Chem. Rev. 2001, 101, 3275-3303
`
`3275
`
`Peroral Route: An Opportunity for Protein and Peptide Drug Delivery†
`
`Anurag Sood and Ramesh Panchagnula*
`Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67,
`S.A.S. Nagar, Punjab 160 062, India
`
`Contents
`I.
`Introduction
`II. Barriers to Peroral Delivery of PP Drugs
`A. Prodrug/Analogue Approach
`1. Pegnology
`B. Physical Barriers to Absorption and
`Absorption Enhancers
`1. Modulation of Transcellular and
`Paracellular Absorption Pathways
`2. Carrier-Mediated Transport
`3. Mucolytic Agents
`C. Enzyme Barrier and Enzyme Inhibitors
`1. Mucoadhesive Polymers for Bioavailability
`Enhancement of PP
`D. Dosage Form Modifications
`1. Matrix Carrier Systems: Nanoparticles,
`Microparticles, and Tablets
`2. Self-Assembling Molecular
`Superstructures: Proteinoids
`3. Vesicular Systems: Liposomes and
`Niosomes
`4. Liquid Emulsions
`5. Colonic Drug Delivery Systems
`III. Future Directions
`IV. Closing Thoughts
`V. Acknowledgments
`VI. Note Added in Proof
`VII. References
`
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`I. Introduction
`The better understanding of endogenous proteins,
`peptides, and peptidergic molecules and their role in
`various body functions and pathological conditions
`in last few decades has resulted in realization of the
`enormous therapeutic potential of proteins and pep-
`tides (PPs). As a consequence, a variety of new PP
`drugs have been developed which offer the advan-
`tages of being very potent and specific therapeutic
`agents.1 Initially, use of PPs as pharmaceuticals was
`severely limited, as they were difficult to produce and
`were isolated from animal sources. These PP prod-
`ucts obtained from animals differed from functional
`molecules present in the human body, and their use
`as therapeutic agents raised concerns with regard to
`their immunogenic potential.2,3 As a result of inten-
`
`† NIPER Communication Number 88.
`* To whom correspondence should be addressed. Phone: 91-172-
`214682/83/84/85/86. Fax: 91-172-214692. E-mail: panchagnula@
`yahoo.com.
`
`Received October 20, 2000
`
`sive research efforts in both academic and industrial
`laboratories, recombinant DNA, protein engineering,
`and tissue culture techniques can now be used to
`obtain PPs, on a commercial scale, which resemble
`endogenous molecules and thus provoke fewer or
`minimal immunological responses. Additionally, due
`to advances in analytical separation technology,
`recombinant proteins can now be purified to unprec-
`edented levels.4 Today, PPs along with informational
`macromolecules normally produced by the body in-
`cluding endorphins, enkephalins, leutinizing hor-
`mone releasing hormone, and interferons form an
`increasingly important class of therapeutic agents.
`Table 1 lists PP products introduced in the market
`over the past few years.5-8
`Though the initial problems related to obtaining
`nonimmunogenic PP drugs in purer form at com-
`mercial scales have been overcome to quite some
`extent,9 their formulation and optimum delivery still
`remain as the biggest challenges to pharmaceutical
`scientists. Use of PPs as therapeutic agents is limited
`due to lack of an effective route and method of
`delivery. Various critical issues associated with PP
`delivery that have drawn the attention of formulation
`scientists include the following. (i) PPs are high
`molecular weight biopolymers which serve as en-
`zymes, structural elements, hormones, or immuno-
`globulins and are involved in several biological
`activities. However, due to their large molecular
`weight and size, they show poor permeability char-
`acteristics through various mucosal surfaces and
`biological membranes.10-12 (ii) Many PP drugs are
`efficacious, in large part because of their tertiary
`structure. The tertiary structure can be lost under
`various physical and chemical environments, result-
`ing in their denaturation or degradation with con-
`sequent loss in biological activity, hence, making
`these molecules inherently unstable.8,13,14 (iii) Many
`PPs have very short biological half-lives in vivo due
`to their rapid clearance in liver and other body
`tissues by proteolytic enzymes.15-17 (iv) As PP drugs
`have very specific actions and are highly potent,
`precise clinical dosing is of utmost importance.18
`The most important consideration when designing
`an effective delivery system for any drug is that of
`achieving a predictable and reproducible absorption
`into systemic circulation with high bioavailability. In
`the case of PP drugs, an interplay of poor perme-
`ability characteristics, luminal, brush border, and
`cytosolic metabolism, and hepatic clearance mecha-
`nisms results in their poor bioavailability from oral
`
`© 2001 American Chemical Society
`10.1021/cr000700m CCC: $36.00
`Published on Web 10/23/2001
`
`Downloaded via UNIV OF TEXAS AT AUSTIN on December 13, 2022 at 16:51:29 (UTC).
