- Email: [email protected]
Stabilization Challenges and Formulation Strategies Associated with Oral Biologic Drug Delivery Systems
Stabilization Challenges and Formulation Strategies Associated with Oral Biologic Drug Delivery Systems Vu Truong-Le, Phillip Lovalenti, Ahmad M. Abdul-Fattah PII: DOI: Reference:
S0169-409X(15)00183-0 doi: 10.1016/j.addr.2015.08.001 ADR 12826
To appear in:
Advanced Drug Delivery Reviews
Received date: Revised date: Accepted date:
2 December 2014 20 July 2015 4 August 2015
Please cite this article as: Vu Truong-Le, Phillip Lovalenti, Ahmad M. Abdul-Fattah, Stabilization Challenges and Formulation Strategies Associated with Oral Biologic Drug Delivery Systems, Advanced Drug Delivery Reviews (2015), doi: 10.1016/j.addr.2015.08.001
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
IP
T
ACCEPTED MANUSCRIPT
MA
NU
SC R
Stabilization Challenges and Formulation Strategies Associated with Oral Biologic Drug Delivery Systems
Vu Truong-Le*, Phillip Lovalenti, and Ahmad M. Abdul-Fattah
Number of pages:
48
AC
CE P
TE
D
Author(s):
*Corresponding author: [email protected], phone: (408)385-1742, fax: (408)9603822
ACCEPTED MANUSCRIPT Table of contents
3.
T
CE P AC
6. 7.
TE
D
5.
MA
NU
4.
SC R
IP
1. 2.
Table of contents .................................................................................................................. 2 List of tables ......................................................................................................................... 3 List of figures........................................................................................................................ 3 Introduction and challenges with oral delivery route .......................................................... 4 Methods employed in preparation of biopharmaceutical oral solid dosage forms .............. 8 2.1 Preparation methods for microspheres and nanoparticles ........................................... 8 2.2 Preparation methods for oral thin films ..................................................................... 18 Stresses encountered during manufacture of oral solid dosage forms and ways to improve in-process stability .............................................................................................. 20 Formulation and stability aspects in oral solid dosage forms for biomacromolecules ...... 26 4.1. Polymers .................................................................................................................... 27 4.1.1. Hydrophobic polymers .................................................................................... 28 4.1.2. Hydrophilic polymers ..................................................................................... 29 4.2. Permeation/absorption enhancers .............................................................................. 31 4.3. Enzyme inhibitors ...................................................................................................... 32 4.4. Other formulation components .................................................................................. 33 Stability post-manufacturing ............................................................................................. 35 5.1 Storage stability ......................................................................................................... 35 5.2 Stability after administration ..................................................................................... 37 Summary and future prospects and trends ......................................................................... 38 References ......................................................................................................................... 40
2
ACCEPTED MANUSCRIPT List of tables Table 1
Routes of administration and dosage forms developed for nonparenteral administration of biopharmaceuticals .................................... 7 A summary of solvent evaporation/extraction methods used to prepare microspheres ......................................................................................... 10 A summary of other techniques used to prepare microspheres ............ 12
Table 2
IP
T
Table 3
List of figures
Sites in the digestive tract that represent opportunities for drug absorption. .............................................................................................. 6 Lab scale spray drying process in environmental enclosure under controlled conditions ............................................................................ 17 Basic steps in a lab-scale thin film manufacturing process and the microstructure of the resulting stabilized vaccine film formulation..... 20 Normalized HRP activity versus time in microparticle formulations of Ac-DEX prepared by three different methods at 5°C (a), 25°C (b) and 45°C (c) [79] ………………………………………............................. 23 Alternative approaches to fabrication of OTF’s for mAb delivery ...... 36
SC R
Figure 1 Figure 2
NU
Figure 3
MA
Figure 4
AC
CE P
TE
D
Figure 5
3
ACCEPTED MANUSCRIPT 1.
Introduction and challenges with oral delivery route
Due to the rapid progress in biotechnology, the industry has produced a large number of therapeutic peptides and proteins on commercial scale. Over 130 biotechnologically derived drug products are approved by the US Food and Drug Administration (FDA) [1]. Most
T
biopharmaceutical drug products (proteins, peptides, and vaccines) are administered
Poor intestinal absorption of these drugs, for
SC R
administration, including the oral route.
IP
parenterally because of their poor bioavailability from different alternate routes of
example, is due to their susceptibility to acid and enzymatic hydrolysis, unfavorable physicochemical properties including size, charge, and hydrophilicity. In addition to hydrolysis in the stomach and gastrointestinal (GI) tract, peptide and protein drugs targeting a local therapeutic
NU
effect in the colon [16] must also survive degradation via bacterial fermentation. Despite these challenges a number of alternate routes of administration for biologic drugs have been
MA
pursued with varying degrees of success. Some examples of these alternative routes that have advanced to human use are provided in Table 1.
Alternate delivery routes can offer
convenience/non-invasiveness, enhanced targeting to diseased tissues, controlled release rate,
TE
D
improved compliance, and can provide a game-changing commercial opportunity.
Development of an effective oral delivery system for biologics requires a detailed
CE P
understanding of the several barriers along the digestive tract (See Figure 1), as well as the mechanisms involved in their absorption across targeted tissues. It is generally believed that the challenges to oral delivery of biopharmaceuticals are significant, and substantial
AC
opportunities remain to optimize delivery approaches, formulation components and processing conditions for each peptide and protein drug.
Obviously the most convenient route for the systemic delivery of pharmaceuticals is oral; however, attempts to deliver large molecular weight proteins and peptides orally have seen limited successes. Bioavailability via this route is poor for molecules of molecular mass greater than a couple hundred Daltons. In addition, proteins are susceptible to hydrolysis and modification at gastric pH levels and can be degraded by proteolytic enzymes in the small intestine. Various approaches currently under investigation include amino acid backbone modifications [2], conjugation of bacterial and viral transcytosis peptide sequences [3,4],
4
ACCEPTED MANUSCRIPT formulation design, chemical modification, use of proteolytic inhibitors, and passive absorption enhancers; all have shown variable successes. Parenteral delivery of proteins and peptides has been the method of choice for systemic delivery mainly for avoidance of biological barriers through which it is difficult for proteins to
T
pass, and the ability to achieve pharmacologic levels of circulating protein over a relatively
IP
short period of time. In addition to parenteral administration, interest has increased in the area of local delivery of proteins through mucosal tissues of the buccal area [15], gut, sinus and
SC R
lungs by both oral and inhalation delivery systems.1 In these applications, proteins typically must be administered in formulations which protect against unwanted proteolysis and target the mucosal tissues. In recent years, there has been a rise in quick dissolving oral thin films in
NU
commercial application ranging from Listerine® breath freshener strips to analgesics, thus warrants some review coverage. There are already a number of excellent reviews on the
MA
biological barriers to oral delivery related to absorption mechanisms, molecular approaches to absorption enhancement, including comprehensive reviews of different oral delivery systems [5]. Using case studies from literature and from our own work on oral thin films, we will
D
continue this special edition’s theme on protein stability and focus on the manufacturing
TE
processes, the in-process stability and storage stability of biopharmaceuticals formulated in delivery systems designed for oral delivery to the gastro-intestinal (GI) tract. Among the
CE P
range of delivery systems, biodegradable polymer systems for protection and controlled release of proteins have been the most studied; hence, we will review these systems in greater depth. These include polymeric biodegradable microspheres or nanospheres that contain
AC
proteins or vaccines, which are designed to minimize both administration frequency and biologically effective protein dosage. Specifically, this review will include a landscape survey of the systems that have been studied, the manufacturing processes involved, stability through the manufacturing process, key pharmaceutical formulation parameters that impact stability of the encased proteins, and storage stability of the encapsulated proteins in these delivery systems.
1
Examples of products in this area include Generex Biotechnology’s insulin buccal spray Oral-lyn™ (in Phase III clinical trials) and the oral thin film products of MonoSol Rx®, which include both the marketed small molecule products Zuplenz® and Suboxone® as well as several complex-molecule-containing oral films in development, such as insulin that is in clinical trials.
5
MA
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
Figure 1. Sites in the digestive tract that represent opportunities for drug absorption. The variety of conditions (pH, chemical exposure and time) in each compartment are barriers that drug delivery systems will need to overcome. From: http://en.wikipedia.org/wiki/Digestive_tract (Copyright-free)
6
ACCEPTED MANUSCRIPT
Table 1. Routes of administration and dosage forms developed for non-parenteral administration of biopharmaceuticals [77]
IP
T
Examples of biopharmaceuticals tested (C – clinical trials, A – approved product) Calcitonin (C) Insulin (C) Exenatide (C) Octreotide (C) Rotavirus vaccine (A) Typhoid vaccine (A) Adenovirus vaccine (A) Anthrax vaccine Cholera vaccine (C) H5N1 Avian flu vaccine (C) Polio vaccine (A) Smallpox vaccine
SC R
MA
Oral
Examples of Dosage forms/Drug delivery vehicles Pills Capsules Microspheres Hydrogels Nanoparticles Enteric coated tablets Enteric coated dry emulsions Liquid dropper
NU
Administration route
Mucoadhesive patches/films Liquid spray
Insulin (C) Interferon (C) Oxytocin
Sublingual
Tablets
Desmopressin (A)
Ocular
Eye drops Injections
VEGF-targeted Fab and IgG1 mAb (A)
Gels
LHRH (luteinizing hormone releasing hormone) analogue (C)
Rectal
TE
CE P AC
Vaginal
D
Buccal
Suppositories
Cutaneous/Topical
Micro-needles Transdermal patches, creams, sprays, gels
Nasal
Aerosol sprays
Inhalable
Aerosol and dry powder sprays
Influenza virus (A) Parathyroid hormone (PTH) (C) Insulin (C) Testosterone (A) Influenza vaccine (A) Salmon calcitonin (A) Insulin (A,C) Dornase alfa (A)
7
ACCEPTED MANUSCRIPT 2.
Methods employed in preparation of biopharmaceutical oral solid dosage forms
T
Proteolysis (e.g. enzymatic, pH-driven, etc.) is a common problem throughout the gastro-
oral bioavailability of proteins and peptides.
IP
intestinal (GI) tract, especially in the stomach and upper GI tract, thus affecting subsequent Overcoming the potential proteolysis, and
SC R
thereby enhancing absorption through the GI epithelium and improving oral bioavailability, has been achieved mainly via protein and peptide encapsulation. For example, polymeric microspheres of poly(methacrylic-g-ethylene glycol) have been used to stabilize insulin in the
NU
GI tract [10]. The gels were swollen in the acidic stomach medium due to intermolecular polymer complex formation without insulin release into the gastric fluid. In the basic and neutral intestinal media, the complexes dissociated, resulting in the release of insulin.
MA
Similarly, Jani and coworkers have studied the uptake of polymeric nanoparticles in the rat GI
D
tract following oral administration as a potential drug carrier [6, 7].
The manufacturing or preparation methods for the variety of encapsulation methods for oral
TE
drug delivery systems range from simple to complex; each involves its own set of physical and chemical stresses to the active pharmaceutical ingredient (API). A better understanding of
CE P
the range of stresses to the API payload during manufacturing may allow for alternative strategies that can be considered to improve protein stability. In this section, we will cover the preparation methods for the most commonly tested solid oral delivery systems for
AC
biopharmaceuticals: microspheres/microparticles, nanoparticles, and oral thin films. Tablets and powder filled capsules are established technologies that are diverse, extensively reviewed elsewhere, and will not be included in our review.
2.1
Preparation methods for microspheres and nanoparticles
The range of the degradable polymeric protein and peptide delivery systems designed for oral delivery that have been reported over recent years is very broad. Recent interest in particulate systems for oral delivery of proteins have focused on targeting [8, 11] and addressing the mucous layer barrier and mucoadhesion in the GI [12, 13, 40]. Requirements for a successful encapsulation process using biodegradable polymers include maintaining stability and biological activity of the incorporated drugs during and after encapsulation, high yield and
8
ACCEPTED MANUSCRIPT encapsulation efficiency, reproducible release profile of the drug without the significant initial burst and desirable product attributes such as appropriate particle size of micro/nanoparticles as well as free-flowing particles [113]. This section will provide an overview of the various preparation methods that have been developed to fabricate nano- and micro-particulate
T
systems that can be delivered orally as suspensions, pressed into oral tablets, or filled into
IP
capsules for oral ingestion. Many methods are well-developed for preparation of hydrophilic
SC R
polymer microspheres and nanoparticles for protein delivery. The majority include solvent evaporation/extraction methods (emulsion formation with organic solvents using water soluble or insoluble polymers), coacervation/precipitation methods, spray drying, ionic gelation methods and the template assembly method. An excellent review of many of these
NU
methods is provided by Yeo and coworkers [26], much of which is summarized in Tables 2 and 3. Some of the most common are further highlighted below. The interested reader may
MA
find greater details on a particular manufacturing method in the references listed in Tables 2
AC
CE P
TE
D
and 3.
9
ACCEPTED MANUSCRIPT
Cryogenic Solvent Extraction Microspheres
Organic solvent (e.g. dichloromethane) with dissolved polymer and drug PLGA
AC
CE P
TE D
MA N
US
CR
IP
T
Table 2. A summary of solvent evaporation/extraction methods used to prepare microspheres Method Solvent Evaporation/Solvent Extraction Spray Desolvation Single Emulsion Single Emulsion Double Emulsion Solvent Evaporation Product Solvent evaporation Microspheres and Microspheres Microspheres preferred for porous nanospheres prepared by solvent fast release prepared by solvent extraction and microspheres, evaporation evaporation solvent extraction preferred for fast release microspheres Dispersed phase Polymer solution in Organic solvent Aqueous phase Aqueous phase organic solvent (e.g. with dissolved or Dichloromethane, dispersed drug ethyl acetate) with dispersed or dissolved drug Example of Polylactic acid Polylactic acid PLGA Polyvinyl alcohol Encapsulation (PLA) and (PLA) and (PVA), PLGA Polymer ploy(lactide-coploy(lactide-coglycolide) (PLGA) glycolide) (PLGA) Continuous phase Mineral oil or O/W emulsion Organic solvent as Aqueous solution acetone, ethanol Emulsifier Span 85 and Same Same N/A sorbitan sesquioleate aluminum tristearate for O/O emulsion. Carbopol 951, methyl cellulose, polyvinyl alcohol (PVA) for O/W 10
Ethanol N/A
ACCEPTED MANUSCRIPT
Insulin, bovine serum albumin (BSA), ovalbumin (OVA), bovine superoxide dismutase (bSOD)
26
CR
IP
T
W/O emulsion (aqueous drug solution emulsified in organic solvent with polymer) added to aqueous phase W/O/W emulsion organic solvent removal/evaporation Carbonic anhydrase, BSA, recombinant human growth hormone (hGH), recombinant human erythropoietin (rhEPO), urease and lysozyme. 17, 26
26
AC
References
CE P
TE D
Example of protein/peptide prepared
Dispersed phase added to continuous phase homogenization solvent removal by drying (e.g. freeze drying)
US
emulsions Dispersed phase added to continuous phase homogenization solvent removal centrifugation or filtration of microspheres freeze drying
MA N
Process
11
Polymer/drug solution Spray onto desolvating liquid precipitation of microspheres filter, collect and dry
Polymer /drug solution Spray onto frozen ethanol precipitation of microspheres filter, collect and dry
BSA
Tetanus toxoid, recombinant human growth hormone (hGH)
26
18, 19, 20
T
ACCEPTED MANUSCRIPT
Interfacial Polymerization
Microspheres Nanoparticles Protein in aqueous solution with diamine monomer Nylon, polyester
AC
CE P
TE D
MA N
US
CR
IP
Table 3. A summary of other techniques used to prepare microspheres Method Emulsion crossCoacervation Non solvent linking with addition Precipitation Complex water soluble coacervation polymers Product Microspheres Microspheres Microspheres Microspheres, Nanospheres Dispersed phase Protein dissolved Protein in Protein in Protein in or dispersed in aqueous aqueous aqueous solution aqueous polymer solution solution with solution one polymer Example of Chitosan, Gelatin Blend of PLA, PLGA, Encapsulation collagen, gelatin Polycation (e.g. gelatin, agarose Polymer Gelatin) + and polyvinyl Polyanion (e.g. alcohol (PVA): Gum Arabic, single polymer or Chondroitin-6- blends sulfate (CS6) Continuous phase Organic solvent Polymer Aqueous First nonor oil solution solution of solvents for the another polymers: polymer Silicone oil, vegetable oil, light liquid paraffin oils. Second nonsolvent : heptane, hexane, petroleum ether Process Aqueous phase Protein Aqueous Slow addition of 12
Biocompatible oil (e.g. mono/diglycerides) containing dichloride monomer and surfactant (e.g. Tween 80)
Aqueous phase
ACCEPTED MANUSCRIPT
with monomer added/emulsified into continuous phase (O) with second monomer to form W/O emulsion Add acid chloride polymerization at interface to form microspheres.
BSA, Insulin, granulocytemacrophage colonystimulating factor (GM-CSF) 26-32
Insulin, BSA
IP
T
first non-solvent to aqueous solution microparticles form add second nonsolvent to harden the microspheres
US
CR
solution containing protein added to second polymer solution polymerpolymer interaction, complex formation phase separation and microsphere precipitation Add glutaraldehyde to harden microcapsules Chondroitin-6sulfate (CS6)
MA N
solution added to polymer solution precipitation of microspheres. Emulsion stabilizers (e.g. sodium sulfate) may be added
References
Insulin, hepatitis B surface antigen (HBsAg)
21, 22
AC
Example of protein/peptide prepared
CE P
TE D
added/emulsified into continuous phase (O) to form W/O emulsion homogenization Add cross linker emulsion solidified into microspheres.
23, 24, 25
34
13
44, 45
ACCEPTED MANUSCRIPT
Solvent evaporation/extraction The solvent evaporation method is widely used to prepare microspheres for delivery of poorly
T
water soluble and hydrophobic small molecules. To prepare microspheres containing peptides
IP
and proteins oil/water (O/W), oil/oil (O/O) and water/oil/water (W/O/W) emulsification
SC R
methods are used. The biodegradable polymers polylactic acid (PLA) and poly(lactide-coglycolide) (PLGA) are most frequently the polymers of choice for encapsulation. The release kinetics of encapsulated drug is governed by the chain length and degree of crosslinking of the PLGA used. PLGA high in glycolide content undergoes more rapid degradation, thus loaded
NU
drugs are released faster, and vice versa. The solvent evaporation approach can be further
MA
divided into single emulsion and double emulsion methods.
Single emulsion method (O/O or O/W)
In this method, the active ingredient is dissolved/suspended in the organic (dispersed) phase
D
containing the polymer. The drug solution/suspension is added to the continuous phase
TE
containing one or a mixture of emulsifiers. The continuous phase can be either mineral oil for an O/O emulsion, or water for an O/W emulsion. Spans and sorbitan sesquioleate aluminum
CE P
tristearate are preferred emulsifiers for O/O emulsions, while Carbopol 951, methyl cellulose, polyvinyl alcohol (PVA) are the emulsifiers of choice for O/W emulsions. After adding the dispersed phased to the continuous phase, the organic solvent is removed by solvent The resulting
AC
evaporation or solvent extraction, after which the emulsion hardens.
microspheres are collected by centrifugation or filtration, and then freeze-dried. This method has been used to encapsulate Insulin (nanospheres), bovine serum albumin (BSA), ovalbumin (OVA) and bovine superoxide dismutase (bSOD), resulting in high encapsulation efficiency with good retention of activity.
Double emulsion method (W/O/W) In a double emulsion method, a water-in-oil emulsion (W/O) is first created from a drugcontaining aqueous phase (W) and an organic phase (O) with dissolved polymer. This emulsion is then added to a second aqueous phase containing emulsifier, to form a W/O/W double emulsion. The microspheres are formed when the organic solvent is extracted into the external aqueous phase, precipitating the polymer. Solvent removal occurs by evaporation.
14
ACCEPTED MANUSCRIPT Carbonic anhydrase and BSA were encapsulated within PLGA microspheres [17]. The protein release from the microspheres was incomplete due to protein aggregation and adsorption within the microspheres. Recombinant human growth hormone (hGH) similarly prepared had
Incompatible polymer addition or salt addition
SC R
IP
Several additional methods exist for producing microparticles:
T
continuous delivery lasting a month following an initial burst release of 20% of its payload.
Incompatible polymer addition or salt addition is another method where two chemically incompatible polymers are dissolved in a common solvent [33]. Here, a polymer is added until
NU
the phase boundary is passed, causing phase separation of the polymer rich phase to form immiscible droplets containing the drug, and coalesce to form microcapsules. Inorganic salts
MA
can be added to aqueous solution of water-soluble polymers to cause phase separation.
Ionic/Ionotropic Gelation (polyelectrolyte complexation)
D
In recent years, the method of ionic gelation (IG) for preparation of hydrophilic polymer
TE
microspheres containing proteins has drawn much interest because of its very simple and mild manufacturing process, which is helpful to retain bioactivity of loaded proteins [39, 40]. In
CE P
this method, the reversible physical cross linking by electrostatic interactions is employed for microsphere formation. Proteins are premixed with a hydrophilic polymer solution, which is then dropped into an ionic solution under constant stirring.
Bodmeier and coworkers
AC
produced tripolyphosphate (TPP)-chitosan microspheres by dropping chitosan solution containing proteins into a TPP solution; many researchers have investigated its use in pharmaceutical applications [39]. Alginate gelation can also be used to produce microspheres [41]. In this process a protein-containing solution of sodium alginate is sprayed as droplets into a cross-linking solution of divalent ions such as Ca2+, Sr2+ or Ba2+. The very mild environment provided by this process generally performs well in preserving the native activity of loaded proteins.
15
ACCEPTED MANUSCRIPT The ability of electrolytes to crosslink in the presence of counter-ions to form hydrogels has been exploited to prepare particles. Alginate, a polysaccharide composed of 1,4-linked betaD-mannuronic acid and alpha D-guluronic acid, is one of the most widely used polyanions for microencapsulation [42]. Other polyelectrolytes tested include chitosan (IG with
T
tripolyphosphate), carboxymethylcellulose (IG with aluminum), pectin (IG with calcium),
IP
gelan gum (IG with calcium), and alginate and cellulose phosphate (IG with calcium),
SC R
polyphosphazene (IG with calcium). For example, calcium alginate hydrogels have been used for delivery of vascular endothelial growth factor (VEGF), achieving an encapsulation efficiency of 30-67% and constant release for up to 14 days [43].
