thoughts from science leaders of tomorrow · 2014-09-11 · thoughts from science leaders of...

catalent institute: thoughts from science leaders of tomorrow 1

THOUGHTS FROM SCIENCE LEADERS OF TOMORROW

www.drugdeliveryinstitute.com2

CONTENTS

FOREWORD

SLIPPING THE BLOOD BRAIN BARRIER WITH CELL PENETRATING PEPTIDE MIMICS

CHALLENGES IN DEVELOPING INHALATION TECHNOLOGY: RIGHT PARTICLE SIZE AND DEVICE SELECTION

PEDIATRIC DRUGSREGULATORY CHALLENGES AND AVAILABLE DOSAGE FORMS

STEM CELLS AS NOVEL CARRIERS FOR CANCER THERAPY

DOUBLE EMULSIONS: SCOPE AND ATTEMPTS

ACKNOWLEDGEMENTS

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FOREWORD

The Catalent Applied Drug Delivery Institute is committed to investing in future life science leaders of tomorrow. The Catalent Institute collaborated with our partners at the American Association of Pharmaceutical Scientists (AAPS) by launching the 3rd Annual Global Academic Competition in the fall of 2013. Graduate students studying life sciences, pharmaceutical sciences, and chemical engineering from universities around the globe had the opportunity to write review articles on one of seven drug delivery topics.

A panel of judges made up of industry and academic scientists selected the five winning entries which you have the opportunity to read and learn from in this publication.We are proud of our finalists representing universities in the Unites States and European Union.

You will see topics related to drug delivery which are quite diverse. The discovery of tumor-homing capacity of stem cells and specifically mesenchymal stem cells (MSC) has raised great interest in the past decade. As a promising alternative to the conventional gene therapy, there are many variables attributed to the application of MSC that need to be standardized for the safety and efficacy of cancer treatment. Learn how cell penetrating peptide mimics (CPPMs) can support treatments that must cross the blood brain barrier

by delivering antibodies to specifically target an intracellular molecule. There is novel guidance from regulatory agencies encouraging the availability and innovation of pediatric dosage forms. Double emulsions are complex heterogeneous systems that may have great potential in pharmaceutical application but their inherent instability poses as the limiting factor.

Various attempts are reviewed to overcome the issue of instability in order to make a robust formulation. Successful inhalational technology requires a good formulation and device. While the right particle size, either solid or liquid form, decides the fate of inhaled pharmaceuticals, the appropriate device drives the formulation to the intended site. Successful teamwork of a formulator and the device engineer along with integrated assistance of software and electronics has resulted in several revolutionary inhalational technologies. These are the exciting subjects that you will discover in this publication.

The Catalent Institute is dedicated to bringing industry and academia together to identify needs and provide resources for the most promising innovations related to improved drug delivery. We hope that initiatives like this annual competition ultimately inspire learning which leads to improved outcomes for patients.

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BY: TERRY ROBINSONExecutive Director


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BY: EMRAH ILKER OZAY

SLIPPING THE BLOOD BRAIN BARRIER WITH CELL PENETRATING PEPTIDE MIMICS


INTRODUCTIONThe blood-brain barrier (BBB) may be the most important barrier protecting the brain against unwanted molecules despite impeding drug transport via the blood circulation. Although advances in medical technology and breakthroughs in nanotechnology-based approaches are progressed, many diseases associated with brain or central nervous system (CNS) remain poorly treated because of the lack of effective therapies. The inability of many therapeutic molecules to cross the BBB creates problems for an efficient therapeutic strategy. In order to overcome this issue, the physiology of the BBB must be well understood for the sake of permeability under different pathological and disease conditions.

The purpose of this review is to tackle the delivery and targeting problems across BBB under normal physiologic and pathological conditions. Opportunities for maximizing the drug delivery are assessed when BBB is exposed to physical or chemical stimuli. Here, cell penetrating peptide mimics are discussed for the better delivery to across the barrier as well as taking the advantage of using biologics for brain-related diseases. The recent approaches and insights into using nanotechnology and ligand conjugation to target the BBB via receptor mediated transport are discussed and compared what is proposed here for the better efficiency and safety.

THE MECHANISM OF PENETRATION INTO THE BRAIN BY USING DELIVERY VEHICLESIt has been reported that many transport routes can help molecules to move across the BBB. There are paracellular and transcellular diffusion into the brain. Small water-soluble molecules easily diffuse through tight junctions as shown in Figure 1. On the other hand, small lipid-soluble molecules such as alcohol and steroid hormones penetrate transcellularly by virtue of their solubilization in lipid plasma membrane. Among the pathways that molecules can cross the BBB, adsorptive-mediated transcytosis has remarkably become important as an effective drug delivery strategy into the brain via cationic proteins, and cell-penetrating peptides. CPPs have amphipathic characteristics with positively charged peptide residues. They have been shown to be able to internalize cargo into living cells without revealing any cytolytic effects.

COURTESY OF HAMISH KIDD


CPPs are successfully used for delivery of P-glycoprotein (P-gp) substrates. People showed that P-gp substrate delivery elevated doxorubicin transport into rat brain up to 30-fold suggesting that CPP carriers were effective since they utilize adsorptive-mediated endocytosis to enter the brain. Moreover, CPPs have been successful to deliver poorly brain-penetrating drugs. For instance, SynB3 (RRLSYSRRRF) was shown to enhance the delivery of morphine-6-glucoronide to the brain in a clinical trial. CPPs share several common features, which include hydrophobicity and helical moment, net positive charge, amphipathic nature, the ability to interact with lipid membranes, and retain a secondary structure while interacting with lipids.

The mechanism of internalization with CPPs is mainly adsorptive-mediated transcytosis (AMT) although TAT (HIV-1 trans-activating transcriptor) derived CPPs enter cells primarily by lipid-raft mediated macropinocytosis. It is noteworthy to imply that AMT is much more useful in diseased states based on reports in cell studies and animal models. It was shown that brain endothelia cells showed an increase in AMT upon tumor necrosis factor- α (TNF-α) and interleukin-6 (IL-6) treatment, which are critical cytokines in several neurological diseases such as MS and AIDS. Furthermore, the presence of TNF-α facilitated transcytosis of lactoferrin (Lf) at the BBB in vitro, which is an iron-binding protein against infection and severe inflammation. Taking these into consideration, maximizing the drug transport into the diseased brain by AMT leverages the rationale behind designing drug delivery systems with cationic proteins or peptides (i.e. cell-penetrating peptides)

CPPS AND SYNTHETIC MIMICSThe first CPP is found in HIV-1-TAT, a small nuclear trans-activator of transcription protein. It was shown that TAT readily crosses cellular membrane and localize into the nucleus of many cell lines. The responsible sequence for the penetration is a small, cation-rich domain between amino acids 49-57 (RKKRRQRRR). Later, its synthetic variant, polyarginine, showed much more uptake efficiency than TAT and entered cells in a length-dependent fashion.

FIGURE 1 DRUG DELIVERY ROUTES INTO THE BRAIN (CHEN & LIU, 2012)


Moreover, β-peptides and several other molecular scaffolds (unique MWs) were designed with guanidine functionality and exerted similar internalization properties. Other extensive studies showed that a Drosophila homeoprotein, Antennapedia, had an amino acid sequence 43-58, RQIKIWFQWRRMKWKK, that was responsible for cellular internalization as well.

Since we know that CPPs contain arginine and lysine residues like Tat, they also contain hydrophobic residues which take part in cellular uptake. This improved design of CPPs suggests better efficiency for cellular uptake compared to Tat and polyarginine. For instance, Transportan (GWTLNS-AGYLLGKINLKALAALAKKIL-NH2), which is a fusion between the neuropeptide galanin-1_13 and wasp venom peptide mastoparan, and Pep-1 (KETWWETWWTEWSQPKKKRKV-cya), which is a fusion between lysine-rich NLS from Simian Virus 40 large T antigen and a tryptophan-rich sequence linked by SQP sequence, are mostly studied amphiphilic CPPs which have block-type arrangements about their topology. Inspired by the abilities of these native and chimeric structures, there are non-peptidic, synthetic CPP mimics (CPPMs) which has improved properties compared to CPPs. CPPMs are fast, efficient, biocompatible and possess easy structures in order to better understand physicochemical properties.

CPPMs are very novel designed molecules for many therapeutic purposes such as siRNA, protein, antibody and plasmid delivery. Especially, cells, which are hard-to-transfect, can be used for any type of intracellular delivery targets in order to achieve a biological response. Another advantage of CPPMs is to have non-covalent interactions cargo so that the dissociation of CPPM/cargo complex is very quick after the internalization. It was reported that CPPMs can deliver siRNA into primary T cells in order to knockdown a specific protein in T cells in a very efficient way. The study showed the ability of silencing the protein for different therapeutic purposes such as cancer treatment and autoimmune suppression. There are also different studies which showed superior efficient delivery of proteins and antibodies into those primary cells.

FIGURE 2 FROM CPPs TO CPPMs (SGOLASTRA ET AL., 2013)


‘The biology is really against drug delivery to the brain, so clever strategies are needed’ says Joan Abbott, a neuroscientist at King’s College London, UK. With the light of the information above, it can be suggested that CPPMs can be used for efficient delivery via AMT or lipid-raft mediated macropinocytosis to cross the BBB in order to achieve any therapeutic purposes for CNS diseases.

SAFETY AND EFFICACY ISSUES FOR DRUG DELIVERY VIA CPPMThe safety and efficacy parameters for delivery into devastated brain were formulated with 10 key development criteria. There are three different topics that the criteria fall upon: (1) targeting blood-brain barrier, (2) drug carrier systems and (3) clinical applications. With regard to the targeting blood brain barrier, both selected receptor and targeting ligand should be non-toxic and they have to be applicable for both acute and chronic conditions in the clinical development. The proper drug delivery system will improve pharmacokinetic profile of an administered drug. If the cargo will be a biologic such as antibodies specific for only one target, then the off-target effects will be excluded and the delivery will have much safer and efficacious. CPPMs are promising for antibody delivery intracellularly so that delivered antibody can only recognize its unique target, involved in disease progression, to interfere with its function. Also, since CPPMs are biodegradable and non-covalently attached to cargo, they release cargo very quickly in order for them to exert their function. Afterwards, CPPMs are degraded suggesting the low possibility of toxicity. Finally, clinical use for drug delivery strategy should be straightforward and low cost. CPPMs are synthesized based on ring opening metathesis polymerization (ROMP) method which allows a fast and simple production if there is going to be a large-scale production. Additionally, the production seems to be low cost since the chemistry behind CPPMs is very easy. Also, toxicity for CPPMs is tested for different cell lines and also ex-vivo delivery strategies. Therefore, it makes those carriers much more promising for clinical studies as well.

COURTESY OF AAD VAN VLIET


CONCLUSIONDrug delivery into the brain is challenging because of the blood brain barrier.CPPMs help to cross the blood brain barrier via AMT. They are capable of internalizing any type of biological cargo into cells without any toxicity. Here, it can be suggested that CPPMs can be used for biologic delivery into the brain with a high efficiency and safety. Humanized antibodies are recently highlighted for many therapeutic interventions. They show highly specific targeting to any molecule and reduced adverse effects. This brings a big plus for clinical implications for humanized antibody delivery. Additionally, CPPMs can carry those antibodies intracellularly so that the therapy could be extremely specific and effective by choosing intracellular targets. This may be a good hope for treating brain diseases while finding specific targets for each disease. In conclusion, CPPMs may be good candidates for therapeutic interventions in the treatment of CNS disorders.


REFERENCESBasavaraj, S.& Betageri, G.V. 2014. Can formulation and drug delivery reduce attrition during drug discovery and development-review of feasibility, benefits and challenges. Acta Pharmaceutica Sinica B 4(1), 3-17.

Chen, Y. & Liu, L. 2012. Modern methods for delivery of drugs across the blood-brain barrier. Advanced Drug Delivery Reviews 64, 640-665.