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`

`

`3276 Chemical Reviews, 2001, Vol. 101, No. 11
`
`Sood and Panchagnula
`
`Anurag Sood was born in 1972. He received his B.Pharm and M.Pharm
`degrees from Dr. Hari Singh Gour Vishwavidyalaya, Sagar, India. After
`finishing his Master’s degree, he joined Dr. Reddy’s Research Foundation,
`Hyderabad, India, as Trainee Pharmacologist, where he was a part of
`team responsible for in vivo characterization of potential drug candidates
`for pharmacokinetics and metabolism. Presently, he is a research scholar
`at National Institute of Pharmaceutical Education and Research (NIPER),
`India. He has been a recipient of a Junior Research Fellowship (1994-
`96) from the University Grants Commission (UGC), India, and a Senior
`Research Fellowship (1998 onward) from the Council of Scientific and
`Industrial Research (CSIR), India. His research activities include design,
`development, and evaluation of conventional and novel peroral-controlled
`release drug delivery systems. Various drug delivery systems, drug release
`kinetics and mechanisms, and analytical method development and
`validation are the topics of his interest. He has 13 publications to his
`credit and has presented his research at various national and international
`conferences. He is a recipient of the G. P. Nair Award of the Indian Drugs
`Manufacturer’s Association (IDMA) for securing first rank in his B.Pharm
`degree course at University. He is fond of music and loves listening to
`Hindi and Urdu Ghazals during his leisure time.
`
`and nonoral mucosal routes.19 Hence, at present
`these drugs are usually administered by parenteral
`route. However, inherent short half-lives of PPs and
`almost warranted chronic therapy requirements in
`a majority of cases make their repetitive dosing
`necessary. Frequent injections, oscillating blood drug
`concentrations, and low patient acceptability make
`even the simple parenteral administration of these
`drugs problematic. This has prompted researchers to
`develop new delivery systems which can effectively
`deliver this important class of drugs.20-30 Although
`there have been reports of successful delivery of
`various PP therapeutics across non-peroral mucosal
`routes,31,32 peroral route continues to be the most
`intensively investigated route for PP administration.
`This interest in the peroral route, despite enormous
`barriers to drug delivery that exist in the gas-
`trointestinal tract (GIT), can be very well appreciated
`from obvious advantages such as ease of administra-
`tion, large patient acceptability, etc. Potential cost
`savings to the health care industry further augment
`the advantages of peroral systems in terms of patient
`compliance and acceptability, since peroral formula-
`tions do not require sophisticated sterile manufactur-
`ing facilities or the direct involvement of health care
`professionals. There have been efforts to circumvent
`the gastrointestinal (GI) absorption barriers to PP
`drugs since the 1920s, when insulin was used first
`as a therapeutic protein, however only with a limited
`success.33-38 After the success of peroral cyclosporin
`formulations,39-41 the efforts in this field have further
`intensified. There are a plethora of attempts and
`
`the Department of
`Ramesh Panchagnula is Professor and Head of
`Pharmaceutics at National
`Institute of Pharmaceutical Education and
`Research (NIPER), India. He received his B.Pharm and M.Pharm degrees
`from Andhra University, India, his M.Sc. degree (Pharmacology) from the
`University of Strathclyde, U.K., and his Ph.D. degree (1990) from the
`University of Cincinnati, U.S. He has 15 years of research and teaching
`experience in pharmaceutics and drug delivery systems and was assistant
`professor at North Dakota State University and the Massachusetts College
`of Pharmacy (1990-94). He has been conferred with many awards such
`as the 2000 scientific prize of IUATLD and PAMDAL-Colorcon Young
`Scientist Award. He has more than 100 publications and presentations to
`his credit, and he is on the Editorial Board of several international journals.