NU
Spray drying
Spray drying is a well-established method commonly used in the pharmaceutical industry for
MA
producing a dry powder from a liquid phase. The resulting powder can be pressed into tablets, filled into capsules, or fabricated into other dosage presentations for oral delivery. The method is illustrated in Figure 2. It consists of drying small atomized droplets of a solution of
D
dissolved or suspended solids by rapid evaporation in a stream of hot dry gas such as air or
TE
nitrogen to form microparticles typically in the size range of 1 to 100 µm. Despite the elevated temperature of the drying gas, the droplets remain colder owing to the cooling effect
CE P
of the evaporating solvent. To date, many hydrophilic polymer microparticles loaded with proteins were successfully prepared by this method, such as chitosan, mannitol, hydroxypropyl methylcellulose and carbopol microspheres [35,36]. This technique shows
AC
some important advantages over other encapsulation techniques. The preparation process is rapid, convenient and generally straightforward to scale up. Furthermore, this process is suitable for most proteins because it does not depend on their solubility parameter. The proteins can be dissolved or dispersed as solids in aqueous or organic solvents, which allows either hydrophilic or hydrophobic polymers to be used. Plasticizers can also be added to improve the sphericity of the resulting microparticles.
As an example, PLGA microparticles containing thyrotropin releasing hormone (TRH) were produced by spray drying a miscible solution of TRH and PLGA in acetonitrile and water [37]. To counteract microparticle agglomeration, mannitol solution was sprayed simultaneously as an anti-adherent. In another example, hyaluronic acid (HA) microhydrogel
16
ACCEPTED MANUSCRIPT was successfully developed using spray drying as a novel drug carrier for a controlled release
D
MA
NU
SC R
IP
T
formulation of human erythropoietin [38].
TE
Figure 2. Lab scale spray drying process in environmental enclosure under controlled conditions (2=drying gas heater, 3=spray nozzle, 4=aerosol drying chamber, 5=centrifugal separator, 6=product collection vessel, 7=fines filter, 8=vacuum pump)
CE P
Spray Freeze drying Spray Freeze drying is another method that has been evaluated to produce protein-containing particles [46]. In this process, an atomization two-fluid nozzle is used to spray the contents
drying.
AC
directly into a liquid nitrogen bath; the resulting frozen particles are subjected to freeze
Spray chilling
Spray chilling involves dispersing drug in a liquid carrier (without solvent) above its melting temperature. The microparticles are formed by spraying the drug dispersion into a cold air stream to solidify the small droplets [47].
17
ACCEPTED MANUSCRIPT 2.2
Preparation methods for oral thin films
Quick dissolving oral thin films (OTF’s) offer a remarkable array of advantages for vaccine applications including compact size and weight (~<1/100th of size & weight of typical
T
vaccine package), making it highly transportable, and simplicity of use, enhancing patient
IP
compliance [48]. Furthermore, there is no reconstitution step, which ensures dosage accuracy, and there are well-established, cost-effective manufacturing processes (e.g. Listerine® breath
SC R
freshener strips). These thin films, which rapidly (< 1 minute) dissolve in saliva once placed on the tongue, have seen a steady rise in the number of medicinal applications in recent years, expanding from a simple breath freshener to over-the-counter medicines (pain reliever,
NU
cold/flu, allergy, teeth whitening, nutraceuticals) to prescription medicines for severe pain [49], nausea [50], allergic rhinitis [51], epilepsy [52], etc. The commonality among these thin
MA
film products is that they are all small molecule drugs, which tend to be significantly more stable than biologics; therefore they are able to withstand the thin film manufacturing process, which involves relatively high heat, pressure, and in some cases organic solvent exposure.2
D
Such manufacturing process conditions are not compatible with thermally labile vaccines and
TE
proteins. Thus, this technology barrier will have to be overcome in order to expand the potential applications of OTF’s into the biologics space. To achieve an optimal technology
CE P
solution, the authors have been successful in incorporating a pharmaceutical stabilization formulation that does not require complex pharmaceutical drying processes to achieve room temperature stability and is compatible with existing thin film manufacturing processes. This process can be as simple as mixing the vaccine with the pharmaceutical stabilizers and thin
films.
AC
film formers in a ‘one-pot’ configuration, then spreading out the mixture for drying into thin
Such a technology can be simple, cost effective, and easy to implement worldwide. For vaccines, an effective OTF formulation could potentially enhance the vaccine coverage by improving patient compliance and facilitate booster immunization campaigns, which ultimately could lead to improved effectiveness of vaccines. It could also enhance utilization of current oral vaccines, which include vaccines for typhoid, cholera, rotavirus, shigellosis, and polio. 2
Some progress in the development of OTF formulations of insulin for buccal delivery has been achieved by MonoSol Rx® as well as by research groups in academia, such as Morales, McConville and coworkers (80).
18
ACCEPTED MANUSCRIPT For example, thin film formulation and manufacturing process was developed that successfully encased a thermally labile live virus vaccine with complete preservation of activity and storage stability at high temperature (45 °C). We combined our liquid vaccine stabilization technology [53] and plasticized glass stabilization technology [54-56] to
T
demonstrate an OTF formulation that successfully encased live rotavirus vaccine with
IP
complete preservation of vaccine potency and improved stability at 45 °C from several days to
SC R
several months.
This formulation technology is compatible with conventional film forming components used in a variety of OTF products and film manufacturing processes such as solvent casting [57]
NU
and film extrusion [58]. Film casting is a process used for preparation of oral thin films (e.g. over-the-counter analgesics and Triaminic® films) and adhesive patches. The formulation
MA
comprises specific GRAS (Generally Recognized as Safe) pharmaceutical excipients, plasticizers, and protein binding ligands added to stabilize a key viral protein. Furthermore, a unique thin film polymer composition has been developed that is compatible with the
D
stabilized formulation and can be subjected to a simple convective drying process to yield a
TE
robust thin film with physical properties that are indistinguishable from a number of commercial oral thin film products. The thin film manufacturing process was developed as a
CE P
‘one-pot’ formulation blending method, whereby all components are blended in a single master mix that are cast and dried into thin films using a simple solvent casting convective air drying process. Shown in Figure 3 is an example of a lab-scale process for fabricating
AC
biologics such as a vaccine into oral thin films.
19
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
D
Stresses encountered during manufacture of oral solid dosage forms and ways to improve in-process stability
TE
3.
MA
Figure 3. Basic steps in a lab-scale thin film manufacturing process and the microstructure of the resulting stabilized vaccine film formulation
During the manufacture of oral solid dosage forms proteins can be subjected to certain
CE P
stresses that threaten their stability, such as extremes in heat, shear or pH, in addition to exposure to aggressive process steps from freezing and drying. This section will review the range of stresses involved. There are already excellent reviews elsewhere in this special focus
AC
edition and in other articles and books on the general topic of the stresses associated with freezing and drying, commonly used in pharmaceutical processes such as lyophilization and spray drying, and the stabilization strategies employed. In this section, we will discuss specific stresses that may be associated with solid oral dosage form manufacturing processes.
20
ACCEPTED MANUSCRIPT In addition to thermal stress [59], proteins are prone to denaturation and aggregation by various factors in processes like the solvent evaporation/extraction methods described above, including exposure to high shear forces during processing (particularly emulsification), exposure to the large interfacial area between the emulsified oil and aqueous phases or at the
T
air-water interface of small droplets, as well as interaction with an encapsulating polymer or
IP
organic solvent. The loss of protein activity by denaturation/aggregation can be minimized by
SC R
using a number of stabilizers such as polyols, albumins and other proteins, surfactants, poloxamers, cyclodextrins, etc, depending on the stresses involved in the manufacturing processes for various oral drug delivery systems, which are further highlighted below.
NU
Organic solvents – Unless the surface of the protein has significant hydrophobicity, proteins are generally vulnerable to denaturation and activity loss in the presence of most organic
MA
solvents used to make polymeric oral delivery systems [60,61]. In general, polar organic solvents that are water-miscible are more enzyme-deactivating than water-immiscible solvents [62]. Proteins often retain the native structure in pure water-immiscible solvents and their
D
activity can be improved by adding very small quantities of water and other excipients [63].
TE
Because water-miscible solvents have a greater likelihood to strip off tightly bound water
CE P
from the protein surface, they can bring about both loss in protein structure and activity [64].
Air/water interface - The liquid and gas phase contacting surface has long been shown to be adverse microenvironment for soluble proteins [65]. The interfacial tension associated with
AC
such phase boundaries (e.g. in the spray drying process) brings about much of the shearrelated protein stress. Proteins that adsorb more readily at air/water interfaces exhibit higher shear rheological properties and are thermodynamically less stable [66]. To solve this challenge, nonionic surfactants such as Tween-20 and Pluronic F-68, added in various fold molar excess over the protein can significantly suppress protein aggregation.
21
ACCEPTED MANUSCRIPT Ice/water interface – Protein denaturation during freezing is found be casually related to surface-induced denaturation as shown in a study using low temperature rheology on highly viscous and elastic behavior of frozen protein formulations [67]. Carpenter has found that a strong correlation exists between the tendency of a protein to freeze denature and its tendency
T
to surface denature [68]. When surface-active agents such as Tween80 and/or cryo-protectant
IP
sugars or polyols are added, strong protection against freeze-induced denaturation at the ice-
SC R
water interface during freeze-drying is observed.
Drying – Unless the formulation is optimized before applying any type of drying process (e.g. freeze drying, spray drying, etc.), the drying step itself poses a stress to the biomacromolecule
NU
during the preparation of micro- or nanocapsules as a result of water removal and lack of adequate water substitution by suitable lyoprotectants [114]. For some molecules, the
MA
application of a freezing step before drying (e.g. freeze drying, spray freeze drying) introduces a combined ice/water interface and drying stresses during the process. Reviews about stability advantages of encapsulation with and without drying are inconclusive. For example, Johnson
D
et al found no significant differences in physical stability (aggregation and fragmentation),
TE
chemical stability (deamidation and oxidation) and bioactivity between freeze dried formulations of human growth hormone with and without encapsulation (biodegradable
CE P
microspheres) [115].
Mixing/homogenization – To obtain a small-size dispersion it often requires significant
drugs.
AC
mixing- or homogenization-generated shear forces, which may degrade protein and peptide For
example,
in
emulsion
preparation
of
rhEPO,
the
W/O
emulsion
homogenization/sonication step caused an aggregates increase from 4% to 15%. Here, protein stabilizers such as BSA, hydroxypropyl-β-cyclodextrin were beneficial in decreasing the aggregates [69].
22
ACCEPTED MANUSCRIPT The effect of various processing steps on protein activity was evaluated by Göpferich and coworkers [78] for solvent evaporation methods using the double emulsion technique to produce proteins encapsulated polymeric microspheres. For example, in the encapsulation of trypsin, a 30 seconds sonication (“mixing shear” effect where eddy size is comparable or
T
smaller than protein size) to prepare the primary emulsion led to 31% reduction in activity,
IP
while vortex mixing, a 3 hour solvent evaporation and post freeze drying led to negligible
SC R
reduction. The addition of stabilizers such as BSA and glycine did not improve the sonication loss, although the loss was reduced with reduced sonication time. Thus they concluded the sonication probe itself (rather than an increase in the interfacial area) provided the major source of activity loss in preparing the microspheres, and may be the result of localized heat,
NU
shear or cavitation.
Kanthamneni et al. prepared microparticles of acetalated dextran (Ac-DEX) encapsulating
MA
horseradish peroxidase (HRP) by a milder homogenization method and by a probe sonication method [79]. Particles were stored at different temperatures and storage stability was assessed at different time points for up to 90 days by measuring HRP activity. Stability of the prepared
D
particles was compared against that of lyophilized HRP. Stability in all three cases was
TE
characterized by an initial decline in HRP activity within the first month of measurement, followed by a levelling off of activity of HRP after that consistent with square root of time
CE P
degradation kinetics in amorphous formulations. Under refrigerated conditions, HRP prepared by sonication underwent more degradation as compared to HRP prepared by homogenization and free lyophilized HRP (Figure 4a). Under more stressful storage conditions (25 and 45°C),
AC
HRP prepared by homogenization was the most stable formulation whereas HRP prepared by sonication was least stable (Figure 4b, 4c). An estimate of the rate of degradation for all formulations at different temperatures using square-root-of-time kinetics (results not shown) yielded a similar outcome. Thus it is clear that sonication-induced stress during encapsulation of HRP impacted long term storage stability of HRP. Furthermore, it appears that encapsulation of HRP via a mild preparation method (homogenization) provided better protection and storage stability of HRP against stresses such as the ice-water interface encountered during lyophilization.
Figure 4: Normalized HRP activity versus time in microparticle formulations of Ac-DEX prepared by three different methods at 5°C (a), 25°C (b) and 45°C (c).[79] Figure 4a
23
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
Figure 4b
Figure 4c
24
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
MA
Yeo and coworkers suggested that the large W/O interface associated with methods such as interfacial polymerization is likely where proteins and enzymes are inactivated [26]. Additionally, the protein may sometimes participate in polymerization reactions, and thus can
TE
D
change its biological activity.
Protein stability under manufacturing conditions can be influenced by the type of solvent used
CE P
to dissolve the polymer. For example, with methylene chloride and ethyl acetate solvents, Gopferich and coworkers studied the impact of 3 different stabilizers (Pluronic F68, PEG 4600 and sodium glutamate) on tetanus toxoid emulsion stability [19]. Here, the use of
AC
stabilizers like Pluronic F68 was shown to prevent protein adsorption to hydrophobic surfaces of the polymer used to form the microspheres.
Although the protein could be successfully encapsulated into hydrophilic polymer microspheres, there still existed a few drawbacks to emulsion cross-linking. The proteins easily aggregate in the interface between the water and oil phase during the emulsifying process, which results in their activity reduction. Moreover, the intense shearing stress brought by a high speed homogenizer or a sonicator also led to protein denaturation and aggregation. In addition, cross linking reactions can also occur between proteins and/or between proteins and polymer, leading to activity reduction of proteins.
25
ACCEPTED MANUSCRIPT 4.
Formulation and stability aspects in oral solid dosage forms for biomacromolecules
A drug product for oral delivery of a biomacromolecule may have a more complex
T
formulation compared to its parenteral counterpart. Formulations for oral protein and peptide
IP
delivery will usually contain the well-known ingredients in parenteral formulations that include the active ingredient, a suitable buffer (e.g. phosphate, citrate, histidine, succinate,
SC R
etc.), a surfactant (e.g. polysorbates, pluronics) and a stabilizer (e.g. sucrose, trehalose). Theories proposed for explaining stabilization in the solid state by the so-called “stabilizers” include the glass dynamics theory as well as the water substitution theory [72]. Other
NU
ingredients in oral dosage forms, similar to those in biopharmaceutical parenteral dosage forms, may include one or more of the following; bulking agents (e.g. mannitol, glycine), salts
MA
(e.g. sodium chloride), antioxidants (e.g. methionine, GSH), stabilizing amino acids (e.g. arginine), antimicrobial preservatives (for multi-dose parenterals e.g. benzyl alcohol) and adjuvants (for vaccine formulations e.g. Alum, Freud incomplete adjuvant, etc.). Additional
D
ingredients included in formulations for oral delivery of proteins and peptides are polymers
TE
and may also include enzyme inhibitors, permeation enhancers and plasticizers. A more complex mixture of inactive ingredients will lead to greater concerns about excipient
CE P
compatibility if not properly selected, as well as more stability concerns for the additional inactive ingredients added in the formulation. Finally, preparation methods will become more complicated as demonstrated in the previous section, and therefore more concerns will emerge around manufacturing the drug product (e.g. interactions with different equipment
AC
components, adsorption, etc.). Thus a more elaborate DoE (Design of Experiment) must be included in the formulation screening and design phase. In turn, this translates into a much elaborate and longer development time frame. This section will provide a high-level overview of the additional excipients used in protein and peptide formulations for oral delivery that are uncommon to the parenteral counterparts, and to highlight degradation issues and stability concerns during formulation and/or delivery, demonstrating with a few case studies in the process.
26
ACCEPTED MANUSCRIPT Note that owing to their potential toxicity, much research has been done to avoid use of enzyme inhibitors and/or permeation enhancers, such as chemical modifications of protein and peptide drugs at the amino acid side chains or at the carbohydrate moieties to improve their resistance to protease degradation, attaching bacterial translocation sequences to shuttle
T
the protein conjugate transcytotically through the gut epithelium, etc. [3].
These molecular
IP
modification approaches have generated much excitement and are further reviewed by Mahato
SC R
and coworkers [14]. However, these technologies will not be examined in detail in the article. More focus will be on discussing permeation enhancement and enzyme inhibition from a
4.1
NU
formulation excipient/additive perspective.
Polymers
MA
The use of polymers in the manufacturing process of a solid oral dosage form and the many previously tested options available were discussed above. The context there was more focused on achieving certain drug delivery aspects and creating the desired physical macroscopic form However, polymers used in a formulation can also have a
D
of the final drug product.
TE
significant impact on stability. In many cases, polymers can be added to improve both process and storage stability through interactions with the active and in altering the The addition of polymers can inhibit
CE P
microstructure of the solids in the drug product.
crystallization during drying processes to produce the solid dosage form, which can also affect stability. In other cases, the selection of the polymer in the final formulation for reasons of manufacturability and drug delivery can have a negative impact on stability requiring
AC
adjustments to the formulation. In the case of PLGA microparticles, for example, generation of deleterious degradation products after administration can alter local pH and require buffering compounds to protect the active during delivery.
27
ACCEPTED MANUSCRIPT In a broad sense, the polymers used can be divided into either hydrophilic or hydrophobic classifications. Other classifications have addressed biodegradable attributes, as well as natural versus synthetic [113]. The following section describes different polymers that have been used in vitro or in animal studies that suggest that the polymer could be used for oral
T
peptide and/or protein delivery. Polymers have been utilized in much of the research for oral
IP
delivery to produce microparticles/nanoparticles. The latter particles can deliver small and
SC R
biomacromolecules in a targeted or localized way, can protect biomacromolecules from the harsh pH environment and enzyme effects at various regions of the GIT, deliver molecules in a sustained manner thus avoiding repeated dose administration, and are considerably the easiest presentation to be taken up by the lining of the GIT [81]. In these studies, focus has
NU
been on fabrication from different polymers, encapsulation efficiency and release profiles. Microparticulate systems include microcapsules, nanocapsules, or hydrogel microparticles.
MA
Overall, little work has been published about solid state stability of the polymers and of the biomacromolecules they carry.
group
comprises
poly(esters),
poly(cyanoacrylate),
poly(ortho
esters)
and
TE
This
D
4.1.1. Hydrophobic polymers
poly(phosphazenes). Examples of poly(esters) include Poly(lactic acid-co-glycolic acid)
CE P
(PLGA), poly(ɛ-caprolactone), poly(β-hydroxybutyric acid) and poly(β-hydroxyvaleric acid). Poly(esters) are excellent candidates for delivery of biomacromolecules in the form of microparticles/nanoparticles based on the ability of their particles to protect the encapsulated active ingredient from degradation in the GIT. Many are approved by the FDA and are
AC
considered Generally Recognized as Safe (GRAS). However, more research is needed to improve particle uptake by the GI lymphoid tissue. Poly(esters) can be used by themselves or in combination with other polymers to prepare microparticles/nanoparticles. Each class has its unique physico-chemical properties that in turn affect its rate of biodegradation to release their contents, rate of uptake at delivery site, rate of chemical degradation (i.e., solid state stability), etc. [82]. PLGAs comprise the broadest poly(ester) type polymers used in research in oral protein and peptide delivery due to desirable properties such as their GRAS status, varying physicochemical properties (e.g mechanical), good sustained release properties, biodegradability, biocompatibility and non-toxic properties. PLGAs are referred to by the percent lactic acid to glycolic acid content present. For example, a PLGA with 25% D-lactic acid, 25% L-lactic
28
ACCEPTED MANUSCRIPT acid and 50% glycolic acid is described as a 50:50 poly(D,L-lactide-co-glycolide). As % poly(glycolic acid) increases, solubility decreases and physico-chemical properties of the PLGA change. However, they have a low encapsulation efficiency and undesirable effect on pH-susceptible encapsulated bio-macromolecules upon storage under certain conditions [83,
T
82]. Recent studies have demonstrated that PLGA copolymers have an adverse effect on the
IP
stability and bioactivity of biomacromolecules due to the hydrophobicity of the polymers and
SC R
the presence of acidic degradation products. [113]. As the polymer degrades, an acidic environment is produced surrounding and within the polymer matrix [84]. As the polymer backbone undergoes hydrolysis, more carboxylic acid groups are produced in the matrix and pH of the surrounding fluid decreases resulting in biodegradation and release of the active
NU
ingredient in vivo. During this degradation process, instability of low pH-susceptible payloads may also result during delivery or storage. High levels of moisture accelerate this undesirable
MA
hydrolytic instability during storage [81]. Water-in-oil-in-water emulsion method is the predominant method used for encapsulation within microparticles. However, loading efficiency of poly(ester) microparticles remain quite low. Sufficient loadings are possible for
D
low dose drugs, but large amounts of drug are needed to get the high loadings necessary.
microparticles [82].
TE
Thus, more research is needed to improve loading efficiency of large doses of drugs within
CE P
Poly(cyanoacrylates) (PCAs) are more popular in preparing microparticles than poly(esters) due to a mild polymerization procedure leading to less activity loss during incorporation of peptides and proteins, in addition to a higher encapsulation efficiency. PCAs are also GRAS
AC
and their rate of degradation is much faster than PLGAs which does not necessitate overloading the subject with excess polymer after initial treatment [85]. PCAs can be easily formed into spheres [86] or capsules [87] to deliver biomacromolecules. Some examples of PCAs include poly(isobutyl cyanoacrylate) (PBCA) and poly(isohexyl cyanoacrylate) (PIHCA) [88].