Gaillard et al. 2012. Enhanced brain drug delivery: safely crossing the blood-brain barrier. Drug Discovery Today:Technologies 9(2), 155-160.

King, A. 2011. Breaking through the barrier. Chemistry World, 36-39.

Pardridge, W.M. 2012. Drug transport across the blood-brain barrier. Journal of Cerebral Blood Flow & Metabolism 32, 1959-72.

Patel et al. 2012. Polymeric nanoparticles for drug delivery to the central nervous system. Advanced Drug Delivery Reviews 64, 701-705.

Sgolastra et al. 2013. Designing mimics of membrane active proteins. Accounts of Chemical Research 46(12), 2977-87.

Sgolastra et al. 2014. Importance of sequence specific hydrophobicity in synthetic protein transduction domain mimics. Biomacromolecules 15, 812-820.

Tezgel et al. 2013. Novel protein transduction domain mimics as nonviral delivery vectors for siRNA targeting NOTCH1 in primary human T cells. Molecular Therapy 21(1), 201-209.

“TOBBB”. (n.d.). Brain drug delivery. Retrieved from http://www.tobbb.com/science/Brain_drug_delivery on April 4th, 2014.


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BY: NILESH GUPTA

CHALLENGES IN DEVELOPING INHALATION TECHNOLOGY: RIGHT PARTICLE SIZE AND DEVICE SELECTION


INTRODUCTIONSince ancient civilization, respiratory route of administration has always drawn the attraction of scientific community, while mechanistic aspects were less known. In the last few decades, merger of different disciplines of science has led to a significant revolution in the field of inhalation technology. Drug delivery via pulmonary route has gone beyond the treatment of respiratory disorders and has opened new avenues for other diseases. A plethora of features make this route superior over other routes

of drug administration. Drug delivery via lungs is patient friendly as it is non-invasive and multiple dosing is possible even without the assistance of health care professionals (1). Extensive vascularization with vast area (~100 m2) and relatively thin epithelial barrier provide better absorption and rapid onset of action, and thus makes it therapeutically relevant route. It avoids first-pass metabolism and hence lower doses are required to show the intended effects which also reduces systemic side effects of the drugs. Its fair play with small and large molecules offers better stability and absorption as compared to other routes (2). However, aerosolization and administration of an inhaled medicine is plagued with technical challenges which limit its development and validation on a regulatory landscape. Drug-device combination is very complex chemistry which interrupts the development process of a successful inhalational product (3). It is worth to mention that the inhalation technology rides on the success of two parallel developmental pathways: (a) improvement of formulations and (b) optimization of novel or current inhaler devices (4). After inhalation, fate depends on the physicochemical properties of drug and the type of device employed. If not successful, drug wastage may lead to narrow therapeutic index of drug, undesirable side effects and premature clearance through the body (see Figure 1 for better understanding of fate of inhaled particles). To this end, we have chosen the drug-device combination as our debate material and attempted to closely review the challenges associated with particle size and the device selection. We have given particular emphasis on the recent developments in the field of particle engineering and device technology based on the thorough review of literature available.

FIGURE 1 FATE OF INHALED PARTICLES


PARTICLE SIZE AND ITS IMPORTANCEParticle size is the most important governing factor that dictates and decides the fate of inhaled pharmaceutical aerosols. It is the critical quality attribute which is difficult to control during the final synthesis of a drug, but have the tremendous potential to affect its performance. Most of the particles which don’t fit into the respirable range simply get discarded by the body through various clearance mechanisms. Think of a big truck moving in a small room which finds it difficult to bend easily. Similarly, for larger and bulkier particles, it is almost impossible to get into the convoluted paths of mouth and throat along with the inhaled air. Also, the particles which are relatively smaller, they get inhaled easily and exhaled even more easily. So, it is very important to design particles of such a size which can enable them to travel easily through the extra-thoracic, tracheobronchial and alveolar regions and eventually settle down (3). However, the percentage of deposition should not be judged just by the particle size as there are several other factors which affect the site and amount of accumulation. Desirable features of an inhalable formulation are optimal particle size, homogenous distribution and improved dispersibility, which in turn favors enhanced drug stability and bioavailability (4).

Specific optimization of the particle size of inhalational products is important due to the below-mentioned reasons:

a. Difference in particle size leads to varying doses deposited in the lungs.b. Modulation of particle size affects the proportion of

drug in the finished product formulation.

c. Micronized drug particles reduce the amount of active drug content required for a therapeutic response and thus diminish the chances of local side effects.

d. Particle size may change due to hygroscopicity or solvent evaporation.

e. Particle size is an important parameter to be evaluated during the development of generic products.

Formulations intended for inhalation are aerosolized as solid/liquid droplets. Particles undergo a series of aerodynamic maneuvers such as changes in airflow and direction, and pass through a number of airway bifurcations and barriers before deposition in a specific region in the lungs. The inhalation potential of aerosolized particles depends on a parameter called mass median aerodynamic diameter (MMAD), which is defined as the diameter of a sphere of unit density that has the same settling velocity as the particle of interest regardless of its shape or density (1). Since most inhalation formulations are hetero-dispersed, it is required by the regulatory agencies to report the MMAD, which is determined by cascade impactor that simulates the respiratory tract.

Because of the influence of various physical forces, deposition of particles in different regions of lungs is significantly affected by the particles size. Particles that are deposited in the respiratory tract have the size ranging from 1 to 10 μm. The respirable fraction is the percentage of an inhaled formulation that penetrates into the alveolar region of the lungs (10 μm). Fine particle fraction is the percentage of inhaled particles below the particle size of 5 μm. Particles larger than 5 μm


will impact in the upper airways and rapidly removed by coughing, swallowing and mucociliary processes. Inhaled particles between 5-10 μm rapidly removed by coughing, swallowing and mucociliary processes. Inhaled particles between 5-10 μm diameters are deposited on the tracheobronchial surface by impaction.

Particles between 0.5-5 μm are deposited in the alveolar levels by impaction and sedimentation. About 50% of 0.5 μm particles are deposited in the alveoli by diffusion and the rest are exhaled or may not deposit at all. Overall, inhaled particles with unit density and diameter between 1-5 μm exhibit efficient deposition and distribution in the lungs. Unlike traditional powders for inhalers, a newer type of formulations called as large porous particles, have been found efficient in getting deposited deep inside the lungs. They are lighter with a mass density of 0.4 g cm-3 compared to 1 gcm-3 for traditional powders. As they are porous, their aerodynamic diameters are smaller than their volume diameters. There is reduced dose requirement due to reduced aggregation and throat deposition. These particles remain longer in the alveolar region and can be prepared from lung surfactants and polymers.

Several techniques (Table 1) have been utilized to reduce the particle size of aerosol formulations but it is not all about obtaining the same size as the technology employed vary the dispersibility of powdered drug. Better flowability always relies on a delicate balance between the adhesive and cohesive forces between particles (4). Micronization, also known as air jet milling, has been utilized for more than 10 decades for size reduction of drug particles, but has several drawbacks:

a. Multiple and repeated steps are required to produce the desired size, which poses a severe risk of metal contamination and loss of product due to sticking or picking up by the machine.

b. High energy used for micronization sometimes destroys the crystal structure of drugs and the resultant amorphous forms may impact significantly the stability and further therapeutic efficacy of product.

c. Generation of ultra-small particles lead to agglomeration due to the cohesive/adhesive forces between them.

d. Carriers such as lactose need to be separated at correct time for the effective and appropriate deposition of actual drug particles.

e. Conventional micronization procedures are not suitable for formulating inhalable biotherapeutics.

TABLE 1 ADVANTAGES AND DISADVANTAGES OF PARTICLE ENGINEERING TECHNOLOGIES


Particle engineering bridges the gap between a drug substance and drug product. Recently, emulsion, supercritical anti-solvent and spray drying have been adopted to address the abovementioned drawbacks of traditional size reduction methodologies. Emulsion approach produces solid spherical particles such as for insulin and alpha-1-antitrypsin using non-miscible solvent emulsions generated through homogenization or sonication; however, discrete particles produced are not homogenous. Similarly, supercritical or near-critical fluid antisolvent and expansion systems have been used to generate inhalation powder formulations through precipitation of proteins such as for anti-CD4 antibodies and trypsinogen. Supercritical CO2-assisted nebulization with a bubble dryer (Supercritical CAN-BD) is a new technology which is used to prepare proteins by passing the emulsions of drug, solvents and supercritical CO2 through a nozzle to generate liquid droplets that are then dried by nitrogen gas (3).

The pulmonary route has been used for the delivery of macromolecules and biologics for more than three decades, while the success rate is very limited. Inhaled insulin, Exubera®, was the first protein drug that was approved by the FDA and commercialized as dry powders in the USA and Europe for the treatment of diabetes. However, because of cumbersome delivery device and high expense, it failed to receive acceptance among patients and physicians that resulted in discontinuation of the product by Pfizer. But this failure did not stop the development of inhaled formulations of proteins and thus many novel strategies came forward to tackle these problems. Currently, Dance pharmaceuticals (CA, USA), is conducting research to address the issues related to the stability, efficacy and untoward side-effects of inhaled insulin. Spray freeze drying (SFD) technology has enabled overcoming many stability problems of biologics. SFD produces spherical particles with uniform and controlled size, and avoids heat or air induced denaturation of proteins. Insulin loaded liposomes, for example, prepared by SFD can be stored for three months and intratracheal instillation of formulation resulted in ~3 fold increase in the bioavailability. Other strategies include precipitation of particles via solvent change method, incorporation of water soluble cyclodextrins to prevent aggregation of proteins, and use of ink-jet devices consisting of a microheater to generate fine uniformed size droplets (5).

Liquidia Technologies has adapted advanced technologies from the microelectronic industry and recently demonstrated a mold-based particle engineering platform, particle replication in non-wetting templates (PRINT). With the advent of this technology, it has been possible to achieve a next generation inhalation powder formulation with uniform particle shape, size and morphology. Mold gets the input in the form of pre-defined particle size and chemical composition which generates particles suitable for direct pulmonary delivery (6).

SELECTION OF APPROPRIATE DELIVERY DEVICEDrugs, delivered as droplets or particles, differ in their physicochemical properties and thus the delivery devices can play a major role in their deposition inside the different regions of lungs. Devices that are available to deliver drugs via pulmonary


route include nebulizers, MDIs, used either alone or attached to spacers or valve holding chambers and DPIs (Table 2). Below, we have listed some of the major factors which need to be considered before choosing the right device for pulmonary drug administration (2):

a. All inhalation devices are not appropriate for all patients because it requires varying level of cognitive ability depending on the device structure, its functioning, cleaning, loading and storage.

b. Patients need to be adequately educated by the physicians or manufacturers on the correct usage. Patients who are taught beforehand, get the desired therapeutic effects, even different doses are required.

c. Nebulizers produce aerosols at a constant rate regardless of patients’ breathing phase, i.e. during exhalation, inhalation and breath holding. This results in huge wastage of drugs.

d. Lack of actuation-inhalation coordination and cold freon effect are the problems associated with MDIs.

e. Usage of DPIs suffers from problems such as failure to use a forceful and deep inhalation and exhalation to functional residual capacity.

f. Different inhaler types should not be mixed for an individual patient as it may cause confusion.

g. Age is an important parameter which needs consideration before choosing the delivery device. Children, younger and older people behave differently with different devices.

h. Nature of drug to be delivered is a crucial criterion to decide the appropriate devices.

Development of an inhalation therapy requires the optimization of whole system, drug, formulation and device. In this part of review, briefly, we have discussed different types and subtypes of devices with an emphasis on their main features responsible for aerosolization and delivery of drugs (3):

Nebulizers convert drug solutions/suspensions, available as unit dose or concentrated solutions for dilutions, into aerosols that can be easily taken by inspiration. Based on the mechanisms of aerosol generations, nebulizers are classified as air-jet and ultrasonic types. Air-jet nebulizers use a pressurized jet air stream delivered by a compressor which enters via a narrow tube that is forced through a constricted opening called the venturi. Ultrasonic nebulizers use piezo-electric transducer to produce high frequency sound waves in the nebulizer solution. Some newer, compact and battery-operated nebulizers have been designed to address the problems associated with cooling or heating of reservoir solution in nebulizers such as crystallization of drugs, decrease in MMAD, instability etc. They are based on mesh technology and are of two types: static (Micro-Air NE-U22V, Omron Healthcare, Inc., IL) and vibrating mesh (Aeroneb Go, Aerogen, Ireland).