`Dr. Ramesh’s research interests span biopharmaceutic and pharmaco-
`kinetic evaluation of drugs, development and evaluation of advanced drug
`delivery systems, and bioavailability and bioequivalence studies. His group
`was instrumental
`in setting up a bioavailability center at NIPER, which
`has been granted accreditation by WHO to evaluate bioavailability and
`bioequivalence of fixed dose combinations of anti-tubercular drugs. At
`present, his research group focuses on development of new drug delivery
`systems based on the Biopharmaceutics Classification System with
`particular emphasis on peroral, transdermal, and liposomal drug delivery
`systems. He enjoys reading and likes Jeffrey Archer novels.
`
`reports wherein the use of different approaches for
`peroral PP delivery has been investigated. The
`purpose of the present review is to examine recent
`developments in peroral PP drug delivery. Various
`barriers to PP drug absorption have been discussed
`in brief with attention particularly focused on drug
`delivery approaches that have been used or are being
`developed to overcome these barriers. The reports of
`successful improvement of peroral bioavailability of
`PPs and mechanisms involved therein are empha-
`sized the most.
`
`II. Barriers to Peroral Delivery of PP Drugs
`The peroral route poses significant challenges for
`PP drug delivery. The barriers to PP absorption from
`GIT are primarily chemical, enzymatic, as well as
`penetration related. Acid-induced hydrolysis in the
`stomach, enzymatic degradation throughout the GIT
`by several proteolytic enzymes, bacterial fermenta-
`tion in the colon, and physical barriers to absorption
`are traditionally believed to prevent the peroral
`delivery of PPs (Table 2). However, the nature of
`these barriers has now been expanded to include
`intracellular metabolism by cytochrome P450-3A4
`as well as apically polarized efflux mediated by ATP-
`dependent P-glycoproteins.42-44 Although, P-glyco-
`protein-mediated efflux systems are most commonly
`observed in tumor cells, they are also present in
`normal intestinal cells and act to reduce the intra-
`
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`Peroral Route
`
`Chemical Reviews, 2001, Vol. 101, No. 11 3277
`
`Table 1. PP Drug Products Approved in the United States over the Last Few Years
`product name
`protein/peptide
`Actimmune
`Interferon gamma-1b
`Activase
`Alteplase recombinant
`Adagen
`Pegademase bovine
`Alferone N
`Interferon alfa-n3
`Avonex
`Interferon beta-1a
`BeneFIX
`Recombinant human factor IX
`Betaserone
`Interferon beta
`BioTropin
`Human growth hormone
`Bioclate
`Recombinant antihemophilic factor
`CEA-Scan
`Technetium-99m-arcitumomab
`Cerezyme
`Recombinant glucocerebrosidase
`Comvax
`Recombinant vaccine
`Crofab
`Crotalidae polyvalent immune Fab (ovine)
`Enbrel
`Recombinant soluble receptor
`Engerix-B
`Hepatitis B vaccine recombinant
`EPOGEN
`Epoetin alfa
`Follistim
`Recombinant follicle-stimulating hormone
`GenoTropin
`Somatropin
`Geref
`Human growth hormone releasing factor
`Gkucagen
`Recombinant glucagons
`Gonal-F
`Recombinant human follicle stimulating hormone
`Helixate
`Recombinant antihemophilic factor
`Herceptin
`Anti-breast cancer MAb3
`Humalog
`Insulin lispro
`Humate-P
`Antihemophilic factor
`Humatrope
`Somatropin
`Humulin
`Human insulin (recombinant DNA origin)
`Infergen
`Interferon alfacon-1
`Intron
`Interferon alfa-2b
`KoGENate
`Recombinant anti hemophilic factor
`Leukine
`GM-colony stimulating factor
`LYMErix
`Recombinant OspA
`MYOBLOC
`Botulinum toxin type B
`MyoScint
`Imiciromab pentetate, Mab
`Nabi-HB
`Hepatitis B immune globulin (human)
`Neumega
`Oprelvekin, Mab
`NEUPOGEN
`Filgrastim
`Norditropin
`Somatropin
`Novolin
`Recombinant insulin
`Nutropin AQ
`Somatropin
`Nutropin Depot
`Nutropin
`OncoScint
`Oncospar
`Ontak
`Orthoclone OKT 3
`PEG-Intron
`Prevnar
`Procrit
`Proleukin
`ProstaScint
`Protropin
`Pulmozyme
`Rebetron
`Recombinate
`RECOMBIVAX HB
`ReFacto
`Refludan