29
ACCEPTED MANUSCRIPT Poly(phosphazenes) are biodegradable polymers that show potential for oral peptide and protein delivery [89, 90, 91] with varying degradation rates [92]. Degradation of poly(phosphazenes) is accompanied by production of ethyl alcohol, phosphate, ammonium salts and pendant groups [93]. Different pendant groups have been used as substituents, which
T
in turn controlled polymer properties, such as amino acid [94], ethylamino [95], imidazolyl,
IP
oligopeptide, amino acid exters, and depsipeptide groups. When the pendant group is an
SC R
amino acid, all degradation products are natural products normally found in the body. Poly(ortho esters) are a group of hydrophobic polymers with limited use in oral peptide and protein delivery. There have been trials to load rods made from the polymers with
NU
recombinant human growth hormone and bovine serum albumin using polymer-protein mixture extrusion at a temperature range of 50-70 °C. However, the large particle size of the
MA
rods has limited the polymer use in oral peptide and protein delivery [96]. 4.1.2. Hydrophilic polymers
This group comprises poly(alkyl methacrylates), poly(methacrylates), poly(acrylates),
D
alginates, chitosan, polyphosphazene hydrogels and poly(ethylene glycols).
TE
Poly(alkyl methacrylates) include Eudragits (e.g. S100, L100), properties of which vary depending on their degree of modifications. For example, dissolution rate of Eudragits and the
CE P
pH at which they dissolve will depend on modification of carboxylic acid groups of methacrylic acid. These properties make Eudragits versatile polymers to prepare microparticles to encapsulate and deliver proteins in the GIT [97]. Entrapment efficiency is
AC
reported to be as high as 78%. Eudragit microparticles have low solubility in the acidic stomach environment and therefore have been shown to be efficient in delivering sensitive proteins such as insulin in the intestines in oral force fed rats.
30
ACCEPTED MANUSCRIPT Poly(methacrylates) and poly(acrylates) are used to form hydrogel particles that swell isotropically at different rates upon exposure to fluids, thus delivering entrapped proteins and peptides in a swelling rate- and mesh size-dependent manner. Poly(methacrylates) and poly(acrylates) also possess specific and non-specific bioadhesive properties [98, 99] to the GI
T
walls. They have been of interest for colon delivery applications since colon specific enzymes,
IP
e.g. azoreductase, act on polymer cross-linkers (such as azo linkers) resulting in degradation
SC R
of the polymer in the colon and thereby releasing the entrapped biomacromolecules, though addition of penetration enhancers is also necessary due to low absorption potential from the colon [100, 101, 102]. Not much is reported in terms of protein/peptide stability in these
NU
types of matrices.
Polymers derived from naturally occurring resources and considered GRAS include alginate and chitosan. Alginate is derived from seaweed whereas chitosan is derived from chitin.
MA
Alginate is a water-soluble physically cross-linked polymer by means of monovalent or divalent cations. Chitosan, on the other hand, is not readily water soluble but can be solubilized. Alginate microparticles with high encapsulation efficiency for biomacromolecules
D
can be formed under mild conditions, and particle size can be easily varied during
TE
formulation. Alginate and chitosan have been used in combination to form complexes to strengthen the resultant microparticles [103]. Alginate microparticles have been produced
CE P
using atomization, emulsification and coacervation [104, 105, 106]. Chitosan microparticles have been produced by emulsion and phase separation methods. Alginate polymers can be modified to impart muco-adhesive abilities and to increase encapsulation efficiency. Chitosan
AC
by itself is both bioadhesive and has chelating properties. Chitosan is reported to be conjugated to antipain, chymostatin, elastanil, ethylene diamine triacetate (EDTA) and others [107].
Polyphosphazene hydrogels have been used to incorporate biomacromolecules in hydrogel microparticles from which release is by diffusion. A coating can be applied to the particles to prevent or control the initial release of encapsulated molecules, as was demonstrated using bovine serum albumin [93]. 4.2. Permeation/absorption enhancers Owing to the large molecular weight of proteins, transcellular or paracellular protein and peptide transport is difficult to achieve. Unless the action is intended to be localized, permeation enhancers are often included in drug carriers to facilitate systemic delivery of
31
ACCEPTED MANUSCRIPT orally administered biopharmaceuticals. . Permeation enhancers can include surfactants, bile salts, Ca2+ chelating agents, fatty acids, medium chain glycerides, acyl carnitine, alkanoyl cholines, chitosans, and phospholipids [9]. The postulated effects of these absorption enhancers include modification of tight junction
T
permeability (chelating calcium ions can disrupt the junction proteins), enhancement in
IP
membrane fluidity, and reduction in mucus viscosity. The use of Ca2+ chelating agents as
SC R
permeation enhancers is exemplified in the case of oral delivery of human calcitonin [14]. Here, several excipients were evaluated with some success, including sodium myristate, sodium taurodeoxycholate, and sodium deoxycholate.
Other low molecular weight
compounds termed “delivery agents” or “carriers” are used that interact weakly and reversibly
NU
with the co-administered biomacromolecules (e.g. recombinant human growth hormone, insulin, heparin, interferon-alpha, and salmon calcitonin).
The carriers are believed to
MA
temporarily expose the hydrophobic side chains (i.e. cause temporary partial protein unfolding), leading to a transient increase in log P values with subsequent transcellular penetration
[108].
Examples
of
these
carriers
include
N-[8-(2-
D
hydroxybenzoyl)aminocaprylate] (SNAC) (Eligen®-based Technology) and N-acylated,
TE
alpha-amino acids [14, 116]. In some cases, such permeation enhancement techniques have
CE P
improved protein stability in oral hard gelatin capsule formulations of insulin [117, 118].
4.3. Enzyme inhibitors
Enzyme inhibitors are included in oral biologic drugs to protect against proteolytic
AC
inactivation in the GI tract. Enzyme inhibitors such as aminopeptidase inhibitors (e.g. puromycin, aprotinin, bestatin, soybean trypsin inhibitor, and amastatin), sodium glycholate, camostat mesilate, 4-(4-isopropylpiperadinocarbonyl)phenyl 1,2,3,4-tetrahydro-1-naphthoate methanesulphonate ( FK-448), camostatmesilate, , and ovalbumin have all shown to improve the stability of orally administered insulin in either man and/or dogs [73,74, 116]. Enzyme inhibitors act on different moieties in the biomacromolecule. For example, proteases act on protease-vulnerable sequences of biologic agents [74]. Toxicity challenges may arise associated with the use of some enzyme inhibitors (especially protease inhibitors), particularly in chronic drug therapies, where repeated administration of the inhibitor could lead to alteration in metabolic pattern and food digestion changes.
32
ACCEPTED MANUSCRIPT In some cases, penetration enhancers can also act as enzyme inhibitors such as sodium glycocholate, which protects insulin from proteolysis [9]. Formulation components that modify local pH have been known to inhibit enzymatic activity. Some polymers inhibit proteolytic enzymes or can be used to augment the activity of
T
proteolytic inhibitors. For example, polyacrylate and polymethacrylate act as inhibitors of
IP
trypsin, chymotrypsin, carboxypepsidase A and elastase. Carboxymethylcellulose inhibits
SC R
elastase and pepsin. Chitosan inhibits trypsin, carboxypepsidase A and aminopepsidase N. The use of small molecule protease inhibitors with peptides and proteins can cause systemic side effects [109, 110, 111]. Protease-inhibiting polymers and polymer-inhibitor conjugates
NU
are now widely investigated for their ability to protect biomacromolecules from proteolysis. These molecules are effective in the immediate vicinity of the delivery device. ChitosanEDTA shows an inhibitory effect towards Ca2+-dependent proteases [112]. When
MA
chitosan/EDTA/BBI conjugate was included in a formulation with insulin, more than half of the insulin activity was maintained when administered orally versus <10% of insulin activity
D
without the conjugate.
This approach has not been successful with all polymers. Poly(ethylene glycol)s (PEGs) are
TE
widely used as “conjugating” polymers to different proteins. As a result of steric hindrance
CE P
resulting from conjugation, proteolysis is minimized. One may think that chances for absorption increase. However, the high molecular weight complex will have difficulty in crossing the GI wall. Additionally, degradation of the conjugated polymer is still possible by alternate proteolytic mechanisms resulting in exposure of the protein/peptide to the acidic
AC
environment of the GIT. Thus use of PEGs is one of the least successfully investigated strategies for oral delivery of biomacromolecules.
4.4. Other formulation components Enteric coating to prevent breakdown of the dosage presentation in the acidic compartment of the stomach has been applied with good success for a variety of tablet and capsule based oral delivery systems [75]. Common enteric coatings that have been used include methyl acrylatemethacrylic acid copolymers, sodium alginate, cellulose acetate succinate, polyvinyl acetate phthalate (PVAP), hydroxy propyl methyl cellulose phthalate, Shellac, cellulose acetate trimellitate, etc. Drugs that can upset or irritate the stomach, such as aspirin, are often coated with some of the excipients above to permit dissolution only in the small intestine.
33
ACCEPTED MANUSCRIPT Plasticizers (e.g. glycerol, sorbitol, DMSO, etc.) are another group of excipients that can be added to special oral dosage forms. For example, they have been included as formulation components in oral thin films to improve tensile strength, improve dissolution time, or change water vapor permeability [70]. Additionally, there is increasing evidence of their role in
T
protein stabilization. Mechanisms proposed suggest stabilization by plasticization of the
IP
amorphous glass to dampen molecular motions (also called ‘fast dynamics’) during thermal
AC
CE P
TE
D
MA
NU
SC R
exposure [71, 72].
34
ACCEPTED MANUSCRIPT 5. 5.1
Stability post-manufacturing Storage stability
Following manufacture, the drug delivery systems is typically stored for some time period prior to administration. While the storage stability of proteins in liquid or lyophilized
T
formulations have been extensively described in the literature, significantly less information is
IP
available on the storage stability of proteins within biodegradable drug delivery systems,
SC R
especially oral delivery systems.
In the authors’ own development of oral thin film (OTF) delivery systems, the storage stability of the drug product was often sensitive to the selection of water-soluble polymer, in
NU
addition to the nature of the protein dispersion in the OTF. For example, in the production of OTF’s containing an IgG monoclonal antibody (mAb), two approaches were taken to disperse
MA
the protein in polymer: Approach 1 involved first spray-drying the mAb (formulated in a sugar matrix of sucrose or trehalose), followed by dispersion of the resulting powder in an organic solution of polymer, before casting and drying the film; Approach 2 was performed
D
by simply mixing formulated aqueous solutions of mAb and polymer, followed by film
TE
casting and drying (See Figure 5). It was found that although the spray-dried IgG mAb itself was stable for over 21 weeks at 37ºC in both Trehalose and Sucrose/Arginine formulations,
CE P
the storage stability of the antibody in the OTF was still dependent on the interaction of the powder with the polymer in the film. A PVA(polyvinyl alcohol)-based film using Approach 2 provided superior storage stability with regard to maintaining purity and antigen recognition
AC
when compared to either an HPC (hydroxypropyl cellulose)-based film using Approach 1 or PVP (polyvinylpyrrolidone)-based film using Approach 2, indicating the existence of stability-impacting polymer-protein interactions. For the HPC-based film, the spray-dried mAb was formulated at 9.8% in sucrose/arginine and was suspended in a polymer solution of isopropanol prior to casting and drying; earlier studies demonstrated the stability of this spraydried formulation in isopropanol. Here, antigen recognition was determined using an antigen binding ELISA assay, a stability indicating assay which determined the functional binding of the mAb (anti-HIV GP60 IgG1) to the target (HIV GP60 protein).
35
SC R
IP
T
ACCEPTED MANUSCRIPT
NU
Figure 5. Alternative approaches to fabrication of OTF’s for mAb delivery. Approach 1 produces a film as a two-phase dispersion; Approach 2 produces a single phase clear film.
MA
Using the aqueous ‘one-pot’ process method described in Section 2.2 (i.e. Approach 2) and screening unique combinations of pharmaceutical stabilizers and plasticizers, the stability of rotavirus vaccine in OTF formulations was substantially improved. This screening process
D
resulted in a lead formulation that was able stabilize three different rotavirus vaccine
TE
serotypes (G1, G2 and G3) at 45 °C for over eight weeks. Further evaluation of formulation and process variables identified plasticizer level and residual moisture content as critical
CE P
factors for controlling vaccine stability in OTF formulations. By controlling these parameters in particular, the low frequency molecular dynamics within the film can be reduced, and thus
AC
decrease the rate of damage to the virus.
36
ACCEPTED MANUSCRIPT 5.2
Stability after administration
Many factors associated with the microenvironment of the polymer encasing the proteins can influence post-administration stability, including monomer reactive sidechains, concentration, pH, viscosity, surface tension, etc. It is useful to understand both the microenvironment of the
T
polymer matrix that the protein is exposed to and the release or erosion mechanism in order to
IP
effectively design the polymer system. Release from non-biodegradable polymer systems is
SC R
typically a diffusion controlled mechanism. However, release from biodegradable polymers is strongly dependent on polymer erosion, which is a more complex process that is typically determined by the specific polymer, its degradation chemistry (type of bonds or sidechains), and environmental parameters (diffusivity of water, other reactants, pH of medium, Gopferich
and
coworkers,
using
NU
crystallinity).
a
system
of
poly(1,3-bis-(p-
carboxyphenoxy)propane) – sebacic acid as an example, showed that the pH in the vicinity of
MA
the polymer surface is determined by the pH of the released mononers, rather than by the pH of the buffer medium. Thus pH sensitive drugs would need to be better protected in such systems after administration when the encapsulating polymer undergoes degradation.
D
Upon oral administration, hydration of the protein trapped in the polymeric system can be
TE
initiated over prolonged periods of time. In such an environment, the proteins are becoming more susceptible to denaturation and aggregation. This is especially a challenge in polymeric
CE P
systems where the monomer breakdown products or byproducts may be causing protein denaturing due to acidic sidechains or reactive groups such as amines, carboxyls, etc. Unwanted covalent reactions between the polymer scaffold and the proteins can lead to
AC
degradation, irreversible protein adsorption to the polymer matrix, aggregation, and denaturation. Case studies of protein adsorption to polymeric implants have been documented [76]. In one example, human serum albumin was observed to undergo a multilayer adsorption to PLA nanospheres, leading to irreversible adsorption to the polymer. On the other hand, Gopferich and coworkers showed that trypsin and heparinase encapsulation in poly(anhydride) microspheres could stabilize protein activity for 1-2 days at biologically relevant conditions: phosphate buffer at pH 7.4 and 37ºC, which was a significant improvement over the free protein under those same conditions [78].
37
ACCEPTED MANUSCRIPT 6.
Summary and future prospects and trends
In spite of the major hurdles in oral delivery of proteins and peptides (in both liquid and solid dosage forms), oral delivery remains the major route of interest (and the holy grail of macromolecule delivery) in developing biologicals after parenterals. Because much of these
T
preparations have not established potential oral bioavailability, work has been halted after
IP
animal testing. Some work has shown promising results with a few molecules, most notably
SC R
with insulin. However, there are many obstacles in oral delivery of relatively smaller biological molecules such as insulin and even greater challenges remain in developing more complex and larger molecules. For future commercial viability of biomacromolecules that have shown clinical potential, one needs to consider that formulation and process of these
NU
preparations are more complex than their traditional parenteral counterparts. Thus, there are many conditions that need to be satisfied beyond oral bioavailability and toxicity. For
MA
example, stability aspects (in process, storage stability and stability during handling and administration), formulation and process development strategies and timelines, scalability, manufacturing costs, etc. One also needs to consider developing new biomaterials that can
D
effectively protect and deliver protein and peptide drugs. Finally, one needs to consider the
TE
upcoming and future trend of personalized delivery systems capable to target site-specific
CE P
receptors that will significantly impact drug administration.
We have reviewed a number of factors impacting the successful development of an oral delivery system for biologic drugs: preparation methods and the stresses involved as well as
AC
stability considerations and formulation strategies. The manufacturing process and the associated stresses to the API are reasonably well known and are points to consider in developing stabilization strategies. Considerations should be given in formulation selection to balance between functionality, interactions between excipients, interaction of excipients with biomolecule (active ingredient), as well as stability during manufacture, storage and administration.
38
ACCEPTED MANUSCRIPT While the biological barriers along the GI tracts remain a formidable challenge to successful oral delivery of proteins and peptides, the fact that oral delivery formats are more appealing in terms of convenience and patient preference will continue to drive further research and development. Although there may remain several significant challenges associated with
T
maintaining biostability during manufacturing, storage, and transit across the GI tract, recent
IP
advances in materials, manufacturing methods and formulations are beginning to improve the
AC
CE P
TE
D
MA
NU
SC R
prospects of successful oral delivery systems for peptides and proteins.
39
ACCEPTED MANUSCRIPT References
T
1. U.S. Food and Drug Administration. “2012 Biological License Application Approvals.” 9 January 2013. Available at www.fda.gov/ BiologicsBloodVaccines/DevelopmentApprovalProcess/BiologicalApprovalsbyYear /ucm289008.htm
SC R
IP
2. Miller S, Reyna J, Ng S, Zuckermann R, Kerr J, Moos W. (2004) Comparison of the proteolytic susceptibilities of homologous l-amino acid, d-amino acid, and Nsubstituted glycine peptide and peptoid oligomers. Drug Dev. Res. 35(1):20–32. 3. Mrsny, R. J., Daugherty, A. L., McKee, M. L. and Fitzgerald, D. J. (2002) Bacterial toxins as tools for mucosal vaccination. Drug Discovery Today, 7 (4), pp. 247-258.
NU
4. Taslim A. Al-Hilal, Farzana Alam, Youngro Byun. (2013) Oral drug delivery systems using chemical conjugates or physical complexes. Advanced Drug Delivery Reviews 65: 845-64.
MA
5. M. Boegh, C. Foged, A. Müllertz, H. Mørck Nielsen (2013) Mucosal drug delivery: barriers, in vitro models and formulation strategies, J. Drug Del. Sci. Tech., 23 (4) 383-391.
D
6. Jani, P, Halbert, G. W.,Langridge, J. and Florence, A. T. (1989) The Uptake and Translocation of Latex Nanospheres and Microspheres after Oral Administration to Rats. Journal of Pharmacy and Pharmacology, 41:809-812.
TE
7. Jani, P, Halbert, G. W.,Langridge, J. and Florence, A. T. (1990) Nanoparticle Uptake by the rat gastrointestinal mucosa: Quantification and particle size dependency. Journal of Pharmacy and Pharmacology, 42:821-826.
CE P
8. Des Rieux A, Pourcelle V, Cani PD, Marchand-Brynaert J, Preat V. (2013) Targeted nanoparticles with novel non-peptidic ligands for oral delivery, Adv Drug Deliv Rev 65 (6) 833-44. 9. P. Jani, P. Manseta, S. Patel (2012). Pharmaceutical Approaches Related To Systemic Delivery Of Protein And Peptide Drugs: An Overview. Inter. J. Pharm. Sci. Rev. and Res., 12(1) Article-007.
AC
7.
10. Lowman AM, Morishita M, Kajita M, Nagai T, Peppas NA. (1999) Oral delivery of insulin using pH-responsive complexation gels. J Pharm Sci. 88:933–937. 11. Yun Y, Cho YW, Park K. (2013) Nanoparticles for oral delivery: targeted nanoparticles with peptidic ligands for oral protein delivery. Adv Drug Deliv Rev 65 (6) 822-32. 12. Ensign LM, Cone R, Hanes J. (2012) Oral drug delivery with polymeric nanoparticles: the gastrointestinal mucus barriers, Adv Drug Deliv Rev. 64 (6) 557-570. 13. Sosnik A, das Neves J, Sarmento B. (2014) Mucoadhesive polymers in the design of nano-drug delivery systems for administration by non-parenteral routes: A review. Prog Polym Sci, 39 (12) 2030-75. 14. Mahato RI, Narang AS, Thoma L, Miller DD. (2003) Emerging trends in oral delivery of peptide and protein drugs. Crit Rev Ther Drug Carrier Syst. 20:153–214.
40
ACCEPTED MANUSCRIPT 15. F. Veuillez, Y.N. Kalia, Y. Jacques, J. Deshusses, P. Buri, (2001). Factors and strategies for improving buccal absorption of peptides. Eur. J. Pharmaceutics and Biopharmaceutics. 51(2):93-109. 16. Chourasia MK, Jain SK. (2003) Pharmaceutical approaches to colon targeted drug delivery systems. J Pharm Sci. 6 (1) 33-66.
IP
T
17. Crotts G, Park TG. (1998) Protein delivery from poly(lactic-co-glycolic acid) biodegradable microspheres: release kinetics and stability issues. J Microencapsul. 15(6):699-713.
SC R
18. Johnson, O. L., W. Jaworowicz, J. L. Cleland, L. Bailey, M. Charnis, E. Duenas, C. Wu, D. Shepard, S. Magil, T. Last, A. J. S. Jones, and S. D. Putney (1997) The stabilization and encapsulation of human growth hormone into biodegradable microspheres. Pharm. Res. 14: 730-735.
NU
19. A. Gopferich, M.J. Alonso, and R. Langer. (1994) Development and characterization of microencapsulated microspheres. Pharm. Res., 11:1568–1574.
MA
20. Kissel T. (2002) Tetanus toxoid microspheres consisting of biodegradable poly(lactide-co-glycolide)- and ABA-triblock-copolymers: immune response in mice. Int J Pharm 234: 75–90. 21. Jose S, Fangueiro JF, Smitha J, Cinu TA, Chacko AJ, Premaletha K, Souto EB. (2013) Predictive modeling of insulin release profile from cross-linked chitosan microspheres. Eur J Med Chem. Feb; 60: 249-53.
TE
D
22. Sivakumar S.M., Sukumaran N., Nirmala L., Swarnalakshmi R., Anilbabu B., Siva L., Anbu J., Shanmugarajan T.S., Ravichandran V. (2010) Immunopotentiation of hepatitis B vaccine using biodegradable polymers as adjuvant. J. Microbiol. Immunol. Infect. 43(4): 265–270.
CE P
23. Nihant, N., S. Stassen, C. Grandfils, R. Jerome, and P. Teyssie (1994) Microencapsulation by coacervation of PLGA: III. Characterization of the final microspheres. Polym. Int. 34: 289-299.
