 (2002) 27–34

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