Metered dose inhalers (MDIs) allow multiple dosing utilizing metering valves in conjunction with propellants. In MDIs, drug is dispersed in propellants and the device facilitates aerosolization of the drug and delivers as a fine mist. The propellant is the driving force or the “heart” of aerosols. Halogenated hydrocarbons, derived from methane and ethane, are used as propellants in inhaled formulations. In recent years, use of chlorofluorocarbons has largely been replaced with hydrofluoroalkane propellants as the later do not have ozone layer depleting characteristics. Some propellant free MDIs such as Respimat® (Boehringer Ingelheim, Germany) are also gaining widespread popularity. Physical form, particle size and dose of drugs are controlled by metered valves. An MDI has three major components: container, metering valve and spray actuator.

Dry powder inhalers (DPIs) are pulmonary drug delivery devices containing solid drug suspended in a dry powder mix that can be fluidized when patient inhales. DPIs offer several advantages over MDIs such as higher formulation stability in dry conditions and environment friendly because of being propellant free. As DPIs contain powder in a static bed, the formulation in DPIs must undergo flow, fluidization and de-aggregation. Formulations are prepared in powdered form either as carrier-based formulations with large inert carrier excipients (Ventolin Diskus, GlaxoSmithKline) or agglomerated drug-only formulations (Pulmicort, Astra Zeneca). Carriers are used to prevent aggregation and improve flow. DPIs are available as either single dose system such as Aerosolizer® (Novartis) and Handihaler® (Boehringer Ingelheim) or multi-dose systems such as Diskhaler®

(GlaxoSmithKline) and Turbuhaler® (Astra Zeneca; Pulvinal®, Chiesi). Based on the mechanisms involved in aerosolization of particles from bulk powder bed, DPIs can be divided into two categories: passive and active DPIs. In case of passive DPIs,

TABLE 2 TYPES AND COMPARISON OF PULMONARY DRUG DELIVERY DEVICES


patients use their inspiratory force to generate aerosols. DPIs that use pneumatic, impact and vibratory force to generate aerosols are known as active DPIs. Currently, under development active dispersion DPIs are Nektar pulmonary inhaler delivery system and Vectura Aspirair®. In both the devices, small in-built pumps are used to generate compressed air in order to disperse the powdered drug (3).

Although pulmonary delivery can be significantly enhanced by more sophisticated inhalers that use electronic synchronization and actuation control, such devices are more complex and expensive. Also, their reliability and practicality have been questioned a lot. The device selection is always guided by convenience, cost and patient preference. Each inhalational device, whether a nebulizer, MDI or DPI, produces its aerosol differently and hence, the particle size, respirable dose, lung deposition and distribution will also differ. Consequently, the same drug at the same nominal dose delivered from different devices or in different formulations may not be bioequivalent. Therefore, the importance of matching a drug with appropriate device should not be ignored.

SUMMARYClinical practice of inhalational drugs has witnessed several revolutionary discoveries over a period of 50 years. Despite the long-term use of inhalational technologies for the therapeutic delivery of small and large molecules, there still is room for improvement in a number of areas. Teamwork of a formulator, particle engineer and device designer is important to come up with a robust and successful inhalational drug delivery system. While the device designer strives to make an inhaler that can efficiently deliver a reproducible and invariable dose, the job of a formulator and particle engineer is to ensure that the dispersion of drug must be homogenous and its deposition is not affected by the anatomical and physiological features of the respiratory system. Advancement in particle engineering has allowed the development of particles with better dispersibility, entrainment and deposition efficiency from different devices. Novel formulation strategies such as large porous particles have enabled sustained drug delivery. Recent trends in device engineering has made the inhalation effortless and gifted the patients with compact, light in weight, sophisticated and efficient devices.


REFERENCES1. Backman P, Adelmann H, Petersson G, Jones CB. Advances in Inhaled Technologies: Understanding the Therapeutic Challenge, Predicting Clinical Performance, and Designing the Optimal Inhaled Product. Clinical pharmacology and therapeutics. 2014.

2. Mitchell JP. What the pulmonary specialist should know about the new inhalation therapies. The European respiratory journal. 2012;39(4):1054-5; authhor reply 5-6.

3. Ruge CA, Kirch J, Lehr CM. Pulmonary drug delivery: from generating aerosols to overcoming biological barriers-therapeutic possibilities and technological challenges. The lancet Respiratory medicine. 2013;1(5):402-13.

4. Weers JG, Bell J, Chan HK, Cipolla D, Dunbar C, Hickey AJ, et al. Pulmonary formulations: what remains to be done? J Aerosol Med Pulm Drug Deliv. 2010;23 Suppl 2:S5-23.

5. Depreter F, Pilcer G, Amighi K. Inhaled proteins: challenges and perspectives. Int J Pharm. 2013;447(1-2):251-80.

6. Garcia A, Mack P, Williams S, Fromen C, Shen T, Tully J, et al. Microfabricated engineered particle systems for respiratory drug delivery and other pharmaceutical applications. Journal of drug delivery. 2012;2012:941243.

7. Gupta N, Patel B, Ahsan F. Pulmonary and Nasal Drug Delivery in Ashim K. Mitra Eds., Drug Delivery, Jones and Bartlett Learning LLC, Burlington, MA. (In Press).


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BY: MAREN PREIS

PEDIATRIC DRUGSREGULATORY CHALLENGES AND AVAILABLE DOSAGE FORMS


INTRODUCTIONThe supply of age-appropriate dosage forms is a major task. With respect to certain patient populations such as children or the elderly, medicinal treatment can be challenging [1,2]. Esophageal diseases and swallowing issues may complicate compliance and adherence of these patients [3], who often face problems regarding the administration of their medicines. With respect to physical attributes, children of younger age-groups might not be able to swallow the same sized dosage forms as adults. Thus, children and older people form a vulnerable and special patient group, making it worthwhile to further develop dosage forms in order to facilitate drug administration. Children and the elderly face similar issues regarding drug therapy. The challenges for accomplishing pediatric and geriatric oral / oromucosal drug delivery are summarized in Table 1 based on the claims of Breitkreutz and Boos (2007). All claims should be considered with respect to both patient groups.

APPROPRIATE DOSAGE FORMSThe logical solution for pediatrics may be to administer liquid formulations [4]. The choice of formulation strongly depends on the clinical state and the age of the child; liquids should be preferred; nevertheless dosing errors and stability issues are more likely than for solid dosage forms. The amount of liquid should however be as small as possible to decrease the amount of administered excipients (e.g. preservatives) and electrolytes that “may be toxic in neonates because of immature metabolism and elimination” [5]. Neonates are the most vulnerable group of the pediatric population (from birth up to and including the age of 27 days). They are moreover treated unauthorized and off-label in almost 90% of the cases due to lacking clinical studies, which are challenging because of “multiple difficulties, such as ethical (high vulnerability) and technical issues (immaturity, prematurity, lack of self assessment, need for specific formulations, high variability, etc)” [5].

Nevertheless, a World Health Organization (WHO) expert forum proposed a shift of paradigm towards solid dosage forms in 2008 [6]. Still, the initial situation has not changed: children are not able to swallow large-sized tablets or capsules and they

Aspects to considerPalatability/acceptable taste

Safety of excipientsHandling of packaging

Precise & clear product informationAcceptable dose uniformity

Size of dosage formEasy and safe administration

Sufficient bioavailability

TABLE 1 CHALLENGES IN PEDIATRIC AND GERIATRIC ORAL AND OROMUCOSAL DRUG THERAPY (BREITKREUTZ AND BOOS, 2007)


may even refuse taking and swallowing solid dosage forms. However, a new trend in dosage form development has taken place in recent years: orodispersible tablets, multiparticulates or the administration of powder in sachets have been investigated with respect to appropriateness for children [7].

The further development of orodispersible tablets (ODT) has led to orally disintegrating mini-tablets (ODMT) [8]. The use of small-sized tablets (1–2 mm in diameter) has been an emerging success in dosage form development. It combines the convenience of tablets, a solid dosage form, with less issues in stability than liquid formulations, and the opportunity to avoid swallowing a large unit, as the ODMTs are intended to disintegrate rapidly once in contact with the saliva wetted tongue or mucosa [9,10]. Furthermore, studies revealed an overall positive response of children in investigations on mini-tablet (MT) acceptance [11,12]. The MTs (2mm) have been favored compared to syrup by the children in trials, and these results could also be affirmed for children of age six months. The tendency of children to sympathize with small-sized dosage forms has been confirmed in a further study using 4 mm tablets compared to syrup, suspension and powder. Parents were asked to administer placebos to their children of one to four years and rate the acceptability and report about (non-) successful intake. The study again revealed predomination of the tablets in being best accepted by the children [13]. Furthermore, the number of fully swallowed tablets was higher than for other dosage forms.

The use of film preparations as an alternative to liquids or tablets is an upcoming field of interest in drug delivery. It can be proposed that if a film preparation, meaning a thin and flexible polymer sheet at maximum the size of a stamp, can be described as a solid dosage form, as the film is a solid preparation prior to administration. Oromucosal film preparations are placed in the mouth to disperse rapidly (orodispersible film, ODF) or are placed on the mucosal tissue and may dissolve (mucoadhesive buccal film, MBF) [14]. Where ODFs may be described as an alternative per oral dosage form, MBFs offer a variety of possibilities in drug delivery. The list of marketed products reveals several ODFs besides different other dosage forms for pediatric use (Table 2).

The above findings demonstrate that children are willing to accept solid dosage forms; moreover, once convinced by the ease of its administration, they may even favor a certain dosage form.

REGULATORY GUIDANCE MEDICINES FOR PEDIATRIC USE Recently, a novel guideline of the European Medicines Agency (EMA) on pharmaceutical development of medicines for pediatric use has been published and came into effect on February 15, 2014 [15]. From that date onwards, the guideline is mandatory to adhere to in pharmaceutical development for children between birth and 18 years of age. The aim of the “Paediatric Regulation” is “to facilitate the development and accessibility” of age-appropriate pediatric medicines. The aim should be achieved “without subjecting children to unnecessary clinical trials and without delaying the authorisation of medicinal products for other age populations”


[16]. The EMA, the Paediatric Committee (PDCO) and the Committee for Medicinal Products for Human Use (CHMP) do not aim to introduce restrictions, but to make guidance available.

The Food and Drug Administration (FDA) of the United States provides regulatory framework on pediatrics since 1979, when the first pediatric labeling requirement has been introduced [17]. In 2010, Zisowsky et al. summarizes regulatory aspects of the drug development for pediatric populations of the European and United States authorities [18]. In 2001 the Best Pharmaceuticals for Children Act (BPCA) was enacted by the FDA to provide financial incentive for voluntary pediatric studies to companies. The Pediatric Research Equity Act (PREA) signed into law in 2003 and required that companies assess studies on safety and effectiveness of medicines in pediatric patients. In 2012, amendments in the BPCA and PREA by the Food and Drug Administration Safety and Innovation Act (FDASIA) passed and are now permanently reauthorized [19]. The FDA encourages pediatric study plans to expand the knowledge and expertise in pediatrics drug therapy. However, there is no such clear guidance on formulations and dosage forms provided like the EMA does in its latest publication [15]. Therefore, the detailed considerations of the latest official framework on medicines for pediatric use will be further discussed, particularly with regard to the appropriateness of dosage forms.