AC
24. Nihant N, Grandfils C, Jérôme R, et al. Microencapsulation by coacervation of poly(lactide-co-glycolide) IV. Effect of the processing parameters on coacervation and encapsulation. J Control Release 1995; 35: 117-25. 25. Ozbas-Turan S, Akbuga J, and Aral C. Controlled release of interleukin-2 from chitosan microspheres. J Pharm Sci 2002; 91: 1245-51. 26. Yeo Y, Baek N, and Park K, (2001) Microencapsulation Methods for Delivery of Protein Drugs, Biotechnol. Bioprocess Eng. 6: 213-230. 27. Wang L, Yang T, Ma G, (2013) Microspheres and microcapsules for protein drug delivery. Chapter in Microspheres and Microcapsules in Biotechnology: Design, Preparation and Applications, Editors Ma G and Su Z, Pan Stanford Publishing, Pte. Ltd. 28. Pettit, D. K., J. R. Lawter, W. J. Huang, S. C. Pankey, N. S. Nightlinger, D. H. Lynch, J. A. Schuh, P. J. Morrissey, and W. R. Gombotz (1997) Characterization of poly(glycolide-co-D,L-lactide)/poly(D,L-lactide) microspheres for controlled release of GM-CSF. Pharm. Res. 14: 1422-1430.
41
ACCEPTED MANUSCRIPT 29. Li, J. K., N. Wang, and X. S. Wu (1997) A novel biodegradable system based on gelatin nanoparticles and poly(lactic-co-glycolic acid) microspheres for protein and peptide drug delivery. J. Pharm. Sci. 86: 891-895. 30. Wang, N. and X. S. Wu (1998) A novel approach to stabilization of protein drugs in PLGA microspheres using agarose hydrogel. Int. J. Pharm.166: 1-14.
IP
T
31. Wang, N., X. S. Wu, and J. K. Li (1999) A heterogeneously structured composite based on poly(lactic-co-glycolic acid) microspheres and poly(vinyl alcohol) hydrogel nanoparticles for long-term protein drug delivery. Pharm. Res. 16: 14301435.
SC R
32. Bakan, J. A. (1986) Microencapsulation. pp. 412-429. In: L. Lachman H. A. Lieberman, and J. L. Kanig (eds.). The Theory and Practice of Industrial Pharmacy. Lea & Febiger, Philadelphia, USA.
NU
33. Bakan, J. A. (1986) Microencapsulation. pp. 412-429. In: L. Lachman H. A. Lieberman, and J. L. Kanig (eds.). The Theory and Practice of Industrial Pharmacy. Lea & Febiger, Philadelphia, USA.
MA
34. Brown, K. E., K. Leong, C. Huang, R. Dalal, G. D. Green, H. B. Haimes, P. A. Jimenez, and J. Bathon (1998) Gelatin/chondroitin 6-sulfate microspheres for the delivery of therapeutic proteins to the joint. Arthritis Rheum. 41: 2185-2195. 35. Bowey K, Neufeld RJ. (2010) Systemic and mucosal delivery of drugs within polymeric microparticles produced by spray drying. BioDrugs. 24(6): 359-77
TE
D
36. Wang Q, Fu A, Li H, Liu J, Guo P, Zhao XS, Xia LH (2014) Preparation of cellulose based microspheres by combining spray coagulating with spray drying. Carbohydr Polym. 13(111):393-9
CE P
37. Takada S, Uda Y, Toguchi H, Ogawa Y (1995) Application of a spray drying technique in the production of TRH-containing injectable sustained-release microparticles of biodegradable polymers. PDA J Pharm Sci Technol. 49(4):180-4
AC
38. Hahn SK, Kim JS, Shimobouji T. (2007) Injectable hyaluronic acid microhydrogels for controlled release formulation of erythropoietin. J Biomed Mater Res. 80(4): 916-24 39. Bodmeier R, Chen HG, Paeratakul O. (1989) A novel approach to the oral delivery of micro- or nanoparticles. Pharm Res. 6(5): 413-7. 40. Sajeesh S, Sharma CP. (2011) Mucoadhesive hydrogel microparticles based on poly (methacrylic acid-vinyl pyrrolidone)-chitosan for oral drug delivery. Drug Deliv. 18(4): 227-35 41. Gonjari ID, Hosmani AH, Karmarkar AB, Kadam SB, Godage AS, Khade TS. (2009) Microspheres of tramadol hydrochloride compressed along with a loading dose: A modified approach for sustaining release. Drug Discov Ther. 3(4): 176-80. 42. King A, Strand B, Rokstad AM, Kulseng B, Andersson A, Skjåk-Braek G, Sandler S. (2003) Improvement of the biocompatibility of alginate/poly-L-lysine/alginate microcapsules by the use of epimerized alginate as a coating. J Biomed Mater Res A. 64(3): 533-9
42
ACCEPTED MANUSCRIPT 43. Hunt NC, Shelton RM, Henderson DJ, Grover LM. (2013) Calcium-alginate hydrogelencapsulated fibroblasts provide sustained release of vascular endothelial growth factor. Tissue Eng Part A. 19(7-8): 905-14. 44. Couvreur P, Barratt G, Fattal E, Legrand P, Vauthier C. (2002) Nanocapsule technology: a review. Crit Rev Ther Drug Carrier Syst. 19(2): 99-134.
IP
T
45. Aboubakar M, Puisieux F, Couvreur P, Vauthier C. (1999) Physico-chemical characterization of insulin-loaded poly(isobutylcyanoacrylate) nanocapsules obtained by interfacial polymerization. Int J Pharm. 183(1): 63-6
SC R
46. Purvis T, Vaughn JM, Rogers TL, Chen X, Overhoff KA, Sinswat P, Hu J, McConville JT, Johnston KP, Williams RO. (2006) Cryogenic liquids, nanoparticles, and microencapsulation. Int J Pharm. Oct 31; 324(1): 43-50. 47. Gibbs BF, Kermasha S, Alli I, Mulligan CN. (1999) Encapsulation in the food industry: a review. Int J Food Sci Nutr. 50(3): 213-24.
NU
48. Chaturvedi A, Srivastava P, Yadav S, Bansal M, Garg G, Sharma PK. (2011). Fast dissolving films: a review. Curr Drug Deliv. 8(4): 373-80
MA
49. Delgado-Guay MO. 2010. Efficacy and safety of fentanyl buccal for cancer pain management by administration through a soluble film: an update. Cancer Manag Res. 2: 303-6.
TE
D
50. Nishigaki M, Kawahara K, Nawa M, Futamura M, Nishimura M, Matsuura K, Kitaichi K, Kawaguchi Y, Tsukioka T, Yoshida K, Itoh Y. 2012. Development of fast dissolving oral film containing dexamethasone as an antiemetic medication: clinical usefulness. Int J Pharm. 424(1-2): 12-7.
CE P
51. Patel JG, Modi AD. 2012. Formulation, optimization and evaluation of levocetirizine dihyrochloride oral thin strip. J Pharm Bioallied Sci. 4(Suppl 1): S35-6. 52. SK Yellanki, S Jagtap, and R Masareddy. 2011. Dissofilm: A Novel Approach for Delivery of Phenobarbital; Design and Characterization. J Young Pharm. 3(3): 181– 188.
AC
53. Truong-Le, V. et al. Formulations for preservation of rotavirus. US Patent # 8,241,886 54. Ohtake S, Martin R, Saxena A, Pham B, Chiueh G, Osorio M, Kopecko D, Xu D, Lechuga-Ballesteros D, Truong-Le V. Room temperature stabilization of oral, live attenuated Salmonella enterica serovar Typhi-vectored vaccines. Vaccine. 2011 Mar 24; 29(15): 2761-71. 55. Ohtake S, Martin RA, Saxena A, Lechuga-Ballesteros D, Santiago AE, Barry EM, Truong-Le V. 2011. Formulation and stabilization of Francisella tularensis live vaccine strain. J Pharm Sci. 100(8): 3076-87. 56. Cicerone, T.C., et al. Substantially improved stability of biological agents in dried form: The role of glassy dynamics in preservation of biopharmaceuticals. 2003. Bioprocess Int. 1: 2-9. 57. Mokarian-Tabari P, Geoghegan M, Howse JR, Heriot SY, Thompson RL, Jones RA. 2010. Quantitative evaluation of evaporation rate during spin-coating of polymer blend films: Control of film structure through defined-atmosphere solvent-casting. Eur Phys J E Soft Matter. 33(4): 283-9.
43
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
58. Cross GL, O'Connell BS, Pethica JB, Rowland H, King WP. (2008) Variable temperature thin film indentation with a flat punch. Rev Sci Instrum. 79(1): 013904. 59. Bischof JC, He X. (2005) Thermal stability of proteins. Ann N Y Acad Sci. 1066: 1233. 60. Klibanov AM. (1997) Why are enzymes less active in organic solvents than in water? Trends Biotechnol. 15: 97–101. 61. Kamal MZ, Yedavalli P, Deshmukh MV, Rao NM. (2013) Lipase in aqueous-polar organic solvents: activity, structure, and stability. Protein Sci. 7: 904-15. 62. Wangikar PP, Michels PC, Clark DS, Dordick JS. (1997) Structure and function of subtilisin BPN' solubilized in organic solvents. J Am Chem Soc. 119: 70–76. 63. Debulis K, Klibanov AM. (1993) Dramatic enhancement of enzymatic activity in organic solvents by lyoprotectants. Biotechnol Bioeng. 41: 566–571. 64. Gorman LA, Dordick JS. (1992) Organic solvents strip water off enzymes. Biotechnol Bioeng. 39: 392–397. 65. Wiesbauer J, Prassl R, Nidetzky B (2013) Renewal of the air-water interface as a critical system parameter of protein stability: aggregation of the human growth hormone and its prevention by surface-active compounds. Langmuir 29(49): 1524050. 66. Mitropoulos V, Mütze A, Fischer P. (2014) Mechanical properties of protein adsorption layers at the air/water and oil/water interface: a comparison in light of the thermodynamic stability of proteins. Adv Colloid Interface Sci. 206: 195-206. 67. Gu JH, Beekman A, Wu T, Piedmonte DM, Baker P, Eschenberg M, Hale M, Goldenberg M. (2013). Beyond glass transitions: studying the highly viscous and elastic behavior of frozen protein formulations using low temperature rheology and its potential implications on protein stability. Pharm Res. 30(2): 387-401. 68. Chang BS, Kendrick BS, Carpenter JF. (1996) Surface-induced denaturation of proteins during freezing and its inhibition by surfactants. J Pharm Sci. 85(12): 132530. 69. He J, Feng M, Zhou X, Ma S, Jiang Y, Wang Y, Zhang H. (2011) Stabilization and encapsulation of recombinant human erythropoietin into PLGA microspheres using human serum albumin as a stabilizer. Int J Pharm. 416(1): 69-76. 70. Leerahawong A1, Tanaka M, Okazaki E, Osako K (2012) Stability of the physical properties of plasticized edible films from squid (Todarodes pacificus) mantle muscle during storage. J Food Sci. 77(6): E159-65. 71. Wang B, Tchessalov S, Cicerone MT, Warne NW, Pikal MJ. (2009) Impact of sucrose level on storage stability of proteins in freeze-dried solids: II. Correlation of aggregation rate with protein structure and molecular mobility. J Pharm Sci. 98(9): 3145-66. 72. Pikal MJ, Rigsbee D, Roy ML, Galreath D, Kovach KJ, Wang B, Carpenter JF, Cicerone MT. (2008) Solid state chemistry of proteins: II. The correlation of storage stability of freeze-dried human growth hormone (hGH) with structure and dynamics in the glassy solid. J Pharm Sci. 97(12): 5106-21.
44
ACCEPTED MANUSCRIPT
TE
D
MA
NU
SC R
IP
T
73. Yamamoto A. (2001). Improvement of transmucosal absorption of biologically active peptide drugs. Yakugaku Zasshi. 2001 Dec;121(12):929-48. 74. Ueno Y, Ohmi T, Yamamoto M, Kato N, Moriguchi Y, Kojima M, Shimozono R, Suzuki S, Matsuura T, Eda H. J (2007) Orally-administered caspase inhibitor PF03491390 is retained in the liver for prolonged periods with low systemic exposure, exerting a hepatoprotective effect against alpha-fas-induced liver injury in a mouse model. Pharmacol Sci. 105(2): 201-5. 75. Felton LA, Porter SC. (2013). An update on pharmaceutical film coating for drug delivery. Expert Opin Drug Deliv. 10(4): 421-35. 76. Cai QX, Zhu KJ, Chen D, Gao LP (2003) Synthesis, characterization and in vitro release of 5-aminosalicylic acid and 5-acetyl aminosalicylic acid of polyanhydride-P(CBFAS). Eur J Pharm Biopharm. 55(2): 203-8. 77. Mitragotri S, Burke PA, Langer R. Overcoming the challenges in administering biopharmaceuticals: formulation and delivery strategies. Nature Reviews Drug Discovery 2014, 13: 655–672. 78. Göpferich A, Gref R, Minamitake Y, Shieh L, Alonso MJ, Tabata Y, Langer R. (1994) Drug Delivery from Bioerodible Polymers: Systemic and Intravenous Administration. ACS Symposium Series on Formulation and Delivery of Proteins and Peptides (Cleland JL editor). Vol. 567, Chapter 15, 241-277. 79. Kanthamneni N, Sharma S, Meenach S, Billet B, Zhao J, Bachelder E, Ainslie K. Enhanced stability of horseradish peroxidase encapsulated in acetalated dextran microparticles stored outside cold chain conditions. Int. J. Pharmaceut. 2012; 431: 101-110.
AC
CE P
80. Morales JO, Huang S, Williams RO, McConville JT. (2014) Films loaded with insulin-coated nanoparticles (ICNP) as potential platforms for peptide buccal delivery. Colloids and Surfaces B: Biointerfaces 122: 38-45. 81. Fonte P, Soares S, Costa A, Andrade J, Seabra V, Reis S, Sarmento B. Effect of cryoprotectants on the porosity and stability of insulin loaded PLGA nanoparticles after freeze-drying. Biomatter 2012; 2:4, 329-339. 82. Gemeinhart R. Polymeric systems for oral protein and peptide delivery in: Pharmaceutical biotechnology, second edition. Editor: Groves M. Publisher: CRC Press, Boca Raton, FL. 2006; 283-306. 83. Danhier F, Ansorena E, Silva JM, Coco R, Le Breton A, Preat V. PLGA-based nanoparticles: an overview of biomedical applications. J Control Release 2012; 161: 505-522. 84. Lu L, Peter S, Lyman M, Lai H, Leite S, Tamada J, Uyama S, Vacanti J, Langer R, Mikos A. In vitro and in vivo degradation of porous poly(DL-lactic-co-glycolic acid) foams. Biomaterials 2000; 21: 1837-1845. 85. Lherm C, Müller R, Puisieux F, Couvreur P. Alkylcyanoacrylate drug carriers: II. Cytotoxicity of cyanoacrylate nanoparticles with different alkyl chain length. Int. J. Pharmaceut. 1992; 84(1): 13–22.
45
ACCEPTED MANUSCRIPT 86. Couvreur P, Roland M, Speiser P. Biodegradable submicroscopic particles containing a biologically active substance and compositions containing them. 1982; US patent 4329332 A.
T
87. Al-Khouri Fallouh N, Roblot-Treupel L, Fessi H, Devissaguet J, Puisieux F. Development of a new process for the manufacture of polyisobutylcyanoacrylate nanocapsules. Int. J. Pharmaceut. 1986; 28: 125-132.
AC
CE P
TE
D
MA
NU
SC R
IP
88. Chouinard F, Kan F, Leroux J, Foucher C, Lemaerts V. Preparation and purification of polyisohexylcyanoacrylate nanocapsules. Int. J. Pharmaceut. 1991; 72: 211-217. 89. Vandorpe J, Schacht E, Stolnik S, Garnett MC, Davies MC, Illum L, Davis SS. Poly(organo phosphazene) nanoparticles surface modified with poly(ethylene oxide). Biotechnol Bioeng. 1996 Oct 5;52(1):89-95. 90. Veronese FM, Marsilio F, Caliceti P, De Filippis P, Giunchedi P, Lora S. Polyorganophosphazene microspheres for drug release: polymer synthesis, microsphere preparation, in vitro and in vivo naproxen release. J Control Release. 1998 Mar 31;52(3):227-37. 91. Passi P, Zadro A, Marsilio F, Lora S, Caliceti P, Veronese FM.Plain and drug loaded polyphosphazene membranes and microspheres in the treatment of rabbit bone defects. J Mater. Sci. Mater Med. 2000 Oct;11(10):643-54. 92. Scopelianos A. Polyphosphazenes as new biomaterials in: Shalaby, S.W. (ed.) Biomedical polymers: Designed to degrade systems. Hanser/Gardner publishers, Inc., Cincinnati, OH (1994), 153-171. 93. Andrianov A, Payne L. Protein release from polyphosphazene matrices. Adv. Drug Delivery Rev. 1998; 31: 185-196. 94. Crommen JH, Schacht EH, Mense EH. Biodegradable polymers. II. Degradation characteristics of hydrolysis-sensitive poly[(organo)phosphazenes]. Biomaterials. 1992;13(9):601-611. 95. Tanigami T, Ohta H, Orii R, Yamaura K, Matsuzawa S. Degradation of poly[bis(ethylamino)phosphazene] in aqueous solution. Journal of Inorganic and Organometallic Polymers. 1995, Volume 5, Issue 2, pp 135-153. 96. Florence A. The oral absorption of micro- and nanoparticulates: neither exceptional nor unusual. Pharm Res. 1997 Mar;14(3):259-66. 97. Morishita I, Morishita M, Machida Y, Nagai T. Controlled release microspheres based on Eudragit L100 for the oral administration of erythromycin. Drug Des Deliv. 1991 Jul;7(4):309-19. 98. Park H, Robinson J. Mechanisms of Mucoadhesion of Poly(acrylic Acid) Hydrogels. Pharmaceutical Research. 1987; 4(6): 457-464. 99. Ponchel G, Irache J. Specific and non-specific bioadhesive particulate systems for oral delivery to the gastrointestinal tract. Adv Drug Deliv Rev. 1998 Dec 1;34(2-3):191219.
46
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
100. Saffran M, Kumar GS, Savariar C, Burnham JC, Williams F, Neckers DC. A new approach to the oral administration of insulin and other peptide drugs. Science. 1986 Sep 5;233(4768):1081-4. 101. Brondsted H, Kopecek J. Hydrogels for site-specific drug delivery to the colon. Pharmaceut. Res. 1992; 9: 1540-1545. 102. Yeh P, Berenson M, Samowitz W, Kopeckova P, Kopecek J. Site-specific drug delivery and penetration enhancement in the gastrointestinal tract. Journal of controlled release. Volume 36, Issues 1–2, September 1995, Pages 109–124. 103. Takahashi T, Takayama K, Machida Y, Nagai T. Characteristics of polyion complexes of chitosan with sodium alginate and sodium polyacrylate. Int. J. Pharmaceut. 1990; 61(1–2): 35–41. 104. Matsumoto S, Kobayashi H, Takashima Y. Production of monodispersed capsules. J Microencapsul. 1986 Jan-Mar;3(1):25-31. 105. Wan LS, Heng PW, Chan LW. Drug encapsulation in alginate microspheres by emulsification. J Microencapsul. 1992 Jul-Sep;9(3):309-16. 106. Arneodo C, Benoit J, Thies C. Characterization of complex coacervates used to form microcapsules. Polym. Mater.Sci. Eng. 1987; 57: 255-259. 107. Bernkop-Schnuerch A, Scerbe-Saiko A. Synthesis and in vitro evaluation of chitosan-EDTA-protease inhibitor conjugates which might be useful in oral delivery of peptides and proteins. Pharmaceut. Res. 1998; 15: 263-369. 108. Arbit E, Majuru S, Orellana I. Oral delivery of biopharmaceuticals using the Eligen technology in: Protein formulation and delivery, second edition. Editor: McNally E, Hastedt J. Publisher: Informa Healthcare USA, Inc. New York, NY. 2008; 285-302. 109. Yagi T, Ishizaki K, Takebe H. Cytotoxic effects of protease inhibitors on human cells. 2. Effect of elastatinal. Cancer Lett. 1980 Oct;10(4):301-7. 110. McCaffrey G, Jamieson JC. Evidence for the role of a cathepsin D-like activity in the release of Gal beta 1-4GlcNAc alpha 2-6sialyltransferase from rat and mouse liver in whole-cell systems. Comp Biochem Physiol B. 1993 Jan;104(1):91-4. 111. Plumpton C, Kalinka S, Martin RC, Horton JK, Davenport AP. Effects of phosphoramidon and pepstatin A on the secretion of endothelin-1 and big endothelin-1 by human umbilical vein endothelial cells: measurement by two-site enzyme-linked immunosorbent assays. Clin Sci (Lond). 1994 Aug;87(2):245-51. 112. Bernkop-Schnuerch A, Krajicek M. Mucoadhesive polymers for peroral peptide delivery: Synthesis and evaluation of chitosan-EDTA conjugates. J. Control. Release. 1998; 50: 215-223. 113. Park JH, Ye M, Park K. Biodegradable Polymers for Microencapsulation of Drugs: Molecules 2005, 10, 146-161. 114. Carpenter JF, Pikal MJ, Chang BS, Randolph TW. Rational Design of Stable Lyophilized Protein Formulations: Some Practical Advice. Pharmaceutical Research August 1997, Volume 14, Issue 8, pp 969-975.
47
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
115. Johnson OL, Jaworowicz W, Cleland JL, Bailey L, Charnis M, Duenas E, Wu C, Shepard D, Magil S, Last T, Jones AJ, Putney SD. The stabilization and encapsulation of human growth hormone into biodegradable microspheres. Pharm Res. 1997 Jun;14(6):730-5. 116. Park K, Kwon I, Park K. Oral protein delivery: current status and future prospect. Reactive and Functional Polymers. 2011 (71): 280-287. 117. Kipnes M, Dandona P, Tripathy D, Still JG, Kosutic G. Control of postprandial plasma glucose by an oral insulin product (HIM2) in patients with type 2 diabetes. Diabetes Care. 2003 Feb;26(2):421-426. 118. Clement S, Still JG, Kosutic G, McAllister RG. Oral insulin product hexyl-insulin monoconjugate 2 (HIM2) in type 1 diabetes mellitus: the glucose stabilization effects of HIM2. Diabetes Technol Ther. 2002;4(4):459-466).