`Regranex
`Remicade
`ReoPro
`Retavase
`Rituxan
`Roferone-A
`Saizen
`Serostim
`Simulect
`Synagis
`Thymoglobulin
`Thyrogen
`TNKase
`Verluma
`Wellferone
`Zenapax
`
`Somatropin
`Satumomab pendetide, Mab
`PEG-L-asparaginase
`Denileukin diftitox
`Muromonab-CD3, Mab
`Peginterferon alfa-2b
`Diphtheria CRM197 Protein
`Epoetin alfa
`Interleukin-2
`Capromab pentitate, Mab
`Somatrem
`Recombinant dornase alfa
`Ribavirin/interferon alfa-2b combination
`Recombinant anti hemophilic factor
`Recombinant hepatitis B vaccine
`Recombinant antihemophilic factor
`Lepuridin
`Becaplermin
`Infliximab, Mab
`Abciximab, anti-platelet Mab
`Reteplase
`Ritiximab, Mab
`Recombinant interferon alfa-2a
`Somatropin
`Somatropin
`Basiliximab, Mab
`Palivizumab, Mab
`Thymocyte globulin, polyclonal antibody
`Thyrotropin alfa
`Tenecteplase
`Nofetumomab, MAB
`Interferon alfa-n1
`Daclizumab, Mab
`
`company
`InterMune Pharmaceuticals
`Genentech
`Enzon
`Interferon Sciences
`Biogen
`Genetics Institute
`Chiron/Berlex
`Bio-Technology General
`Centeon
`Immunomedics
`Genzyme
`Merck
`Protherics
`Immunex
`SmithKline Beecham
`Amgen
`Organon
`Pharmacia & Upjohn
`Serono Laboratories
`Novo Nordisk
`Serono Laboratories
`Centeon
`Genentech
`Eli Lilly
`Centeon
`Eli Lilly
`Eli Lilly
`Amgen
`Schering-Plough
`Bayer Corporation
`Immunex
`SmithKline Beecham
`Elan
`Centocor
`Nabi
`Genetics Institute
`Amgen
`Novo Nordisk
`Novo Nordisk
`Genetech
`
`Genentech
`Cytogen
`Enzon
`Ligand Pharmaceuticals
`Ortho Biotech
`Schering Corporation
`Lederle
`Ortho Biotech
`Chiron
`Cytogen
`Genentech
`Genentech
`Schering-Plough
`Baxter Healthcare
`Merck
`Genetics Institute
`Aventis
`Ortho-McNeil
`Centocor
`Centocor/Eli Lilly
`Centocor
`Genentech
`Hoffmann-La Roche
`Serono laboratories
`Serono Laboratories
`Novartis
`MedImmune
`SangStat
`Genzyme
`Genentech
`DuPont Merck
`Glaxo Wellcome
`Hoffman-La Roche
`
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`3278 Chemical Reviews, 2001, Vol. 101, No. 11
`
`Sood and Panchagnula
`
`Table 2. Various Peroral Absorption Barriers and Their Bearing on PP Drug Absorption from GIT
`barrier nature
`location and description
`effect on PP drug absorption
`acidic environment in stomach (pH 1.2-3.0)
`chemical
`pH-induced oxidation, deamidation, or hydrolysis
`and alkaline environment in intestine (pH 6.5-8.0)
`luminally secreted, membrane-bound, and cytosolic
`proteolytic enzymes throughout the length of GI tract
`microbial flora present in colon
`unstirred aqueous boundary layer and viscous
`mucus layer covering the surface of GI epithelial cell lining
`lipid bilayer of epithelial cell membrane
`
`enzymatic
`
`physical
`
`intercellular spaces (mean pore radii of 0.8, 0.3,
`and 0.3 nm in duodenum, ileum, and colon,
`respectively) gated by closely fitting tight junctions
`(TJ) on apical side of epithelial cells
`p-glycoprotein present on epithelial cell membrane
`
`proteolytic degradation in lumen and during
`absorption through enterocytes
`breakdown PP as part of their metabolic activity
`decreased diffusion to reach absorptive epithelial
`cell membrane
`inhibits absorption of PP drugs that are
`hydrophilic and charged through the cell
`(transcellular transport)
`TJ prevent passage of PP macromolecules
`through the intercellular spaces
`(paracellular transport)
`
`promote apically polarized efflux to remove
`permeated drug molecules
`
`Figure 1. Diagrammatic representation of different barriers to protein and peptide drug absorption from the intestinal
`tract. Shaded square text boxes show the pathways for drug absorption: P, paracellular; T, transcellular; CT, carrier-
`mediated transport. Target sites for different absorption enhanceement strategies are indicated by numerals in
`paranthesis: 1, prodrugs/analogues; 2, protease inhibitors; 3, mucolytic agents; 4, paracellular and transcellular absorption
`enhancers; 5, mucoadesive polymers; 6, dosage form modifications; 7, pH modulation to enzymatic activity minima 8,
`p-glycoprotein inhibitors.