General considerations Active substances Dosage form

„Infants are simply unable to swallow conventionally-sized tablets”

Pharmaceutical design appropriate for use in target group

Development of dosage forms facilitating the administration of a range of doses being

acceptable to different age groups

Consideration of special needs: minimum age, condition-related, pharmacodynamics, dose

regimen, age associated activities, duration and frequency of therapy, environment setting,

characteristics/behavior (child and caregiver)

Liquid medicinal product:

e.g. improved solubility (use of salt

or different salt instead of base)

or “less soluble form (…) to overcome taste

issues, e.g. base instead of salt”

Selection of the form of the active substance

“Route of administration should be discussed and justified for children in each of the target age

group(s)”

To consider: condition(s) to be treated, treatment

duration, properties of active substances, necessity

of particular excipients, measuring and

administration devices, stability issues, dosage requirements, risk of dosing errors, ease of administration and

acceptance

TABLE 3.A REGULATORY GUIDANCE: QUALITY OF MEDICINES FOR PEDIATRIC USE - GENERAL CONSIDERATIONS, ACTIVE SUBSTANCES AND DOSAGE FORMS [15].


DISCUSSIONGENERAL CONSIDERATIONS OF THE NOVEL GUIDELINETable 3a summarizes the general considerations given in the new guideline. It points out once more that the dosage form should enable the administration of variable doses and be suitable for a large range of age groups and their special needs. Furthermore, it becomes evident that the authors of the guideline also kept in mind the competence of the caregivers of children, who are responsible for carrying out the drug administration. To choose the appropriate dosage form, the properties of the active substance should be taken into account to ensure stability. Most certainly, risks regarding dosing errors or measuring devices should be considered.

CHOICE OF DOSAGE FORMIt is stated in the guideline that the use of either solid or liquid dosage forms reveals advantages (Table 3b). Solid, single-unit dosage forms represent an easy dosage approach. However, multiparticulates and liquid preparations may be dosed even more flexible. An interesting statement in the guideline is the demand for investigating and developing several different dosage forms to serve the diversity of preferences. Depending on their experiences, children might refuse a certain dosage form; e.g. the child may have experienced a very poor-tasting medicine liquid and will therefore reject all liquids. Another child may have had the same experience with tablets.

Oral administration Oromucosal preparations

Dosing frequency Modified release

preparations

“Main choice (<) between liquids and solid dosage

forms”

Solids single-unit dosage form: stable and easy dosing

approach

“Oral powders, granules and liquids normally provide greater dosing flexibility”

Liquids: avoiding multiple step procedures (risk of dosing

errors)

Investigation on the feasibility of bringing different dosage

form (<) to the market

“Correct use and acceptability (<)

depend on the age of the child and the ability to keep the preparation in a

specific part of the mouth over a

defined period of time”

Adhesive properties (if necessary)

Suitable applicator needed (e.g. for

dental gels, mouthwashes)

Justification according to:

active substance; pharmacokinetic

profile; indication; convenience;

therapeutic adherence

“Maximum of twice daily dosing is

preferred for out-patient use”

Special attention when medicines used > 2

times a day (suitability of administration when no trained caregiver is

around)

“Should not be restricted to the oral

route”

Prolonged release: reduced dosing frequency (<)

beneficial

“risk of chewing and its impact on the efficacy and

safety”

Impact of physiological

conditions on drug absorption

TABLE 3.B REGULATORY GUIDANCE: QUALITY OF MEDICINES FOR PEDIATRIC USE - ORAL ADMINISTRATION, OROMUCOSAL PREPARATIONS, DOSING FREQUENCY AND MODIFIED RELEASE PREPARATIONS [15].


The guideline further refers to preparations that are intended to stay in the mouth for a certain time; the ability of the dosage form to adhere to a specific site in the mouth should also be considered.

The use of ODFs would circumvent the challenge of correct use as mentioned in the guideline: simple placing in the mouth and subsequent immediate disintegration of the thin film strip does not require the application of the film to a special absorption site and the child would have nothing to accomplish other than the natural swallowing of its saliva, where the film is dispersed [20,21]

The dosing frequency is recommended to be at a maximum two times daily with regard to the background that the medicine shall be taken at home in the morning and in the evening. More doses over the day would imply ease of administration that does not require the help of a trained caregiver.

MODIFIED RELEASEModified release preparations are reasonable not only in terms of oral dosage forms. The advantage of prolonged release drug formulation is a reduction of the dosing frequency facilitating the therapy.

However, the use of such preparations may entail the risk of varying efficacy, for example, when the dosage form is intended to be swallowed or remain in the mouth. The drug release may be influenced by the child chewing on the medicinal product or altering dissolution effects in the child’s gastric area. Furthermore, the gastrointestinal transit times in children are highly variable [1]; a modified release preparations might therefore not follow the expected release kinetic.

Excipients Acceptability Container closure

system Measuring

device

“key element of its pharmaceutical development”

Special safety considerations:

function; safety profile (single or daily

exposure); duration of treatment; severity of

the condition; acceptability including palatability; allergies

and sensitization

Influenced by child’s age, individual health status, behavior,

disabilities, background and culture

“should preferably be studied in children themselves”

“should also be assured during the life-cycle of the product” (e.g.

changes in composition, packaging)

Adolescent children: “discrete and portable, and when reasonable, enable individual

doses”

“...consider novel packaging and administration

strategies”

“Specific attention should be given to the

ease and accuracy of the

administration.”

Oral liquids: oral syringe = “more reliable method

(<) in the youngest age groups than a

spoon or a cup”

TABLE 3.C REGULATORY GUIDANCE: QUALITY OF MEDICINES FOR PEDIIATRIC USE - EXCIPIENTS, ACCEPTABILITY, CONTAINER CLOSURE SYSTEM, MEASURING DEVICE [15].


FIXED DOSE COMBINATIONSCombination drug products are advantageous in the treatment of chronic diseases (e.g. HIV, tuberculosis). The development of age-appropriate fixed dose combinations is encouraged by the European Medicines Agency [15]. However, flexibility and adequate dose adjustment needs to be ensured.

EXCIPIENTSThe choice of excipients is another crucial factor in the development of medicinal products for pediatric use. The guideline provides a detailed decision tool to evaluate the safety profile of excipients. The easiest way to ensure that an excipient can be used is given as when there is a European Food Safety Agency scientific opinion (EFSA) available for the excipient supporting its use in children.

According to the guideline, there is no further justification needed to use a particular excipient. Other routes to justify the use of an excipient are given when the excipient is

- included in a CHMP opinion or- included in a Committee/CHMP/ICH guideline or- approved in current pediatric medicines or- included in the European food legislation

and when the information is still up-to-date, related to the target age group and relevant to the maximum daily exposure/acceptable daily intake (ADI).

If none of this information is available on the particular excipient, additional data is required, e.g. juvenile animal / clinical studies, or there is simply the need to reformulate and choose other excipients. The acquisition of the required additional data is connected to high costs and is not affordable for most suppliers. Consequently, nobody wants to introduce a novel excipient that has not been described elsewhere previously and will therefore consider reformulation and exclusion of the excipient.

ACCEPTABILITY AND PALATABILITYThe acceptability of medicinal products by children is highly dependent on individual conditions. However, there are aspects like the taste of API, the overall palatability, dose regimen and mode of administration that can nonetheless influence acceptability (Table 3c). It is stated in the guideline that an international harmonized method for assessing acceptability is lacking and that the authorities know about variation in the outcome of acceptability trials even when same target groups are investigated. However, when discussed thoroughly, it becomes evident that reasonable benefit-risk consideration may justify the chosen method. The major focus is on palatability (“the overall appreciation of an (often oral) medicinal product in relation to its smell, taste, aftertaste and texture”) and the ease of mixing medicines with food or drinks. Palatability is mainly influenced by the characteristics of the active substance and excipients. The guidance points out that “Information on the palatability of the active substance should consequently be acquired at an early stage in the development of a medicinal product, e.g. from dedicated adult panels or literature” [15]. The assessment of results from human


taste panels and the acquisition of literature on the taste of specific active substances is a major challenge. Furthermore, the taste perceived by adults may significantly differ from the perception of children [22,23]. However, taste assessments in an early stage in development are stated to be important. The use of electronic tongues is an innovative approach to circumvent taste panels in the first place to investigate formulations and to provide pre-selection for further trials before having to employ cost-intensive human trials [24].

The mixing of drugs with food and drinks is accepted as reasonable and desirable in terms of masking the taste and insufficient palatability when a formulation does not provide acceptable palatability. Therefore, effects concerning the mixing with food and drinks should be discussed for a novel formulation with regard to feasibility, stability and compatibility.

CONTAINER CLOSURE SYSTEM AND MEASURING DEVICE The container closure system should enable the appropriate use of the medicinal product. A discrete and portable system should be provided for adolescent children. The authorities encourage to investigate and develop innovative approaches to facilitate administration and to enhance acceptability. Measuring devices like spoons, cups or syringes should ensure adequate dosing and their use has to be justified. Therefore, the measuring device should be qualified with the respective drug formulation.

CONCLUSIONThe overall impression of the novel guidance is that the authorities possess an immense interest in improving and promoting the development of pediatric medicines by providing detailed considerations on how novel products should be designed. Additionally, the guideline implies that innovations in pediatric medicines are intended and should be encouraged. The currently available dosage forms for children are various. The shown examples reveal the potential for suitable immediate release solid dosage forms, but also approaches for modified release products.


REFERENCES1. Breitkreutz, J., Boos, J., 2007. Paediatric and geriatric drug delivery. Exp. Opin. Drug Deliv. 4, 37-45.

2. Cram, A., et al., 2009. Challenges of developing palatable oral paediatric formulations. Int. J. Pharm. 365, 1-3.

3. Stegemann, S. et al., 2012. Swallowing dysfunction and dysphagia is an unrecognized challenge for oral drug therapy. Int. J. Pharm. 430, 197-206.

4. EMA, 2006. European Medicines Agency. Reflection paper: formulations of choice for the paediatric population. EMEA/CHMP/PEG/194810/2005.

5. EMA, 2009. European Medicines Agency. Guideline on the investigation of medicinal products in the term and preterm neonate. EMEA/536810/2008.

6. WHO, 2008. World Health Organization. Report of the Informal Expert Meeting on Dosage Forms of Medicines for Children.

7. Stoltenberg, I., et al., 2010. Solid oral dosage forms for children - Formulations, excipients and acceptance issues. J. Appl. Ther. Res. 7, 141-146.

8. Stoltenberg, I., Breitkreutz, J., 2011. Orally disintegrating mini-tablets (ODMTs) - A novel solid oral dosage form for paediatric use. Eur. J. Pharm. Biopharm. 78, 462-469.

9. Hermes, M., 2012. Dissertation: Kindgerechte, niedrigdosierte Zubereitungen mit Enalapril. University of Düsseldorf, Germany.

10. Stoltenberg, I., 2012. Dissertation: Orodispersible Minitabletten - Entwicklung und Charakterisierung einer neuen festen Darreichungsform für die Pädiatrie. University of Düsseldorf, Germany.

11. Klingmann, V., et al., 2013. Favorable Acceptance of Mini-Tablets Compared with Syrup: A Randomized Controlled Trial in Infants and Preschool Children. J. Ped. 163, 1728–1732.

12. Spomer, N., et al., 2012. Acceptance of uncoated mini-tablets in young children: Results from a prospective exploratory cross-over study. Arch. Dis. Child. 97, 283-286.

13. Van Riet-Nales, D.A., et al., 2013. Acceptability of different oral formulations in infants and preschool children. Arch. Dis. Child. 98, 725-731.


14. Ph.Eur., 2012. Oromucosal Preparations. European Pharmacopoeia (7.4).

15. EMA, 2013. European Medicines Agency. Guideline on pharmaceutical development of medicines for paediatric use. EMA/CHMP/QWP/805880/2012 Rev.2.

16. Council of Europe, 2006. Regulation (EC) No 1901/2006 Off.J.EU.

17. FDA, 2013. U.S. Food and Drug Administration. Pediatric product and formulation development: BPCA and PREA / Presentation by L. Yao.