48
S0169-409X(15)00183-0 doi: 10.1016/j.addr.2015.08.001 ADR 12826
To appear in:
Advanced Drug Delivery Reviews
Received date: Revised date: Accepted date:
2 December 2014 20 July 2015 4 August 2015
Please cite this article as: Vu Truong-Le, Phillip Lovalenti, Ahmad M. Abdul-Fattah, Stabilization Challenges and Formulation Strategies Associated with Oral Biologic Drug Delivery Systems, Advanced Drug Delivery Reviews (2015), doi: 10.1016/j.addr.2015.08.001
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
IP
T
ACCEPTED MANUSCRIPT
MA
NU
SC R
Stabilization Challenges and Formulation Strategies Associated with Oral Biologic Drug Delivery Systems
Vu Truong-Le*, Phillip Lovalenti, and Ahmad M. Abdul-Fattah
Number of pages:
48
AC
CE P
TE
D
Author(s):
*Corresponding author: [email protected], phone: (408)385-1742, fax: (408)9603822
ACCEPTED MANUSCRIPT Table of contents
3.
T
CE P AC
6. 7.
TE
D
5.
MA
NU
4.
SC R
IP
1. 2.
Table of contents .................................................................................................................. 2 List of tables ......................................................................................................................... 3 List of figures........................................................................................................................ 3 Introduction and challenges with oral delivery route .......................................................... 4 Methods employed in preparation of biopharmaceutical oral solid dosage forms .............. 8 2.1 Preparation methods for microspheres and nanoparticles ........................................... 8 2.2 Preparation methods for oral thin films ..................................................................... 18 Stresses encountered during manufacture of oral solid dosage forms and ways to improve in-process stability .............................................................................................. 20 Formulation and stability aspects in oral solid dosage forms for biomacromolecules ...... 26 4.1. Polymers .................................................................................................................... 27 4.1.1. Hydrophobic polymers .................................................................................... 28 4.1.2. Hydrophilic polymers ..................................................................................... 29 4.2. Permeation/absorption enhancers .............................................................................. 31 4.3. Enzyme inhibitors ...................................................................................................... 32 4.4. Other formulation components .................................................................................. 33 Stability post-manufacturing ............................................................................................. 35 5.1 Storage stability ......................................................................................................... 35 5.2 Stability after administration ..................................................................................... 37 Summary and future prospects and trends ......................................................................... 38 References ......................................................................................................................... 40
2
ACCEPTED MANUSCRIPT List of tables Table 1
Routes of administration and dosage forms developed for nonparenteral administration of biopharmaceuticals .................................... 7 A summary of solvent evaporation/extraction methods used to prepare microspheres ......................................................................................... 10 A summary of other techniques used to prepare microspheres ............ 12
Table 2
IP
T
Table 3
List of figures
Sites in the digestive tract that represent opportunities for drug absorption. .............................................................................................. 6 Lab scale spray drying process in environmental enclosure under controlled conditions ............................................................................ 17 Basic steps in a lab-scale thin film manufacturing process and the microstructure of the resulting stabilized vaccine film formulation..... 20 Normalized HRP activity versus time in microparticle formulations of Ac-DEX prepared by three different methods at 5°C (a), 25°C (b) and 45°C (c) [79] ………………………………………............................. 23 Alternative approaches to fabrication of OTF’s for mAb delivery ...... 36
SC R
Figure 1 Figure 2
NU
Figure 3
MA
Figure 4
AC
CE P
TE
D
Figure 5
3
ACCEPTED MANUSCRIPT 1.
Introduction and challenges with oral delivery route
Due to the rapid progress in biotechnology, the industry has produced a large number of therapeutic peptides and proteins on commercial scale. Over 130 biotechnologically derived drug products are approved by the US Food and Drug Administration (FDA) [1]. Most
T
biopharmaceutical drug products (proteins, peptides, and vaccines) are administered
Poor intestinal absorption of these drugs, for
SC R
administration, including the oral route.
IP
parenterally because of their poor bioavailability from different alternate routes of
example, is due to their susceptibility to acid and enzymatic hydrolysis, unfavorable physicochemical properties including size, charge, and hydrophilicity. In addition to hydrolysis in the stomach and gastrointestinal (GI) tract, peptide and protein drugs targeting a local therapeutic
NU
effect in the colon [16] must also survive degradation via bacterial fermentation. Despite these challenges a number of alternate routes of administration for biologic drugs have been
MA
pursued with varying degrees of success. Some examples of these alternative routes that have advanced to human use are provided in Table 1.
Alternate delivery routes can offer
convenience/non-invasiveness, enhanced targeting to diseased tissues, controlled release rate,
TE
D
improved compliance, and can provide a game-changing commercial opportunity.
Development of an effective oral delivery system for biologics requires a detailed
CE P
understanding of the several barriers along the digestive tract (See Figure 1), as well as the mechanisms involved in their absorption across targeted tissues. It is generally believed that the challenges to oral delivery of biopharmaceuticals are significant, and substantial
AC
opportunities remain to optimize delivery approaches, formulation components and processing conditions for each peptide and protein drug.
Obviously the most convenient route for the systemic delivery of pharmaceuticals is oral; however, attempts to deliver large molecular weight proteins and peptides orally have seen limited successes. Bioavailability via this route is poor for molecules of molecular mass greater than a couple hundred Daltons. In addition, proteins are susceptible to hydrolysis and modification at gastric pH levels and can be degraded by proteolytic enzymes in the small intestine. Various approaches currently under investigation include amino acid backbone modifications [2], conjugation of bacterial and viral transcytosis peptide sequences [3,4],
4
ACCEPTED MANUSCRIPT formulation design, chemical modification, use of proteolytic inhibitors, and passive absorption enhancers; all have shown variable successes. Parenteral delivery of proteins and peptides has been the method of choice for systemic delivery mainly for avoidance of biological barriers through which it is difficult for proteins to
T
pass, and the ability to achieve pharmacologic levels of circulating protein over a relatively
IP
short period of time. In addition to parenteral administration, interest has increased in the area of local delivery of proteins through mucosal tissues of the buccal area [15], gut, sinus and
SC R
lungs by both oral and inhalation delivery systems.1 In these applications, proteins typically must be administered in formulations which protect against unwanted proteolysis and target the mucosal tissues. In recent years, there has been a rise in quick dissolving oral thin films in
NU
commercial application ranging from Listerine® breath freshener strips to analgesics, thus warrants some review coverage. There are already a number of excellent reviews on the
MA
biological barriers to oral delivery related to absorption mechanisms, molecular approaches to absorption enhancement, including comprehensive reviews of different oral delivery systems [5]. Using case studies from literature and from our own work on oral thin films, we will
D
continue this special edition’s theme on protein stability and focus on the manufacturing
TE
processes, the in-process stability and storage stability of biopharmaceuticals formulated in delivery systems designed for oral delivery to the gastro-intestinal (GI) tract. Among the
CE P
range of delivery systems, biodegradable polymer systems for protection and controlled release of proteins have been the most studied; hence, we will review these systems in greater depth. These include polymeric biodegradable microspheres or nanospheres that contain
AC
proteins or vaccines, which are designed to minimize both administration frequency and biologically effective protein dosage. Specifically, this review will include a landscape survey of the systems that have been studied, the manufacturing processes involved, stability through the manufacturing process, key pharmaceutical formulation parameters that impact stability of the encased proteins, and storage stability of the encapsulated proteins in these delivery systems.
1
Examples of products in this area include Generex Biotechnology’s insulin buccal spray Oral-lyn™ (in Phase III clinical trials) and the oral thin film products of MonoSol Rx®, which include both the marketed small molecule products Zuplenz® and Suboxone® as well as several complex-molecule-containing oral films in development, such as insulin that is in clinical trials.
5
MA
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
Figure 1. Sites in the digestive tract that represent opportunities for drug absorption. The variety of conditions (pH, chemical exposure and time) in each compartment are barriers that drug delivery systems will need to overcome. From: http://en.wikipedia.org/wiki/Digestive_tract (Copyright-free)
6
ACCEPTED MANUSCRIPT
Table 1. Routes of administration and dosage forms developed for non-parenteral administration of biopharmaceuticals [77]
IP
T
Examples of biopharmaceuticals tested (C – clinical trials, A – approved product) Calcitonin (C) Insulin (C) Exenatide (C) Octreotide (C) Rotavirus vaccine (A) Typhoid vaccine (A) Adenovirus vaccine (A) Anthrax vaccine Cholera vaccine (C) H5N1 Avian flu vaccine (C) Polio vaccine (A) Smallpox vaccine
SC R
MA
Oral
Examples of Dosage forms/Drug delivery vehicles Pills Capsules Microspheres Hydrogels Nanoparticles Enteric coated tablets Enteric coated dry emulsions Liquid dropper
NU
Administration route
Mucoadhesive patches/films Liquid spray
Insulin (C) Interferon (C) Oxytocin
Sublingual
Tablets
Desmopressin (A)
Ocular
Eye drops Injections
VEGF-targeted Fab and IgG1 mAb (A)
Gels
LHRH (luteinizing hormone releasing hormone) analogue (C)
Rectal
TE
CE P AC
Vaginal
D
Buccal
Suppositories
Cutaneous/Topical
Micro-needles Transdermal patches, creams, sprays, gels
Nasal
Aerosol sprays
Inhalable
Aerosol and dry powder sprays
Influenza virus (A) Parathyroid hormone (PTH) (C) Insulin (C) Testosterone (A) Influenza vaccine (A) Salmon calcitonin (A) Insulin (A,C) Dornase alfa (A)
7
ACCEPTED MANUSCRIPT 2.
Methods employed in preparation of biopharmaceutical oral solid dosage forms
T
Proteolysis (e.g. enzymatic, pH-driven, etc.) is a common problem throughout the gastro-
oral bioavailability of proteins and peptides.
IP
intestinal (GI) tract, especially in the stomach and upper GI tract, thus affecting subsequent Overcoming the potential proteolysis, and
SC R
thereby enhancing absorption through the GI epithelium and improving oral bioavailability, has been achieved mainly via protein and peptide encapsulation. For example, polymeric microspheres of poly(methacrylic-g-ethylene glycol) have been used to stabilize insulin in the
NU
GI tract [10]. The gels were swollen in the acidic stomach medium due to intermolecular polymer complex formation without insulin release into the gastric fluid. In the basic and neutral intestinal media, the complexes dissociated, resulting in the release of insulin.
MA
Similarly, Jani and coworkers have studied the uptake of polymeric nanoparticles in the rat GI
D
tract following oral administration as a potential drug carrier [6, 7].
The manufacturing or preparation methods for the variety of encapsulation methods for oral
TE
drug delivery systems range from simple to complex; each involves its own set of physical and chemical stresses to the active pharmaceutical ingredient (API). A better understanding of
CE P
the range of stresses to the API payload during manufacturing may allow for alternative strategies that can be considered to improve protein stability. In this section, we will cover the preparation methods for the most commonly tested solid oral delivery systems for
AC
biopharmaceuticals: microspheres/microparticles, nanoparticles, and oral thin films. Tablets and powder filled capsules are established technologies that are diverse, extensively reviewed elsewhere, and will not be included in our review.
2.1
Preparation methods for microspheres and nanoparticles
The range of the degradable polymeric protein and peptide delivery systems designed for oral delivery that have been reported over recent years is very broad. Recent interest in particulate systems for oral delivery of proteins have focused on targeting [8, 11] and addressing the mucous layer barrier and mucoadhesion in the GI [12, 13, 40]. Requirements for a successful encapsulation process using biodegradable polymers include maintaining stability and biological activity of the incorporated drugs during and after encapsulation, high yield and
8
ACCEPTED MANUSCRIPT encapsulation efficiency, reproducible release profile of the drug without the significant initial burst and desirable product attributes such as appropriate particle size of micro/nanoparticles as well as free-flowing particles [113]. This section will provide an overview of the various preparation methods that have been developed to fabricate nano- and micro-particulate
T
systems that can be delivered orally as suspensions, pressed into oral tablets, or filled into
IP
capsules for oral ingestion. Many methods are well-developed for preparation of hydrophilic
SC R
polymer microspheres and nanoparticles for protein delivery. The majority include solvent evaporation/extraction methods (emulsion formation with organic solvents using water soluble or insoluble polymers), coacervation/precipitation methods, spray drying, ionic gelation methods and the template assembly method. An excellent review of many of these
NU
methods is provided by Yeo and coworkers [26], much of which is summarized in Tables 2 and 3. Some of the most common are further highlighted below. The interested reader may
MA
find greater details on a particular manufacturing method in the references listed in Tables 2
AC
CE P
TE
D
and 3.
9
ACCEPTED MANUSCRIPT
Cryogenic Solvent Extraction Microspheres
Organic solvent (e.g. dichloromethane) with dissolved polymer and drug PLGA
AC
CE P
TE D
MA N
US
CR
IP
T
Table 2. A summary of solvent evaporation/extraction methods used to prepare microspheres Method Solvent Evaporation/Solvent Extraction Spray Desolvation Single Emulsion Single Emulsion Double Emulsion Solvent Evaporation Product Solvent evaporation Microspheres and Microspheres Microspheres preferred for porous nanospheres prepared by solvent fast release prepared by solvent extraction and microspheres, evaporation evaporation solvent extraction preferred for fast release microspheres Dispersed phase Polymer solution in Organic solvent Aqueous phase Aqueous phase organic solvent (e.g. with dissolved or Dichloromethane, dispersed drug ethyl acetate) with dispersed or dissolved drug Example of Polylactic acid Polylactic acid PLGA Polyvinyl alcohol Encapsulation (PLA) and (PLA) and (PVA), PLGA Polymer ploy(lactide-coploy(lactide-coglycolide) (PLGA) glycolide) (PLGA) Continuous phase Mineral oil or O/W emulsion Organic solvent as Aqueous solution acetone, ethanol Emulsifier Span 85 and Same Same N/A sorbitan sesquioleate aluminum tristearate for O/O emulsion. Carbopol 951, methyl cellulose, polyvinyl alcohol (PVA) for O/W 10
Ethanol N/A
ACCEPTED MANUSCRIPT
Insulin, bovine serum albumin (BSA), ovalbumin (OVA), bovine superoxide dismutase (bSOD)
26
CR
IP
T
W/O emulsion (aqueous drug solution emulsified in organic solvent with polymer) added to aqueous phase W/O/W emulsion organic solvent removal/evaporation Carbonic anhydrase, BSA, recombinant human growth hormone (hGH), recombinant human erythropoietin (rhEPO), urease and lysozyme. 17, 26
26
AC
References
CE P
TE D
Example of protein/peptide prepared
Dispersed phase added to continuous phase homogenization solvent removal by drying (e.g. freeze drying)
US
emulsions Dispersed phase added to continuous phase homogenization solvent removal centrifugation or filtration of microspheres freeze drying
MA N
Process
11
Polymer/drug solution Spray onto desolvating liquid precipitation of microspheres filter, collect and dry
Polymer /drug solution Spray onto frozen ethanol precipitation of microspheres filter, collect and dry
BSA
Tetanus toxoid, recombinant human growth hormone (hGH)
26
18, 19, 20
T
ACCEPTED MANUSCRIPT
Interfacial Polymerization
Microspheres Nanoparticles Protein in aqueous solution with diamine monomer Nylon, polyester
AC
CE P
TE D
MA N
US
CR
IP
Table 3. A summary of other techniques used to prepare microspheres Method Emulsion crossCoacervation Non solvent linking with addition Precipitation Complex water soluble coacervation polymers Product Microspheres Microspheres Microspheres Microspheres, Nanospheres Dispersed phase Protein dissolved Protein in Protein in Protein in or dispersed in aqueous aqueous aqueous solution aqueous polymer solution solution with solution one polymer Example of Chitosan, Gelatin Blend of PLA, PLGA, Encapsulation collagen, gelatin Polycation (e.g. gelatin, agarose Polymer Gelatin) + and polyvinyl Polyanion (e.g. alcohol (PVA): Gum Arabic, single polymer or Chondroitin-6- blends sulfate (CS6) Continuous phase Organic solvent Polymer Aqueous First nonor oil solution solution of solvents for the another polymers: polymer Silicone oil, vegetable oil, light liquid paraffin oils. Second nonsolvent : heptane, hexane, petroleum ether Process Aqueous phase Protein Aqueous Slow addition of 12
Biocompatible oil (e.g. mono/diglycerides) containing dichloride monomer and surfactant (e.g. Tween 80)
Aqueous phase
ACCEPTED MANUSCRIPT
with monomer added/emulsified into continuous phase (O) with second monomer to form W/O emulsion Add acid chloride polymerization at interface to form microspheres.
BSA, Insulin, granulocytemacrophage colonystimulating factor (GM-CSF) 26-32
Insulin, BSA
IP
T
first non-solvent to aqueous solution microparticles form add second nonsolvent to harden the microspheres
US
CR
solution containing protein added to second polymer solution polymerpolymer interaction, complex formation phase separation and microsphere precipitation Add glutaraldehyde to harden microcapsules Chondroitin-6sulfate (CS6)
MA N
solution added to polymer solution precipitation of microspheres. Emulsion stabilizers (e.g. sodium sulfate) may be added
References
Insulin, hepatitis B surface antigen (HBsAg)
21, 22
AC
Example of protein/peptide prepared
CE P
TE D
added/emulsified into continuous phase (O) to form W/O emulsion homogenization Add cross linker emulsion solidified into microspheres.
23, 24, 25
34
13
44, 45
ACCEPTED MANUSCRIPT
Solvent evaporation/extraction The solvent evaporation method is widely used to prepare microspheres for delivery of poorly
T
water soluble and hydrophobic small molecules. To prepare microspheres containing peptides
IP
and proteins oil/water (O/W), oil/oil (O/O) and water/oil/water (W/O/W) emulsification
SC R
methods are used. The biodegradable polymers polylactic acid (PLA) and poly(lactide-coglycolide) (PLGA) are most frequently the polymers of choice for encapsulation. The release kinetics of encapsulated drug is governed by the chain length and degree of crosslinking of the PLGA used. PLGA high in glycolide content undergoes more rapid degradation, thus loaded
NU
drugs are released faster, and vice versa. The solvent evaporation approach can be further
MA
divided into single emulsion and double emulsion methods.
Single emulsion method (O/O or O/W)
In this method, the active ingredient is dissolved/suspended in the organic (dispersed) phase
D
containing the polymer. The drug solution/suspension is added to the continuous phase
TE
containing one or a mixture of emulsifiers. The continuous phase can be either mineral oil for an O/O emulsion, or water for an O/W emulsion. Spans and sorbitan sesquioleate aluminum
CE P
tristearate are preferred emulsifiers for O/O emulsions, while Carbopol 951, methyl cellulose, polyvinyl alcohol (PVA) are the emulsifiers of choice for O/W emulsions. After adding the dispersed phased to the continuous phase, the organic solvent is removed by solvent The resulting
AC
evaporation or solvent extraction, after which the emulsion hardens.
microspheres are collected by centrifugation or filtration, and then freeze-dried. This method has been used to encapsulate Insulin (nanospheres), bovine serum albumin (BSA), ovalbumin (OVA) and bovine superoxide dismutase (bSOD), resulting in high encapsulation efficiency with good retention of activity.
Double emulsion method (W/O/W) In a double emulsion method, a water-in-oil emulsion (W/O) is first created from a drugcontaining aqueous phase (W) and an organic phase (O) with dissolved polymer. This emulsion is then added to a second aqueous phase containing emulsifier, to form a W/O/W double emulsion. The microspheres are formed when the organic solvent is extracted into the external aqueous phase, precipitating the polymer. Solvent removal occurs by evaporation.
14
ACCEPTED MANUSCRIPT Carbonic anhydrase and BSA were encapsulated within PLGA microspheres [17]. The protein release from the microspheres was incomplete due to protein aggregation and adsorption within the microspheres. Recombinant human growth hormone (hGH) similarly prepared had
Incompatible polymer addition or salt addition
SC R
IP
Several additional methods exist for producing microparticles:
T
continuous delivery lasting a month following an initial burst release of 20% of its payload.
Incompatible polymer addition or salt addition is another method where two chemically incompatible polymers are dissolved in a common solvent [33]. Here, a polymer is added until
NU
the phase boundary is passed, causing phase separation of the polymer rich phase to form immiscible droplets containing the drug, and coalesce to form microcapsules. Inorganic salts
MA
can be added to aqueous solution of water-soluble polymers to cause phase separation.
Ionic/Ionotropic Gelation (polyelectrolyte complexation)
D
In recent years, the method of ionic gelation (IG) for preparation of hydrophilic polymer
TE
microspheres containing proteins has drawn much interest because of its very simple and mild manufacturing process, which is helpful to retain bioactivity of loaded proteins [39, 40]. In
CE P
this method, the reversible physical cross linking by electrostatic interactions is employed for microsphere formation. Proteins are premixed with a hydrophilic polymer solution, which is then dropped into an ionic solution under constant stirring.
Bodmeier and coworkers
AC
produced tripolyphosphate (TPP)-chitosan microspheres by dropping chitosan solution containing proteins into a TPP solution; many researchers have investigated its use in pharmaceutical applications [39]. Alginate gelation can also be used to produce microspheres [41]. In this process a protein-containing solution of sodium alginate is sprayed as droplets into a cross-linking solution of divalent ions such as Ca2+, Sr2+ or Ba2+. The very mild environment provided by this process generally performs well in preserving the native activity of loaded proteins.
15
ACCEPTED MANUSCRIPT The ability of electrolytes to crosslink in the presence of counter-ions to form hydrogels has been exploited to prepare particles. Alginate, a polysaccharide composed of 1,4-linked betaD-mannuronic acid and alpha D-guluronic acid, is one of the most widely used polyanions for microencapsulation [42]. Other polyelectrolytes tested include chitosan (IG with
T
tripolyphosphate), carboxymethylcellulose (IG with aluminum), pectin (IG with calcium),
IP
gelan gum (IG with calcium), and alginate and cellulose phosphate (IG with calcium),
SC R
polyphosphazene (IG with calcium). For example, calcium alginate hydrogels have been used for delivery of vascular endothelial growth factor (VEGF), achieving an encapsulation efficiency of 30-67% and constant release for up to 14 days [43].