`
`cellular accumulation or the transcellular flux of a
`wide variety of drugs, including peptides.45,46 Figure
`1 shows an overall view of the various barriers to PP
`drug absorption from peroral route and various
`targets for enhancing their absorption. A brief de-
`scription of these barriers has been provided indi-
`vidually at appropriate places in the subsequent
`sections.
`Traditional drug candidates also encounter similar
`barriers, but PP drugs seem to be highly susceptible
`to all these factors, and the options available to
`pharmaceutical scientists are very limited. The syn-
`thetic chemistry approaches that are often successful
`in ameliorating one or more of the barriers and
`resulting in efficacious in vivo absorption of tradi-
`tional, small organic molecules have proved to be of
`little value in the case of PPs due to their much more
`complex chemistry. Various approaches that have
`been taken to overcome barriers with reference to
`
`poor bioavailability of PP drugs from peroral route
`are enumerated as follows and have been described
`later in the review: (i) Chemical modification of the
`protein or peptide lead compoundsprodrug/analogue
`approach; (ii) Use of absorption enhancers such as
`surfactants, bile salts, or calcium chelators; (iii) Use
`of enzyme inhibitors to lower the proteolytic activity;
`(iv) Designing a drug delivery system which is
`targeted to a part of the gut where proteolytic activity
`is relatively low so as to protect PPs from luminal
`proteolytic degradation and release the drug at the
`most favorable site for absorption.
`A. Prodrug/Analogue Approach
`Prodrug or analogue development has probably
`remained one of the most favored approaches in
`solving many drug delivery related problems. The
`most recent example of insulin LysPro, although for
`parenteral administration, has demonstrated the
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`

`Peroral Route
`
`possibility of modifying biopharmaceutic as well as
`pharmacokinetic characteristics of PP drugs by using
`a prodrug/analogue approach. LysPro, a human
`insulin analogue produced by inverting the native
`sequence ProB28, LysB29 in the c-terminal of the
`B-chain of human insulin,47 was developed by Eli
`Lilly and Company and approved for clinical use in
`1996. The sequence inversion results in reduced self-
`association properties of LysPro, making it more
`readily monomeric,48 and consequently LysPro ex-
`hibits different pharmacokinetic properties from
`soluble insulin on subcutaneous administration (rapid
`onset, higher and earlier peak plasma concentrations
`with shorter duration of action).49,50 There are a
`number of other insulin analogues that are presently
`under different phases of investigations for increasing
`its stability and/or modifying its onset and duration
`of activity.51,52 In context to the scope of present
`review, the prodrug/analogue approach can be de-
`fined as conversion of PPs into derivatives (prodrugs
`or analogues) by means of incorporation of sufficient
`modifications so as to engender oral activity.53-58
`Hydrophilic nature and charge of PP drugs are
`because of the polar and ionizable functional groups
`(including terminal amino and carboxyl groups) in
`the molecules. The presence of amide bonds at
`different positions, free N-terminal amino groups,
`and free C-terminal carboxyl groups make them
`susceptible to endopeptidases-, aminopeptidases-,
`and carboxypeptidases-mediated degradation, respec-
`tively. Thus, chemical modification, such as masking
`or blocking polar amide bonds and terminal amino
`and carboxyl groups, primarily brings about an
`alteration in the physicochemical properties of drugs
`such as lipophilicity, hydrogen-bonding capacity,
`charge, molecular size, solubility, configuration, iso-
`electric point, chemical stability, etc., which are
`known to affect their membrane permeability, en-
`zyme liability, and affinity to carrier systems.59,60
`Various structural features of peptides that influence
`their passive diffusion, carrier-mediated transport,
`and efflux mechanisms have been recently reviewed
`by Wang et al.59 and Pauletti et al.61 The lipophilicity
`of various drugs, as expressed in terms of logP
`(logarithm value of octanol-water partition coef-
`ficient) or logD (logarithm value of octanol-pH 7.4
`buffer partition coefficient), can be correlated with
`cell membrane permeability.62 The generalization is
`that within a homologous series, drug absorption
`increases as lipophilicity rises and is maintained at
`a plateau for a few units of logP after which there
`may be a steady decrease, giving a parabolic relation.