18. Zisowsky, J., et al., 2010. Drug development for pediatric populations: Regulatory aspects. Pharmaceutics 2, 364-388.

19. FDA, 2013. U.S. Food and Drug Administration. Using FDASIA to Move Forward with Pediatric Drug Development / Presentation by R. Addy.

20. Hoffmann, E.M., et al., 2011. Advances in orodispersible films for drug delivery. Exp. Opin. Drug Deliv. 8, 299-316.

21. Preis, M., et al., 2013. Oromucosal film preparations: classification and characterization methods. Exp. Opin. Drug Deliv. 10, 1303-1317.

22. Mennella, J.A., Beauchamp, G.K., 2008. Optimizing oral medications for children. Clin.Ther. 30, 2120-2132.

23. Mennella, J.A., et al., 2013. The Bad Taste of Medicines: Overview of Basic Research on Bitter Taste. Clin. Therapeutics 35, 1225-1246.

24. Woertz, K., et al., 2011. Taste sensing systems (electronic tongues) for pharmaceutical applications. Int. J. Pharm. 417, 256-271.


5

BY: TANNAZ RAMEZANLI

STEM CELLS AS NOVEL CARRIERS FOR CANCER THERAPY


INTRODUCTIONCancer was estimated as one of top four cause of human death in the United States in 2010 and it remains one of the most unresponsive diseases to therapy. [1] Even though novel drug delivery systems such as nanocarriers have reached some success in improvement of cancer therapy over traditional methods, there are still some drawbacks with them decreasing their efficacy and hindering their vast application in clinics. The main challenge is drug targeting and penetration to the solid tumors. [2] In addition to the malignant cells, solid tumors are composed of the supporting cells like fibroblast, endothelium, and pericytes, that comprise the tumor stroma. In response to the signals sent by tumor cells, the cells in tumor stroma produce and release components necessary for tumor survival and angiogenesis. [3] Therefore an ideal anticancer treatment should not only be able to kill malignant cells, but also able to specifically migrate to tumor site and suppress the support that is provided by tumor stroma elements.

Inherent tumor tropism and infiltrative potential of stem cells has made them a good vehicle candidate for antitumor therapy. [4] Stem cells are derived from different resources including embryo, fetus, cord blood and several other tissues while all of them possess self-renewal and differentiation properties. [5] Even though some studies have demonstrated promoting effects of mere stem cells on tumor growth, [6] scientists have attempted to confer tumor growth inhibitory properties to stem cells by genetically modify them with therapeutic genes from different categories including suicide genes, immunemodulatory agents (like interleukins), toxic agents, and oncolytic viruses. [7, 8]

In this review different transfection methods of stem cells, the potential and various approaches of using mesenchymal stem cells (MSCs) for cancer treatment, and the challenges exist in stem cell therapy are discussed.

MESENCHYMAL STEM CELLSMesenchymal progenitor cells or MSCs are heterogeneous subset of nonhematopoietic cells that exist within the bone marrow stroma. These adult stem cells can be ex vivo expanded and induced, either in vitro or in vivo, to finally differentiate to cells of diverse lineages. [9] They can be isolated from bone marrow and other tissues and organs such as adipose tissue,[10] muscle, dental pulp,[11] liver,[12] amniotic fluid,[13] menstrual blood[14], and dermis. [15] The following minimum criteria were provided by The International Society for Cellular Therapy for defining multipotent human MSCs: [16] “1) plastic-adherent under standard culture conditions; 2) positive for expression of CD105, CD73, and CD90, and negative for expression of hematopoietic cell surface markers CD34, CD45, CD11a, CD19, and HLA-DR; 3) under specific stimulus, cells should differentiate into osteocytes, chondrocytes, and adipocytes in vitro.”

The biological role of MSC is to repair used and damaged tissues in the body. [17] The increased production of chemokine and inflammatory mediators in the site of injury is believed to be responsible for recruitment of MSCs and stimulate them to migrate to the injured tissue. [18] The use of MSCs for cell therapies relies on their


homing capacity to bone marrow and damaged/injured organs. [17] Additionally, the major histocompatibility complex (MHC)1 is expressed on MSCs, but these cells lack MHC 2 and costimulatory molecules CD80, CD86, and CD40. Thus, as opposed to embryonic stem cells, MSCs can be selected from autologous bone marrow and applied in vivo without immunologic consequences. MSCs can be easily transduced/transfected by viral or non-viral techniques and used in both local and systemic therapies with no serious ethical issues. [19, 20] Recent findings on tumor-directed migration and incorporation of MSCs and their anti-angiogenic effect on melanoma mouse models illustrate the great potential for these stem cells to be utilized as an ideal vehicle for anticancer gene delivery.

GENE TRANSFER TECHNIQUES TO STEM CELLSIn order to genetically engineer stem cells and in particular MSCs with therapeutic genes, efficient gene transfer techniques to these cells are required. Introduction of foreign DNA to most cell lines can be successfully performed using various methods, but transfection of stem cells needs a careful selection of gene transfer techniques. The transfection method should not only provide high transfection efficiency and be non-toxic, but must not alter any of the typical stem cell characteristics, including their differentiation and proliferation potential. [21] Duration required for transgene expression (permanent or transient) is another criterion for choosing the proper gene delivery system. For example, to correct a genetic pathology, gene expression is required for the duration of a patient’s life, but to treat a non-inherited disease such as cancer, the expression of the therapeutic gene is only needed for a short period of time. [22] The strategies that have been used so far to deliver genes into MSCs include applying viral vectors and non-viral gene transfection techniques.

VIRAL VECTORSSeveral types of viruses including adenovirus, herpes simplex virus (HSV), retrovirus, lentivirus, and adeno-associated virus (AAV) have been used to transfer gene to adult stem cells. [23] Lentivirus and retrovirus are both RNA viruses with low packaging capacity that integrate their genome with genome of the host cell. [24] Therefore, they can provide long-term gene expression which makes them suitable vectors for preparing bioengineered stem cells for the purpose of stable and permanent genetic alteration. [21] Lentivirus-based vectors are reported to transduce MSCs most efficiently among all integrating viruses. [25] Retroviral vectors are less effective than lentiviral vectors due to loss of transgene expression overtime. [26]

Adenovirus and HSV are both nonintegrating vectors with double-stranded DNA and are preferred for short-term gene therapy. Most bioengineered oncolytic viruses are either adenovirus or HSV-2. [24] Among all the viruses that are being developed for gene therapy, HSV-1 is the largest and most complex virus. Wild type of this virus has neurotropism and therefore, has been used extensively in clinical trials for


treatment of glioma. [27] HSV-based vectors can efficiently transduce MSCs but in order to develop safe replication-deficient vectors further investigations are needed. [22] Adenoviral vectors are reported as the most efficient class of vector in terms of delivering their genetic cargo to the cell nucleus. [24] Adenovirus can efficiently transduce MSCs and has been a popular vector for gene delivery to the tumor site. In some studies, researchers have modified this viral vector with polyethylene glycol and observed enhanced accumulation of the vector in tumor tissue. [28, 29] However, possibility of a strong immune reaction against adenoviral proteins hinders their overall efficacy and several clinical trials using adenoviral vectors have been terminated because of the unexpected toxicity and immunogenicity. [30, 31]

AAV is single-stranded DNA virus and a member of parvovirus family that needs a helper virus, like adenovirus or HSV for replication. AAV has low DNA packaging capacity but on the other hand no pathology has been reported associated with this virus. [23] Therefore, recombinant AAV vectors (rAAVs) are considered safe for long-term gene transfer and expression in non-proliferating tissues. Many researchers have been tried to transduce hematopoietic and other stem cells with AAV. Many studies reported the feasibility of transuding MSC with AAV serotype 2; however, they left several basic questions regarding transduction efficiency and kinetics unanswered. [25, 32, 33] Reasons for the low transgene expression following AAV mediated gene delivery to MSC include scarcity of the primary cell surface receptor for AAV -which limit the number of endocytosed virions- and rate limiting step of second strand synthesis of single stranded viral genome. [34]

Despite all the limitations and problems reported by using viral vectors, their high transduction efficiency, reproducibility and capability to package single DNA inside their core, have kept them a popular choice for transducing the primary cells. [24, 35]

NON-VIRAL VECTORSDue to the limitations exist for viral vectors including risk of immunogenicity and cost of manufacturing, non-viral vectors have been developed for safe gene transfer to the cells. Ease of preparation, unrestricted plasmid size, and low immune response and are main advantages of non-viral gene transfer methods over using viral vectors. [21, 36] The gene expression by the non-viral system is transient and thus, well suited to be used for cancer therapy. Non-viral techniques used for gene delivery can be divided into two categories: 1) chemical techniques which include using cationic polymer, cationic lipids or calcium phosphate preparation method. 2) physical methods which include needle injection, particle bombardment and electroporation. [37] Liposomal carriers and electroporation are reported as the most efficient techniques/vectors for transfecting primary cells among the current non-viral methods. [22] Therefore, I will only focus on these two methods in this review.

Electroporation was first tested on bacteria and thereafter Neumann et al. [38] used it to deliver a gene to mammalian cells. Since then it has been vastly used for gene transfer to the cells in vitro and in vivo. In this process, after local or systemic injection of the DNA solution, a high-voltage pulse is applied to the cells for fractions of a second and forms temporary micropores in cell membrane, while the following low-voltage pulses drive the nucleic acids through these pores into the cells. [37] Electroporation can be applied to variety of cell lines, is not


limited to plasmid size, and since the uptake of the molecule is immediate it yields greater transfection/transformation efficiencies. However, high cell mortality post-electroporation was reported in many cases. In order to maintain high cell viability, many parameters including electrical parameters must be optimized for each cell size and cell type. [39, 40] Ferreira et al. [22] established optimal electroporation conditions for transfecting MSCs. They were able to deliver lacZ reporter gene to bone-marrow derived rat MSCs by electroporation. They adjusted electric pulse type, pulse electric intensity, electroporation temperature, and electropulsation buffer conductivity to optimize the electrotransfer process.

An improved version of electroporation has been developed in the past few years and is called Nucleofection. This technology was designed for transferring various forms of nucleic acids to primary cells and difficult-to-transfect cell lines. This process is based on using combination of electrical parameters and buffers that should be adjusted for each specific cell type. The ability to deliver nucleic acid to the nucleolus makes this technique highly efficient for transfection of primary cells. [41, 42]

Lipofection is a very popular and affordable transfection method. Cationic lipids have positively charged head groups and hydrophobic tails. By mixing cationic lipids in liposomal form with nucleic acids solution, lipoplexes are formed via electrostatic interaction of nucleic acids and lipids. The positive net charge of lipoplex facilitates their fusion with cell membrane and causes destabilization of endosomal membrane which leads to release of DNA/RNA into cytosol. [37, 43] Cationic liposomes have showed lower transfection efficiency compared to electroporation and viral vectors. Their transfection efficiency can be usually enhanced by applying higher dosage of cationic agents but then it is also associated with toxicity and low cell survival. [44]

In general, non-viral vectors raise less safety concerns, are easier to produce and manufacture in large-scale, and can carry large therapeutic genes; thus, being a more attractive option from a clinical point of view. [45] However, despite recent advances, these systems still show low to moderate gene transfer efficiency when compared with viral vectors. [46] There are very limited investigations where the researchers have compared non-viral with viral techniques for gene delivery to the stem cells. McMahon et al. [47] actually performed a comparative study to transfer genes to the bone marrow-derived rat MSCs via viral and non-viral vectors. Among the viral vectors they used, Lentivirus resulted in transduction efficiencies of up to 95% and was found to be most effective with low levels of cell toxicity. Moderate transfection levels were obtained by lipofection of plasmid DNA and cell mortality was also reported for this method. Electroporation was found to be ineffective at the parameters tested and caused high cell mortality.