NU
Spray drying
Spray drying is a well-established method commonly used in the pharmaceutical industry for
MA
producing a dry powder from a liquid phase. The resulting powder can be pressed into tablets, filled into capsules, or fabricated into other dosage presentations for oral delivery. The method is illustrated in Figure 2. It consists of drying small atomized droplets of a solution of
D
dissolved or suspended solids by rapid evaporation in a stream of hot dry gas such as air or
TE
nitrogen to form microparticles typically in the size range of 1 to 100 µm. Despite the elevated temperature of the drying gas, the droplets remain colder owing to the cooling effect
CE P
of the evaporating solvent. To date, many hydrophilic polymer microparticles loaded with proteins were successfully prepared by this method, such as chitosan, mannitol, hydroxypropyl methylcellulose and carbopol microspheres [35,36]. This technique shows
AC
some important advantages over other encapsulation techniques. The preparation process is rapid, convenient and generally straightforward to scale up. Furthermore, this process is suitable for most proteins because it does not depend on their solubility parameter. The proteins can be dissolved or dispersed as solids in aqueous or organic solvents, which allows either hydrophilic or hydrophobic polymers to be used. Plasticizers can also be added to improve the sphericity of the resulting microparticles.
As an example, PLGA microparticles containing thyrotropin releasing hormone (TRH) were produced by spray drying a miscible solution of TRH and PLGA in acetonitrile and water [37]. To counteract microparticle agglomeration, mannitol solution was sprayed simultaneously as an anti-adherent. In another example, hyaluronic acid (HA) microhydrogel
16
ACCEPTED MANUSCRIPT was successfully developed using spray drying as a novel drug carrier for a controlled release
D
MA
NU
SC R
IP
T
formulation of human erythropoietin [38].
TE
Figure 2. Lab scale spray drying process in environmental enclosure under controlled conditions (2=drying gas heater, 3=spray nozzle, 4=aerosol drying chamber, 5=centrifugal separator, 6=product collection vessel, 7=fines filter, 8=vacuum pump)
CE P
Spray Freeze drying Spray Freeze drying is another method that has been evaluated to produce protein-containing particles [46]. In this process, an atomization two-fluid nozzle is used to spray the contents
drying.
AC
directly into a liquid nitrogen bath; the resulting frozen particles are subjected to freeze
Spray chilling
Spray chilling involves dispersing drug in a liquid carrier (without solvent) above its melting temperature. The microparticles are formed by spraying the drug dispersion into a cold air stream to solidify the small droplets [47].
17
ACCEPTED MANUSCRIPT 2.2
Preparation methods for oral thin films
Quick dissolving oral thin films (OTF’s) offer a remarkable array of advantages for vaccine applications including compact size and weight (~<1/100th of size & weight of typical
T
vaccine package), making it highly transportable, and simplicity of use, enhancing patient
IP
compliance [48]. Furthermore, there is no reconstitution step, which ensures dosage accuracy, and there are well-established, cost-effective manufacturing processes (e.g. Listerine® breath
SC R
freshener strips). These thin films, which rapidly (< 1 minute) dissolve in saliva once placed on the tongue, have seen a steady rise in the number of medicinal applications in recent years, expanding from a simple breath freshener to over-the-counter medicines (pain reliever,
NU
cold/flu, allergy, teeth whitening, nutraceuticals) to prescription medicines for severe pain [49], nausea [50], allergic rhinitis [51], epilepsy [52], etc. The commonality among these thin
MA
film products is that they are all small molecule drugs, which tend to be significantly more stable than biologics; therefore they are able to withstand the thin film manufacturing process, which involves relatively high heat, pressure, and in some cases organic solvent exposure.2
D
Such manufacturing process conditions are not compatible with thermally labile vaccines and
TE
proteins. Thus, this technology barrier will have to be overcome in order to expand the potential applications of OTF’s into the biologics space. To achieve an optimal technology
CE P
solution, the authors have been successful in incorporating a pharmaceutical stabilization formulation that does not require complex pharmaceutical drying processes to achieve room temperature stability and is compatible with existing thin film manufacturing processes. This process can be as simple as mixing the vaccine with the pharmaceutical stabilizers and thin
films.
AC
film formers in a ‘one-pot’ configuration, then spreading out the mixture for drying into thin
Such a technology can be simple, cost effective, and easy to implement worldwide. For vaccines, an effective OTF formulation could potentially enhance the vaccine coverage by improving patient compliance and facilitate booster immunization campaigns, which ultimately could lead to improved effectiveness of vaccines. It could also enhance utilization of current oral vaccines, which include vaccines for typhoid, cholera, rotavirus, shigellosis, and polio. 2
Some progress in the development of OTF formulations of insulin for buccal delivery has been achieved by MonoSol Rx® as well as by research groups in academia, such as Morales, McConville and coworkers (80).
18
ACCEPTED MANUSCRIPT For example, thin film formulation and manufacturing process was developed that successfully encased a thermally labile live virus vaccine with complete preservation of activity and storage stability at high temperature (45 °C). We combined our liquid vaccine stabilization technology [53] and plasticized glass stabilization technology [54-56] to
T
demonstrate an OTF formulation that successfully encased live rotavirus vaccine with
IP
complete preservation of vaccine potency and improved stability at 45 °C from several days to
SC R
several months.
This formulation technology is compatible with conventional film forming components used in a variety of OTF products and film manufacturing processes such as solvent casting [57]
NU
and film extrusion [58]. Film casting is a process used for preparation of oral thin films (e.g. over-the-counter analgesics and Triaminic® films) and adhesive patches. The formulation
MA
comprises specific GRAS (Generally Recognized as Safe) pharmaceutical excipients, plasticizers, and protein binding ligands added to stabilize a key viral protein. Furthermore, a unique thin film polymer composition has been developed that is compatible with the
D
stabilized formulation and can be subjected to a simple convective drying process to yield a
TE
robust thin film with physical properties that are indistinguishable from a number of commercial oral thin film products. The thin film manufacturing process was developed as a
CE P
‘one-pot’ formulation blending method, whereby all components are blended in a single master mix that are cast and dried into thin films using a simple solvent casting convective air drying process. Shown in Figure 3 is an example of a lab-scale process for fabricating
AC
biologics such as a vaccine into oral thin films.
19
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
D
Stresses encountered during manufacture of oral solid dosage forms and ways to improve in-process stability
TE
3.
MA
Figure 3. Basic steps in a lab-scale thin film manufacturing process and the microstructure of the resulting stabilized vaccine film formulation
During the manufacture of oral solid dosage forms proteins can be subjected to certain
CE P
stresses that threaten their stability, such as extremes in heat, shear or pH, in addition to exposure to aggressive process steps from freezing and drying. This section will review the range of stresses involved. There are already excellent reviews elsewhere in this special focus
AC
edition and in other articles and books on the general topic of the stresses associated with freezing and drying, commonly used in pharmaceutical processes such as lyophilization and spray drying, and the stabilization strategies employed. In this section, we will discuss specific stresses that may be associated with solid oral dosage form manufacturing processes.
20
ACCEPTED MANUSCRIPT In addition to thermal stress [59], proteins are prone to denaturation and aggregation by various factors in processes like the solvent evaporation/extraction methods described above, including exposure to high shear forces during processing (particularly emulsification), exposure to the large interfacial area between the emulsified oil and aqueous phases or at the
T
air-water interface of small droplets, as well as interaction with an encapsulating polymer or
IP
organic solvent. The loss of protein activity by denaturation/aggregation can be minimized by
SC R
using a number of stabilizers such as polyols, albumins and other proteins, surfactants, poloxamers, cyclodextrins, etc, depending on the stresses involved in the manufacturing processes for various oral drug delivery systems, which are further highlighted below.
NU
Organic solvents – Unless the surface of the protein has significant hydrophobicity, proteins are generally vulnerable to denaturation and activity loss in the presence of most organic
MA
solvents used to make polymeric oral delivery systems [60,61]. In general, polar organic solvents that are water-miscible are more enzyme-deactivating than water-immiscible solvents [62]. Proteins often retain the native structure in pure water-immiscible solvents and their
D
activity can be improved by adding very small quantities of water and other excipients [63].
TE
Because water-miscible solvents have a greater likelihood to strip off tightly bound water
CE P
from the protein surface, they can bring about both loss in protein structure and activity [64].
Air/water interface - The liquid and gas phase contacting surface has long been shown to be adverse microenvironment for soluble proteins [65]. The interfacial tension associated with
AC
such phase boundaries (e.g. in the spray drying process) brings about much of the shearrelated protein stress. Proteins that adsorb more readily at air/water interfaces exhibit higher shear rheological properties and are thermodynamically less stable [66]. To solve this challenge, nonionic surfactants such as Tween-20 and Pluronic F-68, added in various fold molar excess over the protein can significantly suppress protein aggregation.
21
ACCEPTED MANUSCRIPT Ice/water interface – Protein denaturation during freezing is found be casually related to surface-induced denaturation as shown in a study using low temperature rheology on highly viscous and elastic behavior of frozen protein formulations [67]. Carpenter has found that a strong correlation exists between the tendency of a protein to freeze denature and its tendency
T
to surface denature [68]. When surface-active agents such as Tween80 and/or cryo-protectant
IP
sugars or polyols are added, strong protection against freeze-induced denaturation at the ice-
SC R
water interface during freeze-drying is observed.
Drying – Unless the formulation is optimized before applying any type of drying process (e.g. freeze drying, spray drying, etc.), the drying step itself poses a stress to the biomacromolecule
NU
during the preparation of micro- or nanocapsules as a result of water removal and lack of adequate water substitution by suitable lyoprotectants [114]. For some molecules, the
MA
application of a freezing step before drying (e.g. freeze drying, spray freeze drying) introduces a combined ice/water interface and drying stresses during the process. Reviews about stability advantages of encapsulation with and without drying are inconclusive. For example, Johnson
D
et al found no significant differences in physical stability (aggregation and fragmentation),
TE
chemical stability (deamidation and oxidation) and bioactivity between freeze dried formulations of human growth hormone with and without encapsulation (biodegradable
CE P
microspheres) [115].
Mixing/homogenization – To obtain a small-size dispersion it often requires significant
drugs.
AC
mixing- or homogenization-generated shear forces, which may degrade protein and peptide For
example,
in
emulsion
preparation
of
rhEPO,
the
W/O
emulsion
homogenization/sonication step caused an aggregates increase from 4% to 15%. Here, protein stabilizers such as BSA, hydroxypropyl-β-cyclodextrin were beneficial in decreasing the aggregates [69].
22
ACCEPTED MANUSCRIPT The effect of various processing steps on protein activity was evaluated by Göpferich and coworkers [78] for solvent evaporation methods using the double emulsion technique to produce proteins encapsulated polymeric microspheres. For example, in the encapsulation of trypsin, a 30 seconds sonication (“mixing shear” effect where eddy size is comparable or
T
smaller than protein size) to prepare the primary emulsion led to 31% reduction in activity,
IP
while vortex mixing, a 3 hour solvent evaporation and post freeze drying led to negligible
SC R
reduction. The addition of stabilizers such as BSA and glycine did not improve the sonication loss, although the loss was reduced with reduced sonication time. Thus they concluded the sonication probe itself (rather than an increase in the interfacial area) provided the major source of activity loss in preparing the microspheres, and may be the result of localized heat,
NU
shear or cavitation.
Kanthamneni et al. prepared microparticles of acetalated dextran (Ac-DEX) encapsulating
MA
horseradish peroxidase (HRP) by a milder homogenization method and by a probe sonication method [79]. Particles were stored at different temperatures and storage stability was assessed at different time points for up to 90 days by measuring HRP activity. Stability of the prepared
D
particles was compared against that of lyophilized HRP. Stability in all three cases was
TE
characterized by an initial decline in HRP activity within the first month of measurement, followed by a levelling off of activity of HRP after that consistent with square root of time
CE P
degradation kinetics in amorphous formulations. Under refrigerated conditions, HRP prepared by sonication underwent more degradation as compared to HRP prepared by homogenization and free lyophilized HRP (Figure 4a). Under more stressful storage conditions (25 and 45°C),
AC
HRP prepared by homogenization was the most stable formulation whereas HRP prepared by sonication was least stable (Figure 4b, 4c). An estimate of the rate of degradation for all formulations at different temperatures using square-root-of-time kinetics (results not shown) yielded a similar outcome. Thus it is clear that sonication-induced stress during encapsulation of HRP impacted long term storage stability of HRP. Furthermore, it appears that encapsulation of HRP via a mild preparation method (homogenization) provided better protection and storage stability of HRP against stresses such as the ice-water interface encountered during lyophilization.
Figure 4: Normalized HRP activity versus time in microparticle formulations of Ac-DEX prepared by three different methods at 5°C (a), 25°C (b) and 45°C (c).[79] Figure 4a
23
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
Figure 4b
Figure 4c
24
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
MA
Yeo and coworkers suggested that the large W/O interface associated with methods such as interfacial polymerization is likely where proteins and enzymes are inactivated [26]. Additionally, the protein may sometimes participate in polymerization reactions, and thus can
TE
D
change its biological activity.
Protein stability under manufacturing conditions can be influenced by the type of solvent used
CE P
to dissolve the polymer. For example, with methylene chloride and ethyl acetate solvents, Gopferich and coworkers studied the impact of 3 different stabilizers (Pluronic F68, PEG 4600 and sodium glutamate) on tetanus toxoid emulsion stability [19]. Here, the use of
AC
stabilizers like Pluronic F68 was shown to prevent protein adsorption to hydrophobic surfaces of the polymer used to form the microspheres.
Although the protein could be successfully encapsulated into hydrophilic polymer microspheres, there still existed a few drawbacks to emulsion cross-linking. The proteins easily aggregate in the interface between the water and oil phase during the emulsifying process, which results in their activity reduction. Moreover, the intense shearing stress brought by a high speed homogenizer or a sonicator also led to protein denaturation and aggregation. In addition, cross linking reactions can also occur between proteins and/or between proteins and polymer, leading to activity reduction of proteins.
25
ACCEPTED MANUSCRIPT 4.
Formulation and stability aspects in oral solid dosage forms for biomacromolecules
A drug product for oral delivery of a biomacromolecule may have a more complex
T
formulation compared to its parenteral counterpart. Formulations for oral protein and peptide
IP
delivery will usually contain the well-known ingredients in parenteral formulations that include the active ingredient, a suitable buffer (e.g. phosphate, citrate, histidine, succinate,
SC R
etc.), a surfactant (e.g. polysorbates, pluronics) and a stabilizer (e.g. sucrose, trehalose). Theories proposed for explaining stabilization in the solid state by the so-called “stabilizers” include the glass dynamics theory as well as the water substitution theory [72]. Other
NU
ingredients in oral dosage forms, similar to those in biopharmaceutical parenteral dosage forms, may include one or more of the following; bulking agents (e.g. mannitol, glycine), salts
MA
(e.g. sodium chloride), antioxidants (e.g. methionine, GSH), stabilizing amino acids (e.g. arginine), antimicrobial preservatives (for multi-dose parenterals e.g. benzyl alcohol) and adjuvants (for vaccine formulations e.g. Alum, Freud incomplete adjuvant, etc.). Additional
D
ingredients included in formulations for oral delivery of proteins and peptides are polymers
TE
and may also include enzyme inhibitors, permeation enhancers and plasticizers. A more complex mixture of inactive ingredients will lead to greater concerns about excipient
CE P
compatibility if not properly selected, as well as more stability concerns for the additional inactive ingredients added in the formulation. Finally, preparation methods will become more complicated as demonstrated in the previous section, and therefore more concerns will emerge around manufacturing the drug product (e.g. interactions with different equipment
AC
components, adsorption, etc.). Thus a more elaborate DoE (Design of Experiment) must be included in the formulation screening and design phase. In turn, this translates into a much elaborate and longer development time frame. This section will provide a high-level overview of the additional excipients used in protein and peptide formulations for oral delivery that are uncommon to the parenteral counterparts, and to highlight degradation issues and stability concerns during formulation and/or delivery, demonstrating with a few case studies in the process.
26
ACCEPTED MANUSCRIPT Note that owing to their potential toxicity, much research has been done to avoid use of enzyme inhibitors and/or permeation enhancers, such as chemical modifications of protein and peptide drugs at the amino acid side chains or at the carbohydrate moieties to improve their resistance to protease degradation, attaching bacterial translocation sequences to shuttle
T
the protein conjugate transcytotically through the gut epithelium, etc. [3].
These molecular
IP
modification approaches have generated much excitement and are further reviewed by Mahato
SC R
and coworkers [14]. However, these technologies will not be examined in detail in the article. More focus will be on discussing permeation enhancement and enzyme inhibition from a
4.1
NU
formulation excipient/additive perspective.
Polymers
MA
The use of polymers in the manufacturing process of a solid oral dosage form and the many previously tested options available were discussed above. The context there was more focused on achieving certain drug delivery aspects and creating the desired physical macroscopic form However, polymers used in a formulation can also have a
D
of the final drug product.
TE
significant impact on stability. In many cases, polymers can be added to improve both process and storage stability through interactions with the active and in altering the The addition of polymers can inhibit
CE P
microstructure of the solids in the drug product.
crystallization during drying processes to produce the solid dosage form, which can also affect stability. In other cases, the selection of the polymer in the final formulation for reasons of manufacturability and drug delivery can have a negative impact on stability requiring
AC
adjustments to the formulation. In the case of PLGA microparticles, for example, generation of deleterious degradation products after administration can alter local pH and require buffering compounds to protect the active during delivery.
27
ACCEPTED MANUSCRIPT In a broad sense, the polymers used can be divided into either hydrophilic or hydrophobic classifications. Other classifications have addressed biodegradable attributes, as well as natural versus synthetic [113]. The following section describes different polymers that have been used in vitro or in animal studies that suggest that the polymer could be used for oral
T
peptide and/or protein delivery. Polymers have been utilized in much of the research for oral
IP
delivery to produce microparticles/nanoparticles. The latter particles can deliver small and
SC R
biomacromolecules in a targeted or localized way, can protect biomacromolecules from the harsh pH environment and enzyme effects at various regions of the GIT, deliver molecules in a sustained manner thus avoiding repeated dose administration, and are considerably the easiest presentation to be taken up by the lining of the GIT [81]. In these studies, focus has
NU
been on fabrication from different polymers, encapsulation efficiency and release profiles. Microparticulate systems include microcapsules, nanocapsules, or hydrogel microparticles.
MA
Overall, little work has been published about solid state stability of the polymers and of the biomacromolecules they carry.
group
comprises
poly(esters),
poly(cyanoacrylate),
poly(ortho
esters)
and
TE
This
D
4.1.1. Hydrophobic polymers
poly(phosphazenes). Examples of poly(esters) include Poly(lactic acid-co-glycolic acid)
CE P
(PLGA), poly(ɛ-caprolactone), poly(β-hydroxybutyric acid) and poly(β-hydroxyvaleric acid). Poly(esters) are excellent candidates for delivery of biomacromolecules in the form of microparticles/nanoparticles based on the ability of their particles to protect the encapsulated active ingredient from degradation in the GIT. Many are approved by the FDA and are
AC
considered Generally Recognized as Safe (GRAS). However, more research is needed to improve particle uptake by the GI lymphoid tissue. Poly(esters) can be used by themselves or in combination with other polymers to prepare microparticles/nanoparticles. Each class has its unique physico-chemical properties that in turn affect its rate of biodegradation to release their contents, rate of uptake at delivery site, rate of chemical degradation (i.e., solid state stability), etc. [82]. PLGAs comprise the broadest poly(ester) type polymers used in research in oral protein and peptide delivery due to desirable properties such as their GRAS status, varying physicochemical properties (e.g mechanical), good sustained release properties, biodegradability, biocompatibility and non-toxic properties. PLGAs are referred to by the percent lactic acid to glycolic acid content present. For example, a PLGA with 25% D-lactic acid, 25% L-lactic
28
ACCEPTED MANUSCRIPT acid and 50% glycolic acid is described as a 50:50 poly(D,L-lactide-co-glycolide). As % poly(glycolic acid) increases, solubility decreases and physico-chemical properties of the PLGA change. However, they have a low encapsulation efficiency and undesirable effect on pH-susceptible encapsulated bio-macromolecules upon storage under certain conditions [83,
T
82]. Recent studies have demonstrated that PLGA copolymers have an adverse effect on the
IP
stability and bioactivity of biomacromolecules due to the hydrophobicity of the polymers and
SC R
the presence of acidic degradation products. [113]. As the polymer degrades, an acidic environment is produced surrounding and within the polymer matrix [84]. As the polymer backbone undergoes hydrolysis, more carboxylic acid groups are produced in the matrix and pH of the surrounding fluid decreases resulting in biodegradation and release of the active
NU
ingredient in vivo. During this degradation process, instability of low pH-susceptible payloads may also result during delivery or storage. High levels of moisture accelerate this undesirable
MA
hydrolytic instability during storage [81]. Water-in-oil-in-water emulsion method is the predominant method used for encapsulation within microparticles. However, loading efficiency of poly(ester) microparticles remain quite low. Sufficient loadings are possible for
D
low dose drugs, but large amounts of drug are needed to get the high loadings necessary.
microparticles [82].
TE
Thus, more research is needed to improve loading efficiency of large doses of drugs within
CE P
Poly(cyanoacrylates) (PCAs) are more popular in preparing microparticles than poly(esters) due to a mild polymerization procedure leading to less activity loss during incorporation of peptides and proteins, in addition to a higher encapsulation efficiency. PCAs are also GRAS
AC
and their rate of degradation is much faster than PLGAs which does not necessitate overloading the subject with excess polymer after initial treatment [85]. PCAs can be easily formed into spheres [86] or capsules [87] to deliver biomacromolecules. Some examples of PCAs include poly(isobutyl cyanoacrylate) (PBCA) and poly(isohexyl cyanoacrylate) (PIHCA) [88].