DIFFERENT APPROACHES TO USE MESENCHYMAL STEM CELLS FOR CANCER THERAPYDELIVERY OF INTERLEUKINSInterleukins are one group of cytokines that regulate inflammatory and immune responses. The rational for delivery of interleukins to the tumor site is to boost endogenous immune response by activating natural killer cells and cytotoxic lymphocytes and take advantage of their tumoricidal effects. Bioengineered MSCs that secrete interleukins can insure tumor-targeted delivery of these cytokines and improve the immune surveillance against tumor cells, hence they have synergistic therapeutic benefits. [7] Gao et al. [48] transduced human bone marrow-derived MSCs with adenoviral vector expressing the murine interleukin-12 (IL-12) and administered them systemically into a renal cell carcinoma mouse model. IL-12 secreting MSCs resulted in reduction of tumor growth and enhanced mouse survival. In another study, rat MSCs producing interlukin-18 (IL-18) was injected intratumorally in glioma rat models and was associated with inhibition of tumor growth. The prolonged survival of glioma-bearing rats is due to enhanced T cell infiltration and long-term anti-tumor immunity. [49]

DELIVERY OF INTERFERONAnti-proliferative and proapoptotic effects of Interferon (INF)-β on malignant cells and its antiangiogenic properties have been shown in previous studies. [50] However, systemic administration of effective dose of INF-β can generate toxicity. By manipulating MSCs to produce INF-β one can benefit from tumor tropism of MSCs and provide targeted delivery of INF-β to tumors and metastatic cells. Bone marrow-derived MSCs that were transduced with adenoviral vector expressing INF-β could engraft at the site of tumor and produced INF-β locally in melanoma mice model. However, this treatment could not significantly prolong the survival. [51] Ren et al. [52] used the same approach in a mouse prostate cancer lung metastatic model. In that study, INF-β-expressing MSC therapy was able to promote tumor cell apoptosis, enhance natural killer cells activity, and finally reduce the size of the tumor in lungs significantly without increasing systemic level of INF-β in mouse. This group also tested the antitumor effect of INF-α-expressing MSCs in mouse melanoma lung metastasis model, which resulted in prolongation of mouse survival but could not completely cure the cancer. [53]

GENE-DIRECTED ENZYME PRODRUG THERAPY (GDEPT)This technique uses MSC as a cell-based vehicle for gene delivery to the tumor site. This gene is supposed to encode an enzyme to convert a prodrug to its active form that is toxic to the rapidly dividing cells. The active drug can kill the stem cells and the neighboring tumor cells due to bystander effect. [18] One example of GDEPT system is herpes simplex virus thymidine kinase (HSV-TK)-expressing MSCs


that acts on gancyclovir. This enzyme can selectively phosphorylate gancyclovir to its toxic metabolite, gancyclovir triphosphate. Many groups have used this system for cancer therapy. Uchibori et al. [54] isolated MSCs from rat bone marrow and transduced them with retroviral vector to express HSV-TK. The modified MSCs were injected into systemic circulation of glioma mouse model, which resulted in tumor-specific transduction of HSV-TK and significant suppression of tumor growth compared to the controls. Another popular example of GDEPT system is cytosine deaminase (CD) expressed by MSCs that can convert nontoxic prodrug 5-fluorocytosine (5-FC) to cytotoxic form 5-fluorouracil (5-FU). The short-term efficacy of this system as an antitumor treatment has been tested in vitro and in vivo on many cancer models, including prostate tumor-bearing mice, [55] and xenografts of human melanoma in nude mice, [56] and mouse gastric cancer xenograft. [57] Chang et al. [58] demonstrated that single treatment of MSC expressing CD in presence of 5-FC can suppress brain tumor growth in early tumor stages, but multiple transplantations of these MSCs are required to for a successful therapy in later stages of brain tumor.

ONCOLYTIC VIRUSESOncolytic viruses (OV) are natural or genetically modified viruses that can selectively replicate in cells and as a result destroy them while sparing normal cells. [7] Stem cell-based delivery system not only acts as host cell and a carrier for local delivery of OV to tumor microenvironment, but also shields OV from host defense and increase antitumor immune response. Reovirus, adenovirus, and HSV are examples of viruses that have been tested as oncolytic agents. [59] Feasibility of this approach for tumor-targeted delivery of oncolytic adenovirus has been studied in metastatic ovarian cancer, [60, 61] lung metastatic breast cancer, [61] and glioma. [62] Even though promising results have been achieved using stem cell vehicle systems, some groups have reported lack of specificity of tumor homing of OV loaded-MSCs following intravenous injection, which can be resolved in some cases by direct administration of MSCs into arterial systems of the targeted organ. [63]

DELIVERY OF OTHER THERAPEUTIC GENES AND PROAPOPTOTIC PROTEINSDelivery of antiangiogenic factors has also been studied to normalize the abnormal structure of blood vessels at tumor site. The rational is by blocking angiogenesis, expansion and metastasis of tumor can be prevented. MSCs are known to localize to tumor vasculature following intratumoral implantation, thus offering potential to be used as carrier for delivery of antiangiogenic agents. [7] Eekelen et al. reported sustained delivery of antiangiogenic protein thrombospondin type-I (aaTSP-1) by bioengineered human neural stem cells, which lead to reduction of tumor vessel density and prolongation of survival in glioma-bearing mice. [64]

Other examples of new therapeutic approaches to deliver MSC expressing genes include targeted delivery of NK4, a competitive antagonist of hepatocyte growth factor to inhibit angiogenesis and tumor invasion; and targeted delivery of tumor necrosis factor-related apoptosis induced ligand (TRAIL) that activates intracellular caspases leading to apoptosis preferentially in cancer cells. Human MSCs are found to be resistant to TRAIL- mediated apoptosis.[7, 65]


DELIVERY OF ANTICANCER DRUG-LOADED NANOPARTICLESAnticancer drug-loaded nanoparticle delivery has been a hot topic in the past three decades. Lipid/polymeric particles can help with enhancing stability, solubility and targeting of antineoplastic agents. However, rapid clearance of some nanoparticles from systemic circulation and their low uptake by tumor cells are the main challenges in passive delivery of this system. Inherent migratory properties of MSCs towards tumor make them a good candidate to serve as vehicle for drug delivery in cancer therapy. Internalization of various lipid and polymeric particles in stem cells has been examined. There are some major factors that one needs to take into account when using this therapeutic approach: 1) The process of nanoparticle uptake in MSCs has been shown to have time, dose, and structure-dependent behavior. 2) Nanoparticle system should be able to escape from endosomal degradation. 3) In order to keep the vitality of stem cells after nanoparticle uptake, antineoplastic agent loaded in nanoparticles should not affect stem cell proliferation. Up to now, limited studies have been performed on drug-loaded nanoparticles incorporated in MSCs for cancer therapy. [66] Li et al. [67] were among the first groups who utilized this approach. They prepared doxorubicin-silica nanoparticles and bioconjugated them with a monoclonal antibody that selectively bind to MSCs’ membrane proteins and generate cellular endocytosis of doxorubicin-nanoparticles. Following intratumoral administration of this system into glioma xenogratfs in mice, high distribution of doxorubicin was found in tumor tissue -compared to the mice treated with free doxorubicin- and tumoral apoptosis was reported after 7 days.

MSCS MORE THAN AN INERT VEHICLE IN CANCER THERAPYStem cell-based cancer gene therapy is driven by the tumor tropism of MSCs. These cells can migrate to the tumor site via a similar mechanism involved in their migration to the wounds. Various tumor-secreted chemokines, which have receptors on MSCs, have been discovered to take role in this process. [20] Interestingly, controversial findings exist in the literature on role of MSCs in tumor development. In this section some of the research done on MSCs to study whether they suppress or support tumor proliferation are summarized and analyzed.

MSCs appear to have intrinsic antitumor properties. These stem cells are reported to arrest lymphoma and hepatoma cells at G0/G1 which would reduce proliferation and enhance cancer cell apoptosis. [68] Nakamura et al. [69] also reported inhibition of tumor growth and survival improvement of rats with glioma following intratumoral injection of MSCs. Khakoo et al. [70] demonstrated another mechanism for antitumor activity of MSCs, which is inhibition of Akt protein kinase activity. This inhibition in a mouse model of Kaposi’s sarcoma required the MSCs to make direct cell–cell contact.

The release of soluble factors from MSCs can also led to tumor growth suppression. Qiai et al. [71] reported that conditioned media from MSCs was associated with down regulation of nuclear factor kappaB in hepatoma and breast cancer cells and as a result reduction of their proliferation in vitro. They also described a further


mechanism with down regulation of Wnt pathway in breast cancer cells following release of dickkopf proteins from MSCs, which resulted in reduction of tumor proliferation. [72]

Even though MSCs-based anticancer delivery systems represent an exciting therapeutic approach, a better understanding of their tumor homing/engrafting mechanism and their impact on tumor behavior is crucial before we can understand the risks and full potential of using MSC carrier system in a clinical setting. Several published studies in the past few years have revealed MSC’s stimulatory effects on tumor growth. The immunosuppressive effect of MSCs is one of the main issues that needs to be resolved before using them for cancer therapy. Immunosuppressive properties of MSCs on T-lymphocyte and B-lymphocyte proliferation were found to be mediated by the production of cytokines and some growth factors such as hepatocyte growth factor and transforming growth factor-β1. [73] Djouad et al. [74] investigated immunosuppressive effect of MSCs and its role in tumor growth using mixed lymphocyte reaction assay. They reported that immunosuppressive properties of murine MSCs exhibited in mixed lymphocyte reaction were mediated by soluble factors, secreted only on activation of MSCs in the presence of splenocytes. Furthermore, B16 melanoma cells transplanted into allogeneic mice formed tumors only when MSCs were coinjected.

Production of trophic factors by MCSs has also been associated with increasing tumor growth and spread. MSCs acquired expression of tumor-associated fibroblasts antigens, such as α-smooth muscle actin, fibroblast-specific protein, vimentin, and SDF-1 after exposure to the tumor microenvironment in vivo and in vitro. [75] Karnoub et al. [76] prepared a xenograft model with bone marrow-derived human MSCs with green fluorescent protein-labeled human breast cancer cells (MCF/Ras, MDA-MB-231, MDA-MB-435, and HMLER) in a ratio of 3:1 and injected subcutaneously into immunocompromised mice. The MSCs exhibited causing acceleration of tumor growth only in MCF/Ras cells and not affecting local tumor growth in the other cell types. However, in all the investigated cell lines, coinjection with MSCs enhanced the number of breast cancer metastases in the lungs. Their further screening for the change in the levels of various cytokines, chemokines and growth factors following MSC and MDA-MB-231 co-culture revealed that release of CCL5 from MSCs is the reason for promotion of invasion and metastasis of breast cancer cells. In another study, Sasser et al. [77] introduced interleukin-6 as a key mediator released by MSCs in presence of human breast tumor cell lines, that induced phosphorylation of signal transducer and activator of transcription 3 (STAT3) and terminally enhanced tumor cell proliferation. Further stimulatory effects of MSC on tumor growth were demonstrated with PC3 prostate cancer cells. [78] Tumor vascularization mediated by expression of FGF2 was the proposed mechanism for tumor progression.

Another concern for the use of MSCs as an anticancer delivery carrier is the fate of these cells in vivo. Unlimited potential for proliferation could also lead to transformation of MSCs into malignant tissue. Rubio et al. [79] demonstrated that even though human MSCs can be managed safely during 6-8 weeks of the standard ex vivo expansion period, following long-term in vitro culture (a few months) they might undergo spontaneous transformation.