29
ACCEPTED MANUSCRIPT Poly(phosphazenes) are biodegradable polymers that show potential for oral peptide and protein delivery [89, 90, 91] with varying degradation rates [92]. Degradation of poly(phosphazenes) is accompanied by production of ethyl alcohol, phosphate, ammonium salts and pendant groups [93]. Different pendant groups have been used as substituents, which
T
in turn controlled polymer properties, such as amino acid [94], ethylamino [95], imidazolyl,
IP
oligopeptide, amino acid exters, and depsipeptide groups. When the pendant group is an
SC R
amino acid, all degradation products are natural products normally found in the body. Poly(ortho esters) are a group of hydrophobic polymers with limited use in oral peptide and protein delivery. There have been trials to load rods made from the polymers with
NU
recombinant human growth hormone and bovine serum albumin using polymer-protein mixture extrusion at a temperature range of 50-70 °C. However, the large particle size of the
MA
rods has limited the polymer use in oral peptide and protein delivery [96]. 4.1.2. Hydrophilic polymers
This group comprises poly(alkyl methacrylates), poly(methacrylates), poly(acrylates),
D
alginates, chitosan, polyphosphazene hydrogels and poly(ethylene glycols).
TE
Poly(alkyl methacrylates) include Eudragits (e.g. S100, L100), properties of which vary depending on their degree of modifications. For example, dissolution rate of Eudragits and the
CE P
pH at which they dissolve will depend on modification of carboxylic acid groups of methacrylic acid. These properties make Eudragits versatile polymers to prepare microparticles to encapsulate and deliver proteins in the GIT [97]. Entrapment efficiency is
AC
reported to be as high as 78%. Eudragit microparticles have low solubility in the acidic stomach environment and therefore have been shown to be efficient in delivering sensitive proteins such as insulin in the intestines in oral force fed rats.
30
ACCEPTED MANUSCRIPT Poly(methacrylates) and poly(acrylates) are used to form hydrogel particles that swell isotropically at different rates upon exposure to fluids, thus delivering entrapped proteins and peptides in a swelling rate- and mesh size-dependent manner. Poly(methacrylates) and poly(acrylates) also possess specific and non-specific bioadhesive properties [98, 99] to the GI
T
walls. They have been of interest for colon delivery applications since colon specific enzymes,
IP
e.g. azoreductase, act on polymer cross-linkers (such as azo linkers) resulting in degradation
SC R
of the polymer in the colon and thereby releasing the entrapped biomacromolecules, though addition of penetration enhancers is also necessary due to low absorption potential from the colon [100, 101, 102]. Not much is reported in terms of protein/peptide stability in these
NU
types of matrices.
Polymers derived from naturally occurring resources and considered GRAS include alginate and chitosan. Alginate is derived from seaweed whereas chitosan is derived from chitin.
MA
Alginate is a water-soluble physically cross-linked polymer by means of monovalent or divalent cations. Chitosan, on the other hand, is not readily water soluble but can be solubilized. Alginate microparticles with high encapsulation efficiency for biomacromolecules
D
can be formed under mild conditions, and particle size can be easily varied during
TE
formulation. Alginate and chitosan have been used in combination to form complexes to strengthen the resultant microparticles [103]. Alginate microparticles have been produced
CE P
using atomization, emulsification and coacervation [104, 105, 106]. Chitosan microparticles have been produced by emulsion and phase separation methods. Alginate polymers can be modified to impart muco-adhesive abilities and to increase encapsulation efficiency. Chitosan
AC
by itself is both bioadhesive and has chelating properties. Chitosan is reported to be conjugated to antipain, chymostatin, elastanil, ethylene diamine triacetate (EDTA) and others [107].
Polyphosphazene hydrogels have been used to incorporate biomacromolecules in hydrogel microparticles from which release is by diffusion. A coating can be applied to the particles to prevent or control the initial release of encapsulated molecules, as was demonstrated using bovine serum albumin [93]. 4.2. Permeation/absorption enhancers Owing to the large molecular weight of proteins, transcellular or paracellular protein and peptide transport is difficult to achieve. Unless the action is intended to be localized, permeation enhancers are often included in drug carriers to facilitate systemic delivery of
31
ACCEPTED MANUSCRIPT orally administered biopharmaceuticals. . Permeation enhancers can include surfactants, bile salts, Ca2+ chelating agents, fatty acids, medium chain glycerides, acyl carnitine, alkanoyl cholines, chitosans, and phospholipids [9]. The postulated effects of these absorption enhancers include modification of tight junction
T
permeability (chelating calcium ions can disrupt the junction proteins), enhancement in
IP
membrane fluidity, and reduction in mucus viscosity. The use of Ca2+ chelating agents as
SC R
permeation enhancers is exemplified in the case of oral delivery of human calcitonin [14]. Here, several excipients were evaluated with some success, including sodium myristate, sodium taurodeoxycholate, and sodium deoxycholate.
Other low molecular weight
compounds termed “delivery agents” or “carriers” are used that interact weakly and reversibly
NU
with the co-administered biomacromolecules (e.g. recombinant human growth hormone, insulin, heparin, interferon-alpha, and salmon calcitonin).
The carriers are believed to
MA
temporarily expose the hydrophobic side chains (i.e. cause temporary partial protein unfolding), leading to a transient increase in log P values with subsequent transcellular penetration
[108].
Examples
of
these
carriers
include
N-[8-(2-
D
hydroxybenzoyl)aminocaprylate] (SNAC) (Eligen®-based Technology) and N-acylated,
TE
alpha-amino acids [14, 116]. In some cases, such permeation enhancement techniques have
CE P
improved protein stability in oral hard gelatin capsule formulations of insulin [117, 118].
4.3. Enzyme inhibitors
Enzyme inhibitors are included in oral biologic drugs to protect against proteolytic
AC
inactivation in the GI tract. Enzyme inhibitors such as aminopeptidase inhibitors (e.g. puromycin, aprotinin, bestatin, soybean trypsin inhibitor, and amastatin), sodium glycholate, camostat mesilate, 4-(4-isopropylpiperadinocarbonyl)phenyl 1,2,3,4-tetrahydro-1-naphthoate methanesulphonate ( FK-448), camostatmesilate, , and ovalbumin have all shown to improve the stability of orally administered insulin in either man and/or dogs [73,74, 116]. Enzyme inhibitors act on different moieties in the biomacromolecule. For example, proteases act on protease-vulnerable sequences of biologic agents [74]. Toxicity challenges may arise associated with the use of some enzyme inhibitors (especially protease inhibitors), particularly in chronic drug therapies, where repeated administration of the inhibitor could lead to alteration in metabolic pattern and food digestion changes.
32
ACCEPTED MANUSCRIPT In some cases, penetration enhancers can also act as enzyme inhibitors such as sodium glycocholate, which protects insulin from proteolysis [9]. Formulation components that modify local pH have been known to inhibit enzymatic activity. Some polymers inhibit proteolytic enzymes or can be used to augment the activity of
T
proteolytic inhibitors. For example, polyacrylate and polymethacrylate act as inhibitors of
IP
trypsin, chymotrypsin, carboxypepsidase A and elastase. Carboxymethylcellulose inhibits
SC R
elastase and pepsin. Chitosan inhibits trypsin, carboxypepsidase A and aminopepsidase N. The use of small molecule protease inhibitors with peptides and proteins can cause systemic side effects [109, 110, 111]. Protease-inhibiting polymers and polymer-inhibitor conjugates
NU
are now widely investigated for their ability to protect biomacromolecules from proteolysis. These molecules are effective in the immediate vicinity of the delivery device. ChitosanEDTA shows an inhibitory effect towards Ca2+-dependent proteases [112]. When
MA
chitosan/EDTA/BBI conjugate was included in a formulation with insulin, more than half of the insulin activity was maintained when administered orally versus <10% of insulin activity
D
without the conjugate.
This approach has not been successful with all polymers. Poly(ethylene glycol)s (PEGs) are
TE
widely used as “conjugating” polymers to different proteins. As a result of steric hindrance
CE P
resulting from conjugation, proteolysis is minimized. One may think that chances for absorption increase. However, the high molecular weight complex will have difficulty in crossing the GI wall. Additionally, degradation of the conjugated polymer is still possible by alternate proteolytic mechanisms resulting in exposure of the protein/peptide to the acidic
AC
environment of the GIT. Thus use of PEGs is one of the least successfully investigated strategies for oral delivery of biomacromolecules.
4.4. Other formulation components Enteric coating to prevent breakdown of the dosage presentation in the acidic compartment of the stomach has been applied with good success for a variety of tablet and capsule based oral delivery systems [75]. Common enteric coatings that have been used include methyl acrylatemethacrylic acid copolymers, sodium alginate, cellulose acetate succinate, polyvinyl acetate phthalate (PVAP), hydroxy propyl methyl cellulose phthalate, Shellac, cellulose acetate trimellitate, etc. Drugs that can upset or irritate the stomach, such as aspirin, are often coated with some of the excipients above to permit dissolution only in the small intestine.
33
ACCEPTED MANUSCRIPT Plasticizers (e.g. glycerol, sorbitol, DMSO, etc.) are another group of excipients that can be added to special oral dosage forms. For example, they have been included as formulation components in oral thin films to improve tensile strength, improve dissolution time, or change water vapor permeability [70]. Additionally, there is increasing evidence of their role in
T
protein stabilization. Mechanisms proposed suggest stabilization by plasticization of the
IP
amorphous glass to dampen molecular motions (also called ‘fast dynamics’) during thermal
AC
CE P
TE
D
MA
NU
SC R
exposure [71, 72].
34
ACCEPTED MANUSCRIPT 5. 5.1
Stability post-manufacturing Storage stability
Following manufacture, the drug delivery systems is typically stored for some time period prior to administration. While the storage stability of proteins in liquid or lyophilized
T
formulations have been extensively described in the literature, significantly less information is
IP
available on the storage stability of proteins within biodegradable drug delivery systems,
SC R
especially oral delivery systems.
In the authors’ own development of oral thin film (OTF) delivery systems, the storage stability of the drug product was often sensitive to the selection of water-soluble polymer, in
NU
addition to the nature of the protein dispersion in the OTF. For example, in the production of OTF’s containing an IgG monoclonal antibody (mAb), two approaches were taken to disperse
MA
the protein in polymer: Approach 1 involved first spray-drying the mAb (formulated in a sugar matrix of sucrose or trehalose), followed by dispersion of the resulting powder in an organic solution of polymer, before casting and drying the film; Approach 2 was performed
D
by simply mixing formulated aqueous solutions of mAb and polymer, followed by film
TE
casting and drying (See Figure 5). It was found that although the spray-dried IgG mAb itself was stable for over 21 weeks at 37ºC in both Trehalose and Sucrose/Arginine formulations,
CE P
the storage stability of the antibody in the OTF was still dependent on the interaction of the powder with the polymer in the film. A PVA(polyvinyl alcohol)-based film using Approach 2 provided superior storage stability with regard to maintaining purity and antigen recognition
AC
when compared to either an HPC (hydroxypropyl cellulose)-based film using Approach 1 or PVP (polyvinylpyrrolidone)-based film using Approach 2, indicating the existence of stability-impacting polymer-protein interactions. For the HPC-based film, the spray-dried mAb was formulated at 9.8% in sucrose/arginine and was suspended in a polymer solution of isopropanol prior to casting and drying; earlier studies demonstrated the stability of this spraydried formulation in isopropanol. Here, antigen recognition was determined using an antigen binding ELISA assay, a stability indicating assay which determined the functional binding of the mAb (anti-HIV GP60 IgG1) to the target (HIV GP60 protein).
35
SC R
IP
T
ACCEPTED MANUSCRIPT
NU
Figure 5. Alternative approaches to fabrication of OTF’s for mAb delivery. Approach 1 produces a film as a two-phase dispersion; Approach 2 produces a single phase clear film.
MA
Using the aqueous ‘one-pot’ process method described in Section 2.2 (i.e. Approach 2) and screening unique combinations of pharmaceutical stabilizers and plasticizers, the stability of rotavirus vaccine in OTF formulations was substantially improved. This screening process
D
resulted in a lead formulation that was able stabilize three different rotavirus vaccine
TE
serotypes (G1, G2 and G3) at 45 °C for over eight weeks. Further evaluation of formulation and process variables identified plasticizer level and residual moisture content as critical
CE P
factors for controlling vaccine stability in OTF formulations. By controlling these parameters in particular, the low frequency molecular dynamics within the film can be reduced, and thus
AC
decrease the rate of damage to the virus.
36
ACCEPTED MANUSCRIPT 5.2
Stability after administration
Many factors associated with the microenvironment of the polymer encasing the proteins can influence post-administration stability, including monomer reactive sidechains, concentration, pH, viscosity, surface tension, etc. It is useful to understand both the microenvironment of the
T
polymer matrix that the protein is exposed to and the release or erosion mechanism in order to
IP
effectively design the polymer system. Release from non-biodegradable polymer systems is
SC R
typically a diffusion controlled mechanism. However, release from biodegradable polymers is strongly dependent on polymer erosion, which is a more complex process that is typically determined by the specific polymer, its degradation chemistry (type of bonds or sidechains), and environmental parameters (diffusivity of water, other reactants, pH of medium, Gopferich
and
coworkers,
using
NU
crystallinity).
a
system
of
poly(1,3-bis-(p-
carboxyphenoxy)propane) – sebacic acid as an example, showed that the pH in the vicinity of
MA
the polymer surface is determined by the pH of the released mononers, rather than by the pH of the buffer medium. Thus pH sensitive drugs would need to be better protected in such systems after administration when the encapsulating polymer undergoes degradation.
D
Upon oral administration, hydration of the protein trapped in the polymeric system can be
TE
initiated over prolonged periods of time. In such an environment, the proteins are becoming more susceptible to denaturation and aggregation. This is especially a challenge in polymeric
CE P
systems where the monomer breakdown products or byproducts may be causing protein denaturing due to acidic sidechains or reactive groups such as amines, carboxyls, etc. Unwanted covalent reactions between the polymer scaffold and the proteins can lead to
AC
degradation, irreversible protein adsorption to the polymer matrix, aggregation, and denaturation. Case studies of protein adsorption to polymeric implants have been documented [76]. In one example, human serum albumin was observed to undergo a multilayer adsorption to PLA nanospheres, leading to irreversible adsorption to the polymer. On the other hand, Gopferich and coworkers showed that trypsin and heparinase encapsulation in poly(anhydride) microspheres could stabilize protein activity for 1-2 days at biologically relevant conditions: phosphate buffer at pH 7.4 and 37ºC, which was a significant improvement over the free protein under those same conditions [78].
37
ACCEPTED MANUSCRIPT 6.
Summary and future prospects and trends
In spite of the major hurdles in oral delivery of proteins and peptides (in both liquid and solid dosage forms), oral delivery remains the major route of interest (and the holy grail of macromolecule delivery) in developing biologicals after parenterals. Because much of these
T
preparations have not established potential oral bioavailability, work has been halted after
IP
animal testing. Some work has shown promising results with a few molecules, most notably
SC R
with insulin. However, there are many obstacles in oral delivery of relatively smaller biological molecules such as insulin and even greater challenges remain in developing more complex and larger molecules. For future commercial viability of biomacromolecules that have shown clinical potential, one needs to consider that formulation and process of these
NU
preparations are more complex than their traditional parenteral counterparts. Thus, there are many conditions that need to be satisfied beyond oral bioavailability and toxicity. For
MA
example, stability aspects (in process, storage stability and stability during handling and administration), formulation and process development strategies and timelines, scalability, manufacturing costs, etc. One also needs to consider developing new biomaterials that can
D
effectively protect and deliver protein and peptide drugs. Finally, one needs to consider the
TE
upcoming and future trend of personalized delivery systems capable to target site-specific
CE P
receptors that will significantly impact drug administration.
We have reviewed a number of factors impacting the successful development of an oral delivery system for biologic drugs: preparation methods and the stresses involved as well as
AC
stability considerations and formulation strategies. The manufacturing process and the associated stresses to the API are reasonably well known and are points to consider in developing stabilization strategies. Considerations should be given in formulation selection to balance between functionality, interactions between excipients, interaction of excipients with biomolecule (active ingredient), as well as stability during manufacture, storage and administration.
38
ACCEPTED MANUSCRIPT While the biological barriers along the GI tracts remain a formidable challenge to successful oral delivery of proteins and peptides, the fact that oral delivery formats are more appealing in terms of convenience and patient preference will continue to drive further research and development. Although there may remain several significant challenges associated with
T
maintaining biostability during manufacturing, storage, and transit across the GI tract, recent
IP
advances in materials, manufacturing methods and formulations are beginning to improve the
AC
CE P
TE
D
MA
NU
SC R
prospects of successful oral delivery systems for peptides and proteins.
39
ACCEPTED MANUSCRIPT References
T
1. U.S. Food and Drug Administration. “2012 Biological License Application Approvals.” 9 January 2013. Available at www.fda.gov/ BiologicsBloodVaccines/DevelopmentApprovalProcess/BiologicalApprovalsbyYear /ucm289008.htm
SC R
IP
2. Miller S, Reyna J, Ng S, Zuckermann R, Kerr J, Moos W. (2004) Comparison of the proteolytic susceptibilities of homologous l-amino acid, d-amino acid, and Nsubstituted glycine peptide and peptoid oligomers. Drug Dev. Res. 35(1):20–32. 3. Mrsny, R. J., Daugherty, A. L., McKee, M. L. and Fitzgerald, D. J. (2002) Bacterial toxins as tools for mucosal vaccination. Drug Discovery Today, 7 (4), pp. 247-258.
NU
4. Taslim A. Al-Hilal, Farzana Alam, Youngro Byun. (2013) Oral drug delivery systems using chemical conjugates or physical complexes. Advanced Drug Delivery Reviews 65: 845-64.
MA
5. M. Boegh, C. Foged, A. Müllertz, H. Mørck Nielsen (2013) Mucosal drug delivery: barriers, in vitro models and formulation strategies, J. Drug Del. Sci. Tech., 23 (4) 383-391.
D
6. Jani, P, Halbert, G. W.,Langridge, J. and Florence, A. T. (1989) The Uptake and Translocation of Latex Nanospheres and Microspheres after Oral Administration to Rats. Journal of Pharmacy and Pharmacology, 41:809-812.
TE
7. Jani, P, Halbert, G. W.,Langridge, J. and Florence, A. T. (1990) Nanoparticle Uptake by the rat gastrointestinal mucosa: Quantification and particle size dependency. Journal of Pharmacy and Pharmacology, 42:821-826.
CE P
8. Des Rieux A, Pourcelle V, Cani PD, Marchand-Brynaert J, Preat V. (2013) Targeted nanoparticles with novel non-peptidic ligands for oral delivery, Adv Drug Deliv Rev 65 (6) 833-44. 9. P. Jani, P. Manseta, S. Patel (2012). Pharmaceutical Approaches Related To Systemic Delivery Of Protein And Peptide Drugs: An Overview. Inter. J. Pharm. Sci. Rev. and Res., 12(1) Article-007.
AC
7.
10. Lowman AM, Morishita M, Kajita M, Nagai T, Peppas NA. (1999) Oral delivery of insulin using pH-responsive complexation gels. J Pharm Sci. 88:933–937. 11. Yun Y, Cho YW, Park K. (2013) Nanoparticles for oral delivery: targeted nanoparticles with peptidic ligands for oral protein delivery. Adv Drug Deliv Rev 65 (6) 822-32. 12. Ensign LM, Cone R, Hanes J. (2012) Oral drug delivery with polymeric nanoparticles: the gastrointestinal mucus barriers, Adv Drug Deliv Rev. 64 (6) 557-570. 13. Sosnik A, das Neves J, Sarmento B. (2014) Mucoadhesive polymers in the design of nano-drug delivery systems for administration by non-parenteral routes: A review. Prog Polym Sci, 39 (12) 2030-75. 14. Mahato RI, Narang AS, Thoma L, Miller DD. (2003) Emerging trends in oral delivery of peptide and protein drugs. Crit Rev Ther Drug Carrier Syst. 20:153–214.
40
ACCEPTED MANUSCRIPT 15. F. Veuillez, Y.N. Kalia, Y. Jacques, J. Deshusses, P. Buri, (2001). Factors and strategies for improving buccal absorption of peptides. Eur. J. Pharmaceutics and Biopharmaceutics. 51(2):93-109. 16. Chourasia MK, Jain SK. (2003) Pharmaceutical approaches to colon targeted drug delivery systems. J Pharm Sci. 6 (1) 33-66.
IP
T
17. Crotts G, Park TG. (1998) Protein delivery from poly(lactic-co-glycolic acid) biodegradable microspheres: release kinetics and stability issues. J Microencapsul. 15(6):699-713.
SC R
18. Johnson, O. L., W. Jaworowicz, J. L. Cleland, L. Bailey, M. Charnis, E. Duenas, C. Wu, D. Shepard, S. Magil, T. Last, A. J. S. Jones, and S. D. Putney (1997) The stabilization and encapsulation of human growth hormone into biodegradable microspheres. Pharm. Res. 14: 730-735.
NU
19. A. Gopferich, M.J. Alonso, and R. Langer. (1994) Development and characterization of microencapsulated microspheres. Pharm. Res., 11:1568–1574.
MA
20. Kissel T. (2002) Tetanus toxoid microspheres consisting of biodegradable poly(lactide-co-glycolide)- and ABA-triblock-copolymers: immune response in mice. Int J Pharm 234: 75–90. 21. Jose S, Fangueiro JF, Smitha J, Cinu TA, Chacko AJ, Premaletha K, Souto EB. (2013) Predictive modeling of insulin release profile from cross-linked chitosan microspheres. Eur J Med Chem. Feb; 60: 249-53.
TE
D
22. Sivakumar S.M., Sukumaran N., Nirmala L., Swarnalakshmi R., Anilbabu B., Siva L., Anbu J., Shanmugarajan T.S., Ravichandran V. (2010) Immunopotentiation of hepatitis B vaccine using biodegradable polymers as adjuvant. J. Microbiol. Immunol. Infect. 43(4): 265–270.
CE P
23. Nihant, N., S. Stassen, C. Grandfils, R. Jerome, and P. Teyssie (1994) Microencapsulation by coacervation of PLGA: III. Characterization of the final microspheres. Polym. Int. 34: 289-299.