MSCs interact with tumor cells in a numerous ways, which may either promote or inhibit tumor growth. Moreover, MSCs interact with their tumor-resident neighbors, like immune cells and fibroblasts. [20] Appropriate cell surface marker expression


is one the main requirements before using MSCs for in vivo studies. There are inconsistent reports about the effect of MSCs on malignant cells and that is likely due to the heterogeneity in MSC. To address this issue, cell surface markers, like NG2 that permit isolation of a more homogenous population of MSCs are required. [80] Confluence and Passage number can impact MSC function. Some authors have reported growing MSCs in media supplemented with high serum or growth factor, which may change the MSCs phenotype. Variable dose of MSCs delivered may also contribute to these discrepant results. In general, the groups who reported tumor growth enhancement with MSCs have used a higher MSC to cancer cell ratio. Yu et al. [81] revealed that using higher percentage of MSCs with glioma cell line increased tumor growth. This suggests that MSCs may affect tumor growth in a dose-dependent manner. Another factor influencing MSCs’ performance is the timing at which these stem cells are introduced into the tumor microenvironment. In the literature, scientists reporting tumor growth inhibition by MSCs, have used different methods and timing of MSCs delivery. Some groups prepared a xenograft model containing both MSCs and cancer cells [72],[68]. Implantation into a gelatin matrix [82], intravenous delivery of MSCs [70], and administration of MSCs into an established tumor [83] are other approaches that could minimize the direct contact of MSCs with cancer cells during tumor formation. On the other hand, most of the studies reporting tumor growth promotion, mixed tumor cells with MSCs ex vivo and coinjected the cells in animals. The presence of MSCs during early cancer cell divisions in vivo may act in favor of tumor growth by inducing angiogenesis, which is required for tumor initiation. Lin et al. [78] was among the rare groups who injected MSCs a week after establishment of tumor in mice and observed tumor progression.

CONCLUSIONThe homing of stem cells to the tumor tissue is expected to cause less host toxicity and higher local delivery of the anticancer agents. Scientists have faith in using stem cells as vehicle for cancer treatment and it possess definite potential for translation to clinical medicine. Nevertheless, the malignancy potential of these cells and their unresolved role in tumor growth and metastasis are the major concerns that are needed to be addressed before using them in human subjects. It is important to note that to our knowledge, stimulatory effects of MSCs on tumor development reported in literature were observed only when used as isolated therapy. The studies in which MSCs were utilized as gene careers to deliver specific anticancer agents to tumor cells mostly documented success in reducing tumor size and increasing the survival. In addition, tumor tropism of MSCs has been shown to significantly increase with the application of radiation in glioma, breast and colon cancer xenograft models. [84, 85] This indicates the possibility of using stem cells in combination with radiotherapy for cancer treatment. Finally, many scientific questions about stem cell delivery, including the number of cells needed and their optimal passage number, timing, and route of delivery must be answered before stem cell-based cancer gene therapy becomes a realistic possibility.


ACKNOWLEDGEMENTThe author would like to acknowledge Faranak Salman Nouri for her valuable input in the topic.


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6

BY: PRACHI SHAH

DOUBLE EMULSIONS: SCOPE AND ATTEMPTS


ABSTRACTDouble emulsions are complex heterogeneous systems where smaller droplets are dispersed in an already dispersed phase. They were first noticed in 1925 by Seifriz. However they have attracted attention only in the last 30 years. Double emulsion may have a great potential in pharmaceutical application. However, there is very limited information about the formulation of such a dosage form. The limitation of Double emulsions is their inherent stability. These systems have a tendency for flocculation and creaming making them unsuitable for storage. They also tend to release the entrapped phase in an uncontrolled manner. Various attempts have been made over the last 30 years to overcome this problem. Double emulsion is a relatively new and scarcely tapped area, and the dynamics of these complex systems need better understanding.

INTRODUCTIONEmulsions are heterogeneous systems, where one immiscible liquid is dispersed in another in the form of small droplets. In medical and pharmaceutical practices, emulsions have been used since the earliest days for the administration of oils and drugs. They are classified as either oil in water (o/w) or water in oil (w/o) based on the continuous phase. An emulsifier or surface acting agent is present to stabilize the system. A lipophilic surfactant (low HLB 2-6) is used for a w/o system and a hydrophilic (high HLB above 8) is used for o/w systems. The emulsifier acts as a film former and reduces the interfacial tension and provides a mechanical barrier against coalescence thus stabilizing the system. Emulsions can be administered orally, parenterally and topically. These are complex systems and a change in the stability can affect drug release.

Multiple emulsion systems are more complex than simple emulsion as in these finer droplets are dispersed in an already dispersed phase. In a lot of cases, the innermost dispersed phase is either miscible or in some cases identical to the continuous phase. Also multiple surfactants of varying hydrophilicity are present to stabilize each dispersed phase. In some disciplines, certain multiple emulsions have been termed “liquid membrane” systems, as the liquid film which separates the other liquid phases acts as a thin semi-permeable film through which solute must diffuse moving from one phase to another [1].

Multiple emulsions were first described by Seifriz in 1925 [2]. He noticed tiny water droplets dispersed in the oil phase of an o/w system. Later he also noticed oil droplets dispersed in the aqueous phase of a w/o system. He referred to these systems as bimultiple systems. Further on, he found that even more complex, trimultiple, quatremultiple and quinquemultiple systems existed. Even though multiple emulsions have a history that dates far back, the potentials of these systems have been explored mainly in the last 30 years. A lot of research has been carried out on the practical and theoretical aspects of emulsions. However, of the many systems studied, a very small amount of attention has been paid to multiple emulsions. These systems have shown application in many fields, particularly in separation sciences,


in the separation of hydrocarbons and removal of toxins from waste water [3]. These systems also have potential in biopharmaceutical applications such as adjuvant vaccines [4], prolonged drug delivery systems [5], as sorbent reservoirs for treatment of drug overdose [6], for immobilization of enzymes [7]. Multiple emulsions have also been formulated as cosmetics (British Patent 1979) and in formulation of daily usable products like wax polish [8]. It is thus established that there are many practical applications of multiple emulsions. The main problem associated with multiple emulsions is their inherent instability [9]. For successful pharmaceutical applications, the formulation of a stable and reproducible system is critical. The parameters related to these systems have yet to be studied and understood.

The two major types of double emulsions are w/o/w where the internal and external aqueous phases are separated by an oil layer and o/w/o where the oily phases are separated by an aqueous layer. W/o/w emulsions have the same advantages of a w/o emulsion like taste masking, controlled release, protecting a hydrophilic drug in the GIT [10] and in addition have lower viscosity owing to the lower viscosity of the continuous aqueous phase. Lower viscosity makes them easier to handle and administer orally or parenterally.

Over the years, theory of multiple emulsions has been applied to make nano-particles. Briefly, in this method, a polymer is dissolved in an organic solvent. A drug solution is dispersed in the polymer solution to form a primary emulsion. This primary emulsion is then dispersed in an aqueous solution to make a double emulsion. After the formation of the double emulsion, the organic solvent is evaporated and nano-particles of drug entrapped in polymer matrix are formed [11].

NOMENCLATUREIt is useful to have an unambiguous system of nomenclature to avoid any confusion between the phases. For instance, in a w/o/w emulsion, the continuous oil phase of a primary w/o emulsion used to prepare the multiple emulsion, becomes the dispersed oil phase of the multiple emulsion. Also, the aqueous phase could refer to either the dispersed droplets in the primary emulsion or the continuous phase of the double emulsion. Thus the system can be notated as w1/o/w1. In those cases where the two aqueous phases are not identical, the system can be called w1/o/w2. .

FORMATION OF DOUBLE EMULSIONSThere are reports in literature on the accidental formation of multiple emulsions during normal emulsification procedures. More often than not, there is no reproducibility in these “unexpected preparations”. In most cases, the content of multiple drops is quite low and the products have poor stability. There is a chance that multiple structures are formed in many liquid-liquid interactions but they disappear before they can be observed due to their poor stability. The occurrence


of these systems have been noticed in cases where the disperse phase volumes are over 74% [12] and during phase inversion while the surfactants partition between two phases [13]. In these cases some of the original structure gets trapped in the final emulsion [14].

MAKING A W/O/W EMULSIONDouble emulsions are prepared in the laboratory by re-emulsification of a primary emulsion. A two-step procedure is therefore necessary. The first step involves making a primary w/o emulsion. The second step involves further emulsification of the primary emulsion in water to make a w/o/w emulsion.

The primary emulsion can be prepared by using a vortex mixer, ultrasonicator, high speed homogenizer and the likes. A lipophilic surfactant is used as it favors a w/o emulsion. This primary emulsion is added to an aqueous solution of a hydrophilic surfactant to promote o/w formation. This step of re-emulsification is critical. Excess mixing or high shear mixing can rupture the oil droplets releasing the aqueous phase within to mix with the continuous aqueous phase to form a simple o/w emulsion. In this step high shear mixers and sonicators are unsuitable. A small, low-shear mixer may be employed or the mixture may be shaken by hand. However, no matter what emulsification method is used for the second step, some of the internal aqueous phase is unavoidably lost to the external aqueous phase [9].

COMPONENTS OF DOUBLE EMULSIONLIPIDS The nature of the lipids can seriously affect the behavior of the multiple systems. However, most lipids will form multiple emulsions in correct conditions [9]. A mixture of lipids can be used to modify viscosity. Liquid paraffin was used by Matsumoto et al [15]. Estelle Sigward et al used medium chain triglycerides [16], and Suzuki et al, used triolein [17].

SURFACTANTSBoth ionic and nonionic surfactants can be used to yield multiple emulsions. However their selection would depend on the intended function of the system. In food, drugs and cosmetic purposes, toxicity of surfactants must be paid attention to. Nonionic surfactants are used as they are less likely to interact with ionic compounds and owing to their lower toxicity. It seems necessary to use two surfactants, one capable of emulsifying a w/o system and one capable to emulsify an o/w system. The optimum surfactant required to emulsify a given lipid can be chosen based on its hydrophilic-lipophilic balance (HLB) system, suggested by Griffin. In general, lipophilic surfactants with HLB value in the range of 2-6 can be used for


the primary w/o emulsion whereas hydrophilic surfactants with HLB value from 8-16 can be used for w/o/w emulsion. Equilibration of the systems after mixing will undoubtedly result in the transfer of surfactants between aqueous and non- aqueous components. Estimates of stability can be made by visual inspection of a series of emulsions prepared with blends of surfactants having different HLB values. There should be an optimal concentration range of surfactants that can be used to stabilize the system. Low concentration runs the risk of rapid degradation of the double emulsion and high concentration may increase the viscosity and toxicity.

PHASE VOLUME According to Matsumoto et al [15], internal phase volume has no bearing on the yield of w/o/w emulsions. It would appear that w/o/w emulsions can be prepared using a wide range of internal phase volumes.

NATURE OF THE ENTRAPPED MATERIALThe nature of the entrapped material may affect the stability of the system. Due to the nature of the multiple emulsions, the middle phase may act as a membrane and osmotic effects may become significant. Entrapped drug may interact with the surfactant or get adsorbed at the interface. Both these phenomena can result in decreased stability [9].

TYPES OF DOUBLE EMULSIONS There can be two major types of double emulsions. One in which an aqueous phase is dispersed in a lipid and the lipid is again dispersed in an aqueous media (w/o/w). The aqueous phase may or may not be the same, making either w1/o/w1 or w1/o/w2. Another type is where the aqueous phase separates two lipid phases. The lipids in both the phases may or may not be the same giving either o1/w/o1 or o1/w/o2 emulsions.

STABILITY OF A DOUBLE EMULSIONDouble emulsions are regarded as thermodynamically unstable. This can be responsible for various problems after being prepared such as phase separation, leakage of the innermost phase from the droplets, flocculation of the droplets in the innermost phase.


Three methods have been suggested by Florence and Whitehall [9] for stabilization of double emulsions. They suggest (I) Using a highly viscous oil to prevent diffusion of individual surfactant and water molecules, (II) Polymerization of interfacially adsorbed surfactant molecules and (III) Gelation of the oil or aqueous phases of the emulsion.