AC
24. Nihant N, Grandfils C, Jérôme R, et al. Microencapsulation by coacervation of poly(lactide-co-glycolide) IV. Effect of the processing parameters on coacervation and encapsulation. J Control Release 1995; 35: 117-25. 25. Ozbas-Turan S, Akbuga J, and Aral C. Controlled release of interleukin-2 from chitosan microspheres. J Pharm Sci 2002; 91: 1245-51. 26. Yeo Y, Baek N, and Park K, (2001) Microencapsulation Methods for Delivery of Protein Drugs, Biotechnol. Bioprocess Eng. 6: 213-230. 27. Wang L, Yang T, Ma G, (2013) Microspheres and microcapsules for protein drug delivery. Chapter in Microspheres and Microcapsules in Biotechnology: Design, Preparation and Applications, Editors Ma G and Su Z, Pan Stanford Publishing, Pte. Ltd. 28. Pettit, D. K., J. R. Lawter, W. J. Huang, S. C. Pankey, N. S. Nightlinger, D. H. Lynch, J. A. Schuh, P. J. Morrissey, and W. R. Gombotz (1997) Characterization of poly(glycolide-co-D,L-lactide)/poly(D,L-lactide) microspheres for controlled release of GM-CSF. Pharm. Res. 14: 1422-1430.
41
ACCEPTED MANUSCRIPT 29. Li, J. K., N. Wang, and X. S. Wu (1997) A novel biodegradable system based on gelatin nanoparticles and poly(lactic-co-glycolic acid) microspheres for protein and peptide drug delivery. J. Pharm. Sci. 86: 891-895. 30. Wang, N. and X. S. Wu (1998) A novel approach to stabilization of protein drugs in PLGA microspheres using agarose hydrogel. Int. J. Pharm.166: 1-14.
IP
T
31. Wang, N., X. S. Wu, and J. K. Li (1999) A heterogeneously structured composite based on poly(lactic-co-glycolic acid) microspheres and poly(vinyl alcohol) hydrogel nanoparticles for long-term protein drug delivery. Pharm. Res. 16: 14301435.
SC R
32. Bakan, J. A. (1986) Microencapsulation. pp. 412-429. In: L. Lachman H. A. Lieberman, and J. L. Kanig (eds.). The Theory and Practice of Industrial Pharmacy. Lea & Febiger, Philadelphia, USA.
NU
33. Bakan, J. A. (1986) Microencapsulation. pp. 412-429. In: L. Lachman H. A. Lieberman, and J. L. Kanig (eds.). The Theory and Practice of Industrial Pharmacy. Lea & Febiger, Philadelphia, USA.
MA
34. Brown, K. E., K. Leong, C. Huang, R. Dalal, G. D. Green, H. B. Haimes, P. A. Jimenez, and J. Bathon (1998) Gelatin/chondroitin 6-sulfate microspheres for the delivery of therapeutic proteins to the joint. Arthritis Rheum. 41: 2185-2195. 35. Bowey K, Neufeld RJ. (2010) Systemic and mucosal delivery of drugs within polymeric microparticles produced by spray drying. BioDrugs. 24(6): 359-77
TE
D
36. Wang Q, Fu A, Li H, Liu J, Guo P, Zhao XS, Xia LH (2014) Preparation of cellulose based microspheres by combining spray coagulating with spray drying. Carbohydr Polym. 13(111):393-9
CE P
37. Takada S, Uda Y, Toguchi H, Ogawa Y (1995) Application of a spray drying technique in the production of TRH-containing injectable sustained-release microparticles of biodegradable polymers. PDA J Pharm Sci Technol. 49(4):180-4
AC
38. Hahn SK, Kim JS, Shimobouji T. (2007) Injectable hyaluronic acid microhydrogels for controlled release formulation of erythropoietin. J Biomed Mater Res. 80(4): 916-24 39. Bodmeier R, Chen HG, Paeratakul O. (1989) A novel approach to the oral delivery of micro- or nanoparticles. Pharm Res. 6(5): 413-7. 40. Sajeesh S, Sharma CP. (2011) Mucoadhesive hydrogel microparticles based on poly (methacrylic acid-vinyl pyrrolidone)-chitosan for oral drug delivery. Drug Deliv. 18(4): 227-35 41. Gonjari ID, Hosmani AH, Karmarkar AB, Kadam SB, Godage AS, Khade TS. (2009) Microspheres of tramadol hydrochloride compressed along with a loading dose: A modified approach for sustaining release. Drug Discov Ther. 3(4): 176-80. 42. King A, Strand B, Rokstad AM, Kulseng B, Andersson A, Skjåk-Braek G, Sandler S. (2003) Improvement of the biocompatibility of alginate/poly-L-lysine/alginate microcapsules by the use of epimerized alginate as a coating. J Biomed Mater Res A. 64(3): 533-9
42
ACCEPTED MANUSCRIPT 43. Hunt NC, Shelton RM, Henderson DJ, Grover LM. (2013) Calcium-alginate hydrogelencapsulated fibroblasts provide sustained release of vascular endothelial growth factor. Tissue Eng Part A. 19(7-8): 905-14. 44. Couvreur P, Barratt G, Fattal E, Legrand P, Vauthier C. (2002) Nanocapsule technology: a review. Crit Rev Ther Drug Carrier Syst. 19(2): 99-134.
IP
T
45. Aboubakar M, Puisieux F, Couvreur P, Vauthier C. (1999) Physico-chemical characterization of insulin-loaded poly(isobutylcyanoacrylate) nanocapsules obtained by interfacial polymerization. Int J Pharm. 183(1): 63-6
SC R
46. Purvis T, Vaughn JM, Rogers TL, Chen X, Overhoff KA, Sinswat P, Hu J, McConville JT, Johnston KP, Williams RO. (2006) Cryogenic liquids, nanoparticles, and microencapsulation. Int J Pharm. Oct 31; 324(1): 43-50. 47. Gibbs BF, Kermasha S, Alli I, Mulligan CN. (1999) Encapsulation in the food industry: a review. Int J Food Sci Nutr. 50(3): 213-24.
NU
48. Chaturvedi A, Srivastava P, Yadav S, Bansal M, Garg G, Sharma PK. (2011). Fast dissolving films: a review. Curr Drug Deliv. 8(4): 373-80
MA
49. Delgado-Guay MO. 2010. Efficacy and safety of fentanyl buccal for cancer pain management by administration through a soluble film: an update. Cancer Manag Res. 2: 303-6.
TE
D
50. Nishigaki M, Kawahara K, Nawa M, Futamura M, Nishimura M, Matsuura K, Kitaichi K, Kawaguchi Y, Tsukioka T, Yoshida K, Itoh Y. 2012. Development of fast dissolving oral film containing dexamethasone as an antiemetic medication: clinical usefulness. Int J Pharm. 424(1-2): 12-7.
CE P
51. Patel JG, Modi AD. 2012. Formulation, optimization and evaluation of levocetirizine dihyrochloride oral thin strip. J Pharm Bioallied Sci. 4(Suppl 1): S35-6. 52. SK Yellanki, S Jagtap, and R Masareddy. 2011. Dissofilm: A Novel Approach for Delivery of Phenobarbital; Design and Characterization. J Young Pharm. 3(3): 181– 188.
AC
53. Truong-Le, V. et al. Formulations for preservation of rotavirus. US Patent # 8,241,886 54. Ohtake S, Martin R, Saxena A, Pham B, Chiueh G, Osorio M, Kopecko D, Xu D, Lechuga-Ballesteros D, Truong-Le V. Room temperature stabilization of oral, live attenuated Salmonella enterica serovar Typhi-vectored vaccines. Vaccine. 2011 Mar 24; 29(15): 2761-71. 55. Ohtake S, Martin RA, Saxena A, Lechuga-Ballesteros D, Santiago AE, Barry EM, Truong-Le V. 2011. Formulation and stabilization of Francisella tularensis live vaccine strain. J Pharm Sci. 100(8): 3076-87. 56. Cicerone, T.C., et al. Substantially improved stability of biological agents in dried form: The role of glassy dynamics in preservation of biopharmaceuticals. 2003. Bioprocess Int. 1: 2-9. 57. Mokarian-Tabari P, Geoghegan M, Howse JR, Heriot SY, Thompson RL, Jones RA. 2010. Quantitative evaluation of evaporation rate during spin-coating of polymer blend films: Control of film structure through defined-atmosphere solvent-casting. Eur Phys J E Soft Matter. 33(4): 283-9.
43
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
58. Cross GL, O'Connell BS, Pethica JB, Rowland H, King WP. (2008) Variable temperature thin film indentation with a flat punch. Rev Sci Instrum. 79(1): 013904. 59. Bischof JC, He X. (2005) Thermal stability of proteins. Ann N Y Acad Sci. 1066: 1233. 60. Klibanov AM. (1997) Why are enzymes less active in organic solvents than in water? Trends Biotechnol. 15: 97–101. 61. Kamal MZ, Yedavalli P, Deshmukh MV, Rao NM. (2013) Lipase in aqueous-polar organic solvents: activity, structure, and stability. Protein Sci. 7: 904-15. 62. Wangikar PP, Michels PC, Clark DS, Dordick JS. (1997) Structure and function of subtilisin BPN' solubilized in organic solvents. J Am Chem Soc. 119: 70–76. 63. Debulis K, Klibanov AM. (1993) Dramatic enhancement of enzymatic activity in organic solvents by lyoprotectants. Biotechnol Bioeng. 41: 566–571. 64. Gorman LA, Dordick JS. (1992) Organic solvents strip water off enzymes. Biotechnol Bioeng. 39: 392–397. 65. Wiesbauer J, Prassl R, Nidetzky B (2013) Renewal of the air-water interface as a critical system parameter of protein stability: aggregation of the human growth hormone and its prevention by surface-active compounds. Langmuir 29(49): 1524050. 66. Mitropoulos V, Mütze A, Fischer P. (2014) Mechanical properties of protein adsorption layers at the air/water and oil/water interface: a comparison in light of the thermodynamic stability of proteins. Adv Colloid Interface Sci. 206: 195-206. 67. Gu JH, Beekman A, Wu T, Piedmonte DM, Baker P, Eschenberg M, Hale M, Goldenberg M. (2013). Beyond glass transitions: studying the highly viscous and elastic behavior of frozen protein formulations using low temperature rheology and its potential implications on protein stability. Pharm Res. 30(2): 387-401. 68. Chang BS, Kendrick BS, Carpenter JF. (1996) Surface-induced denaturation of proteins during freezing and its inhibition by surfactants. J Pharm Sci. 85(12): 132530. 69. He J, Feng M, Zhou X, Ma S, Jiang Y, Wang Y, Zhang H. (2011) Stabilization and encapsulation of recombinant human erythropoietin into PLGA microspheres using human serum albumin as a stabilizer. Int J Pharm. 416(1): 69-76. 70. Leerahawong A1, Tanaka M, Okazaki E, Osako K (2012) Stability of the physical properties of plasticized edible films from squid (Todarodes pacificus) mantle muscle during storage. J Food Sci. 77(6): E159-65. 71. Wang B, Tchessalov S, Cicerone MT, Warne NW, Pikal MJ. (2009) Impact of sucrose level on storage stability of proteins in freeze-dried solids: II. Correlation of aggregation rate with protein structure and molecular mobility. J Pharm Sci. 98(9): 3145-66. 72. Pikal MJ, Rigsbee D, Roy ML, Galreath D, Kovach KJ, Wang B, Carpenter JF, Cicerone MT. (2008) Solid state chemistry of proteins: II. The correlation of storage stability of freeze-dried human growth hormone (hGH) with structure and dynamics in the glassy solid. J Pharm Sci. 97(12): 5106-21.
44
ACCEPTED MANUSCRIPT
TE
D
MA
NU
SC R
IP
T
73. Yamamoto A. (2001). Improvement of transmucosal absorption of biologically active peptide drugs. Yakugaku Zasshi. 2001 Dec;121(12):929-48. 74. Ueno Y, Ohmi T, Yamamoto M, Kato N, Moriguchi Y, Kojima M, Shimozono R, Suzuki S, Matsuura T, Eda H. J (2007) Orally-administered caspase inhibitor PF03491390 is retained in the liver for prolonged periods with low systemic exposure, exerting a hepatoprotective effect against alpha-fas-induced liver injury in a mouse model. Pharmacol Sci. 105(2): 201-5. 75. Felton LA, Porter SC. (2013). An update on pharmaceutical film coating for drug delivery. Expert Opin Drug Deliv. 10(4): 421-35. 76. Cai QX, Zhu KJ, Chen D, Gao LP (2003) Synthesis, characterization and in vitro release of 5-aminosalicylic acid and 5-acetyl aminosalicylic acid of polyanhydride-P(CBFAS). Eur J Pharm Biopharm. 55(2): 203-8. 77. Mitragotri S, Burke PA, Langer R. Overcoming the challenges in administering biopharmaceuticals: formulation and delivery strategies. Nature Reviews Drug Discovery 2014, 13: 655–672. 78. Göpferich A, Gref R, Minamitake Y, Shieh L, Alonso MJ, Tabata Y, Langer R. (1994) Drug Delivery from Bioerodible Polymers: Systemic and Intravenous Administration. ACS Symposium Series on Formulation and Delivery of Proteins and Peptides (Cleland JL editor). Vol. 567, Chapter 15, 241-277. 79. Kanthamneni N, Sharma S, Meenach S, Billet B, Zhao J, Bachelder E, Ainslie K. Enhanced stability of horseradish peroxidase encapsulated in acetalated dextran microparticles stored outside cold chain conditions. Int. J. Pharmaceut. 2012; 431: 101-110.
AC
CE P
80. Morales JO, Huang S, Williams RO, McConville JT. (2014) Films loaded with insulin-coated nanoparticles (ICNP) as potential platforms for peptide buccal delivery. Colloids and Surfaces B: Biointerfaces 122: 38-45. 81. Fonte P, Soares S, Costa A, Andrade J, Seabra V, Reis S, Sarmento B. Effect of cryoprotectants on the porosity and stability of insulin loaded PLGA nanoparticles after freeze-drying. Biomatter 2012; 2:4, 329-339. 82. Gemeinhart R. Polymeric systems for oral protein and peptide delivery in: Pharmaceutical biotechnology, second edition. Editor: Groves M. Publisher: CRC Press, Boca Raton, FL. 2006; 283-306. 83. Danhier F, Ansorena E, Silva JM, Coco R, Le Breton A, Preat V. PLGA-based nanoparticles: an overview of biomedical applications. J Control Release 2012; 161: 505-522. 84. Lu L, Peter S, Lyman M, Lai H, Leite S, Tamada J, Uyama S, Vacanti J, Langer R, Mikos A. In vitro and in vivo degradation of porous poly(DL-lactic-co-glycolic acid) foams. Biomaterials 2000; 21: 1837-1845. 85. Lherm C, Müller R, Puisieux F, Couvreur P. Alkylcyanoacrylate drug carriers: II. Cytotoxicity of cyanoacrylate nanoparticles with different alkyl chain length. Int. J. Pharmaceut. 1992; 84(1): 13–22.
45
ACCEPTED MANUSCRIPT 86. Couvreur P, Roland M, Speiser P. Biodegradable submicroscopic particles containing a biologically active substance and compositions containing them. 1982; US patent 4329332 A.
T
87. Al-Khouri Fallouh N, Roblot-Treupel L, Fessi H, Devissaguet J, Puisieux F. Development of a new process for the manufacture of polyisobutylcyanoacrylate nanocapsules. Int. J. Pharmaceut. 1986; 28: 125-132.
AC
CE P
TE
D
MA
NU
SC R
IP
88. Chouinard F, Kan F, Leroux J, Foucher C, Lemaerts V. Preparation and purification of polyisohexylcyanoacrylate nanocapsules. Int. J. Pharmaceut. 1991; 72: 211-217. 89. Vandorpe J, Schacht E, Stolnik S, Garnett MC, Davies MC, Illum L, Davis SS. Poly(organo phosphazene) nanoparticles surface modified with poly(ethylene oxide). Biotechnol Bioeng. 1996 Oct 5;52(1):89-95. 90. Veronese FM, Marsilio F, Caliceti P, De Filippis P, Giunchedi P, Lora S. Polyorganophosphazene microspheres for drug release: polymer synthesis, microsphere preparation, in vitro and in vivo naproxen release. J Control Release. 1998 Mar 31;52(3):227-37. 91. Passi P, Zadro A, Marsilio F, Lora S, Caliceti P, Veronese FM.Plain and drug loaded polyphosphazene membranes and microspheres in the treatment of rabbit bone defects. J Mater. Sci. Mater Med. 2000 Oct;11(10):643-54. 92. Scopelianos A. Polyphosphazenes as new biomaterials in: Shalaby, S.W. (ed.) Biomedical polymers: Designed to degrade systems. Hanser/Gardner publishers, Inc., Cincinnati, OH (1994), 153-171. 93. Andrianov A, Payne L. Protein release from polyphosphazene matrices. Adv. Drug Delivery Rev. 1998; 31: 185-196. 94. Crommen JH, Schacht EH, Mense EH. Biodegradable polymers. II. Degradation characteristics of hydrolysis-sensitive poly[(organo)phosphazenes]. Biomaterials. 1992;13(9):601-611. 95. Tanigami T, Ohta H, Orii R, Yamaura K, Matsuzawa S. Degradation of poly[bis(ethylamino)phosphazene] in aqueous solution. Journal of Inorganic and Organometallic Polymers. 1995, Volume 5, Issue 2, pp 135-153. 96. Florence A. The oral absorption of micro- and nanoparticulates: neither exceptional nor unusual. Pharm Res. 1997 Mar;14(3):259-66. 97. Morishita I, Morishita M, Machida Y, Nagai T. Controlled release microspheres based on Eudragit L100 for the oral administration of erythromycin. Drug Des Deliv. 1991 Jul;7(4):309-19. 98. Park H, Robinson J. Mechanisms of Mucoadhesion of Poly(acrylic Acid) Hydrogels. Pharmaceutical Research. 1987; 4(6): 457-464. 99. Ponchel G, Irache J. Specific and non-specific bioadhesive particulate systems for oral delivery to the gastrointestinal tract. Adv Drug Deliv Rev. 1998 Dec 1;34(2-3):191219.
46
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
100. Saffran M, Kumar GS, Savariar C, Burnham JC, Williams F, Neckers DC. A new approach to the oral administration of insulin and other peptide drugs. Science. 1986 Sep 5;233(4768):1081-4. 101. Brondsted H, Kopecek J. Hydrogels for site-specific drug delivery to the colon. Pharmaceut. Res. 1992; 9: 1540-1545. 102. Yeh P, Berenson M, Samowitz W, Kopeckova P, Kopecek J. Site-specific drug delivery and penetration enhancement in the gastrointestinal tract. Journal of controlled release. Volume 36, Issues 1–2, September 1995, Pages 109–124. 103. Takahashi T, Takayama K, Machida Y, Nagai T. Characteristics of polyion complexes of chitosan with sodium alginate and sodium polyacrylate. Int. J. Pharmaceut. 1990; 61(1–2): 35–41. 104. Matsumoto S, Kobayashi H, Takashima Y. Production of monodispersed capsules. J Microencapsul. 1986 Jan-Mar;3(1):25-31. 105. Wan LS, Heng PW, Chan LW. Drug encapsulation in alginate microspheres by emulsification. J Microencapsul. 1992 Jul-Sep;9(3):309-16. 106. Arneodo C, Benoit J, Thies C. Characterization of complex coacervates used to form microcapsules. Polym. Mater.Sci. Eng. 1987; 57: 255-259. 107. Bernkop-Schnuerch A, Scerbe-Saiko A. Synthesis and in vitro evaluation of chitosan-EDTA-protease inhibitor conjugates which might be useful in oral delivery of peptides and proteins. Pharmaceut. Res. 1998; 15: 263-369. 108. Arbit E, Majuru S, Orellana I. Oral delivery of biopharmaceuticals using the Eligen technology in: Protein formulation and delivery, second edition. Editor: McNally E, Hastedt J. Publisher: Informa Healthcare USA, Inc. New York, NY. 2008; 285-302. 109. Yagi T, Ishizaki K, Takebe H. Cytotoxic effects of protease inhibitors on human cells. 2. Effect of elastatinal. Cancer Lett. 1980 Oct;10(4):301-7. 110. McCaffrey G, Jamieson JC. Evidence for the role of a cathepsin D-like activity in the release of Gal beta 1-4GlcNAc alpha 2-6sialyltransferase from rat and mouse liver in whole-cell systems. Comp Biochem Physiol B. 1993 Jan;104(1):91-4. 111. Plumpton C, Kalinka S, Martin RC, Horton JK, Davenport AP. Effects of phosphoramidon and pepstatin A on the secretion of endothelin-1 and big endothelin-1 by human umbilical vein endothelial cells: measurement by two-site enzyme-linked immunosorbent assays. Clin Sci (Lond). 1994 Aug;87(2):245-51. 112. Bernkop-Schnuerch A, Krajicek M. Mucoadhesive polymers for peroral peptide delivery: Synthesis and evaluation of chitosan-EDTA conjugates. J. Control. Release. 1998; 50: 215-223. 113. Park JH, Ye M, Park K. Biodegradable Polymers for Microencapsulation of Drugs: Molecules 2005, 10, 146-161. 114. Carpenter JF, Pikal MJ, Chang BS, Randolph TW. Rational Design of Stable Lyophilized Protein Formulations: Some Practical Advice. Pharmaceutical Research August 1997, Volume 14, Issue 8, pp 969-975.
47
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
115. Johnson OL, Jaworowicz W, Cleland JL, Bailey L, Charnis M, Duenas E, Wu C, Shepard D, Magil S, Last T, Jones AJ, Putney SD. The stabilization and encapsulation of human growth hormone into biodegradable microspheres. Pharm Res. 1997 Jun;14(6):730-5. 116. Park K, Kwon I, Park K. Oral protein delivery: current status and future prospect. Reactive and Functional Polymers. 2011 (71): 280-287. 117. Kipnes M, Dandona P, Tripathy D, Still JG, Kosutic G. Control of postprandial plasma glucose by an oral insulin product (HIM2) in patients with type 2 diabetes. Diabetes Care. 2003 Feb;26(2):421-426. 118. Clement S, Still JG, Kosutic G, McAllister RG. Oral insulin product hexyl-insulin monoconjugate 2 (HIM2) in type 1 diabetes mellitus: the glucose stabilization effects of HIM2. Diabetes Technol Ther. 2002;4(4):459-466).
48