ATTEMPTS TO IMPROVE STABILITY OF MULTIPLE EMULSIONSEven though double emulsions were first observed in 1925, it is only in the last 30 years that they have attracted attention in pharmaceutical research. Various studies have been carried out to stabilize multiple emulsions. Hino et al [18] stabilized double emulsions by three methods. They used a hypertonic solution for the inner aqueous phase. Making the inner aqueous phase hypertonic prevented phase separation and prolonged the release of drug with low partition coefficient between oil and water. The second approach was addition of chitosan to the inner phase and thirdly by phase inversion of the primary emulsion using porous membrane. Omotosho et al [19] suggested that stability of double emulsions can be increased by interfacial adsorption of albumin. Further on, they used polyvinylpyrrolidone, acacia and gelatin in an attempt to increase stability of multiple emulsions [20]. Colloidal microcrystalline cellulose was used by Oza et al. [21]. Cole et al related the droplet size to release of theophylline from the emulsion. The droplet size was further found to be related to the lipophilic surfactant selected [22].

In more recent times, Qi et al used the a self-emulsification approach drug to stabilize a formulation containing pidotimod, a peptide like drug with high solubility and low permeability [23]. Lastly Sigward et al stabilized a double emulsion system using a high ratio of hydrophilic surfactant. The droplet size achieved was in the nano range [16].

ANALYSIS OF DROPLET RELEASE IN DOUBLE EMULSIONSThere could be many possible pathways leading to the instability of multiple emulsions. This is well represented in figure 2. The aqueous droplets dispersed in the oil droplet could coalescence or diffuse through the oil membrane. Additionally, the larger oil droplet could coalescence to decrease the stability of the system. The mechanism of droplet release from the primary emulsion depends on the inherent properties of the component used. Jager-Lezer et al. [24] studied the kinetics of release of droplets from double emulsion from a formulation having a lipophilic surfactant. Their results indicate that the release was due to swelling of internal aqueous droplets which led to the breakdown of the oil globules and the lipophilic surfactant


used increases the swelling capacity of the globules. On the other hand, in the formulation prepared by Bonnet et al. [25] the release of the water soluble drug took place without rupturing of the oil globule. Leakage occurred by diffusion through the lipid membrane.

ANALYSIS OF DOUBLE EMULSIONSIn most cases, the double emulsions formulated are in the macro size and can be easily observed under an optical microscope. Sigward et al used the Transmission Electron Microscope (TEM) [16] to study the droplets in the nano range.

CONCLUSIONIt can be seen that very limited research has been carried out in the area of multiple emulsions. Their potential is not completely explored. Various parameters like effect of dilution, pH dependence are not yet clear. Stability still remains an obstacle that needs to be overcome. Mechanism of release of the innermost droplets is not clear and this field calls for more research.


FIGURESFIGURE 1 SCHEMATIC REPRESENTATION OF A W/O/W DOUBLE EMULSION DROPLET (ADAPTED FROM [16])

FIGURE 2 POSSIBLE PATHWAYS LEADING TO INSTABILITY OF MULTIPLE EMULSIONS (ADAPTED FROM [26])


FIGURE 3 ILLUSTRATION OF TWO STEP EMULSIFICATION PROCESS TO MAKE A DOUBLE EMULSION (ADAPTED FROM [9])


REFERENCES1. Yung-Chi Lee, Samuel H Yalkowsky, Effect of formulation on the systemic absorption of insulin from enhancer-free ocular devices, Int J of Pharm; 1999; 185(2): 199–204.

2. Seifriz W. Studies in Emulsions. III-V. J Phys Chem 1925;29(6):738-749.

3. Li NN. Separating hydrocarbons with liquid membranes 1968.

4. Taylor P, Miller CL, Pollock T, Perkins F, Westwood M. Antibody response and reactions to aqueous influenza vaccine, simple emulsion vaccine and multiple emulsion vaccine. A report to the Medical Research Council Committee on influenza and other respiratory virus vaccines. J Hyg 1969;67(03):485-490.

5. Elson LA, Mitchlev BC, Collings AJ, Schneider R. Chemotherapeutic effect of a water-oil-water emulsion of methotrexate on the mouse L1210 leukaemia. Rev Eur Etud Clin Biol 1970 Jan;15(1):87-90.

6. Chiang C, Fuller GC, Frankenfeld JW, Rhodes C. Potential of liquid membranes for drug overdose treatment: in vitro studies. J Pharm Sci 1978;67(1):63-66.

7. May S, Li N. Encapsulation of enzymes in liquid membrane emulsions. Enzyme Engineering Volume 2: Springer; 1974. p. 77-82.

8. Mackles L. Wax composition and method for making the same 1968.

9. Florence A, Whitehill D. The formulation and stability of multiple emulsions. Int J Pharm 1982;11(4):277-308.

10. Constantinides PP, Scalart J. Formulation and physical characterization of water-in-oil microemulsions containing long-versus medium-chain glycerides. Int J Pharm 1997;158(1):57-68.

11. Cohen-Sela E, Chorny M, Koroukhov N, Danenberg HD, Golomb G. A new double emulsion solvent diffusion technique for encapsulating hydrophilic molecules in PLGA nanoparticles. J Controlled Release 2009;133(2):90-95.

12. Sherman P. Emulsion science. : Academic Press New York; 1968.

13. Lin T, Kurihara H, Ohta H. Effect of surfactant migration on the stability of emulsions. J.Soc.Cosmet.Chem 1973;24:797-814.


14. Becher P. The effect of the nature of the emulsifying agent on emulsion inversion. J.Soc.Cosmetic Chem 1958;9:141-145.

15. Matsumoto S, Kita Y, Yonezawa D. An attempt at preparing water-in-oil-in-water multiple-phase emulsions. J Colloid Interface Sci 1976;57(2):353-361.

16. Sigward E, Mignet N, Rat P, Dutot M, Muhamed S, Guigner JM, et al. Formulation and cytotoxicity evaluation of new self-emulsifying multiple W/O/W nanoemulsions. Int J Nanomedicine 2013;8:611-625.

17. Suzuki A, Morishita M, Kajita M, Takayama K, Isowa K, Chiba Y, et al. Enhanced colonic and rectal absorption of insulin using a multiple emulsion containing eicosapentaenoic acid and docosahexaenoic acid. J Pharm Sci 1998;87(10):1196-1202.

18. Hino T, Kawashima Y, Shimabayashi S. Basic study for stabilization of w/o/w emulsion and its application to transcatheter arterial embolization therapy. Adv Drug Deliv Rev 2000;45(1):27-45.

19. Omotosho, JA; Law, TK; Whateley, TL; Florence, AT; The stabilization of W/O/W emulsions by interfacial interaction between albumin and non-ionic surfactants; Colloids and surfaces, 1986, 20, 1, 133-144.

20. Omotosho, JA; The effect of acacia, gelatin and polyvinylpyrrolidone on chloroquine transport from multiple w/o/w emulsions; Int.J.Pharm., 1990, 62, 1, 81-84.

21. Oza, Kamlesh P; Frank, Sylvan G, Multiple emulsions stabilized by colloidal microgrystalline cellulose; J of Dispersion Science and Technology, 1989, 10, 2, 163-185.

22. Leadi Cole, Mohammed; L Whateley, Tony; Release rate profiles of theophylline and insulin from stable multiple w/o/w emulsions; J.Controlled Release, 1997, 49, 1, 51-58.

23. Qi X, Wang L, Zhu J, Hu Z, Zhang J. Self-double-emulsifying drug delivery system (SDEDDS): A new way for oral delivery of drugs with high solubility and low permeability. Int J Pharm 2011;409(1):245-251.

24. Jager-Lezer, N; Terrisse, I; Bruneau, F; Tokgoz, S; Ferreira, L; Clausse, D; Seiller, M; Grossiord, JL; Influence of lipophilic surfactant on the release kinetics of water-soluble molecules entrapped in a W/O/W multiple emulsion; J.Controlled Release, 1997, 45, 1, 1-13.


25. Bonnet, Marie; Cansell, Maud; Placin, Frédéric; Monteil, Julien; Anton, Marc; Leal-Calderon, Fernando; Influence of the oil globule fraction on the release rate profiles from multiple W/O/W emulsions; Colloids and Surfaces B: Biointerfaces, 2010, 78, 1, 44-52. 26. Garti, Nissim; Double emulsions—scope, limitations and new achievements; Colloids Surf.Physicochem.Eng.Aspects, 1997, 123, 233-246.


acknowledgements

contributors

kurt nielsen, ph.d. Chairman, Catalent Institute; Chief Technology Officer & Senior Vice President, Innovation and Growth, Catalent Pharma Solutions

cornell stamoran, ph.d. Founding Executive Board Member, Catalent Institute; Vice President, Strategy & Corporate Development, Catalent Pharma Solutions

terry robinson Executive Director, Catalent Institute; Catalent Pharma Solutions

elliott berger Board Member, Catalent Institute; Vice President, Global Marketing & Strategy, Catalent Pharma Solutions

joe montano Catalent Pharma Solutions

graphic designer

rachel santomieri Catalent Pharma Solutions

students

maren preis Heinrich-Heine Universität Düsseldorf

tannaz ramezanli Rutgers University

emrah ilker ozay UMass Amherst

nilesh gupta Texas Tech University

prachi shah St. John's University

7

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api properties formulation technologies

BCS Class I Wet granulation, direct compression, dry granulation

BCS Class II Consider ionizable salts or co-crystals.

Lipid-based delivery systems, SEDDS, spray-dried dispersions, nanocrystals, buccal delivery, hot melt extrusion, cyclodextrin inclusion complexes

BCS Class III Consider pro-drugs or permeability enhancers.

Wet granulation, direct compression, dry granulation

BCS Class IV Consider ionizable salts or co-crystals.

Lipid-based delivery systems, SEDDS, spray-dried dispersions, nanocrystals, buccal delivery, hot melt extrusion, cyclodextrin inclusion complexes, pro-drugs

High Dose Manufacturability concerns.

Wet granulation, dry granulation, dry coating, hot melt extrusion, SEDDS, spray-dried dispersions, ODTs, hot melt extrusion, cyclodextrin inclusion complexes

BCS Class I or BCS Class III low dose

Content uniformity concerns.

Wet granulation, direct compression, dry granulation, dry coating, hot melt extrusion, SEDDS, spray-dried dispersions, nanocrystals, ODTs, ODTs for buccal delivery, hot melt extrusion, cyclodextrin inclusion complexes

BCS Class II or BCS Class IV low dose

Content uniformity concerns. Consider liquid formulations in soft shell capsules.

Also, wet granulation, direct compression, dry granulation, dry coating, hot melt extrusion, lipid-based delivery systems, SEDDS, spray-dried dispersions, nanocrystals, ODTs, ODTs for buccal delivery, hot melt extrusion, cyclodextrin inclusion complexes

pH Sensitive Solvent selection is key. Stabilize and/or enteric coat.

Wet granulation, direct compression, dry granulation, dry coating, spray-dried dispersions, nanocrystals, ODTs, buccal delivery, lipid-based delivery systems, hot melt extrusion, cyclodextrin inclusion complexes

Heat Sensitive Direct compression, dry granulation, dry coating, freeze-dried ODTs, lipid-based delivery systems, SEDDS, spray-dried dispersions, nanocrystals, buccal delivery

Light Sensitive Wet granulation, direct compression, dry granulation, dry coating, lipid-based delivery systems, SEDDS, spray-dried dispersions, nanocrystals, hot melt extrusion, cyclodextrin inclusion complexes.

Consider opaque soft gelatin or soft shell capsules.

Moisture (Water) Sensitive

Direct compression, dry granulation, dry coating, SEDDS, spray-dried dispersions, nanocrystals, hot melt extrusion

Moisture-resistant coating on tablet dosage form

Oxidation Degradation Wet granulation, direct compression, dry granulation, spray-dried dispersions, nanocrystals, ODTs, buccal delivery, lipid-based delivery systems, hot melt extrusion, cyclodextrin inclusion complexes

Consider soft gelatin or soft shell capsules. Avoid excipients with peroxide impurities.

ORAL FORMULATION STRATEGIESWe have synthesized the experiences of industry-leading drug development experts to create this one-page reference guide, which provides suggested formulation technologies that are best suited to help solve specific API challenges.

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