`However, in the case of PP drugs, logP or logD values
`may not always correlate well with drug perme-
`ability.63 In a study with a series of six model
`peptides, prepared from D-phenylalanine and glycine,
`Conradi et al. observed that the permeability of
`peptides across Caco-2 cell monolayers was inversely
`related to the number of hydrogen-bonding groups
`in the structure as these hydrogen bonds must be
`broken for the solute to transfer into the interior of
`cell membrane.64 They showed that although addition
`of amino acid with a large hydrocarbon chain (phen-
`ylalanine) to the peptidic chain resulted in increased
`
`Chemical Reviews, 2001, Vol. 101, No. 11 3279
`
`lipophilicity of modified peptides, their permeability
`was affected adversely. The effect was explained to
`be due to introduction of very polar amide bonds,
`capable of forming strong hydrogen-bonding interac-
`tions with water, in the peptide chain with the
`addition of hydrophobic amino acid residue. In an-
`other study with a tetrapeptide, Conradi et al.
`showed that methylation of amide nitrogens resulted
`in a substantial increase in transport across the
`Caco-2 cell monolayer but without any significant
`change in the octanol-water partition coefficient,
`suggesting that a reduction in the overall hydrogen-
`bonding potential is more important than an increase
`in lipophilicity.65 Similarly, Saitoh and Aungst showed
`that lipophilicity and charge of DMP-728 (a potent
`GP IIb/IIIa receptor antagonist) prodrugs did not
`influence intestinal permeability determined in vitro
`using rat jejunum in diffusion cells; instead, N-
`methyl-substituted analogues exhibited 2-fold greater
`jejunal permeability than DMP-728.66 However, these
`observations were not always consistent with the
`hypothesis that reducing the hydrogen-bonding ca-
`pacity of peptides can increase permeability and
`suggested that this could be because of confounding
`influence of secretory transport by P-glycoprotein.
`Additionally, there are a number of reports where
`an increase in lipophilicity, as indicated by partition
`coefficient values of PP molecules by means of chemi-
`cal modification, has been shown to improve their
`membrane permeability.53,67
`As explained earlier, PP molecules harbor more
`than one polar and ionizable group that contributes
`to the total charge and polarity of molecules and/or
`serves as a site for enzymatic attacks. A chemical
`modification at one site may not always be sufficient
`to significantly improve permeability characteristics
`and/or reduce liability to enzymatic degradation in
`vivo, especially when there are multiple enzymes
`involved in degradation at different sites. In such
`instances, various strategies have been tried which
`allow simultaneous masking of more than one func-
`tional group. Borchardt, Wang, Pauletti, and co-
`workers59,68-75 described preparation of cyclic pro-
`drugs which allow for simultaneous masking of an
`amino and a carboxyl group of peptide drug. These
`cyclic prodrug systems can be prepared by using
`acyloxyalkoxy-, phenolpropionic acid- or coumarine-
`based prodrug moieties (Table 3). Wang et al.59
`explained that cyclization of linear peptides by using
`these prodrug moieties results in significantly altered
`physicochemical properties (due to derivatization of
`carboxyl and amino groups into ester and amide,
`respectively), altered effective size and shape along
`with restricted conformational freedom of the cyclic
`peptide, which consequently reduces the charge on
`peptide and promotes intramolecular hydrogen bond-
`ing within the peptide molecule rather than inter-
`molecular hydrogen bonding between peptide func-
`tional groups and solvent. These prodrugs have
`reduced susceptibility to peptidase metabolism; how-
`ever, they are esterase sensitive and release the
`parent peptide under esterase activity. To achieve
`similar results, chemical modifications at two or three
`functional groups in the PP molecules have also been
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`3280 Chemical Reviews, 2001, Vol. 101, No. 11
`
`Sood and Panchagnula
`
`Table 3. Various Approaches for Derivatization of Peptides and Proteins To Make Produgs/Analogues
`
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`Peroral Route
`
`Chart 1
`
`Chemical Reviews, 2001, Vol. 101, No. 11 3281
`
`suggested, e.g., Weller and co-workers76 prepared
`prodrugs of Lamifiban (Ro 44-9883; I) by one modi-
`fication (modification of either carboxyl or amidino
`group; II, III), two modifications (modification of both
`carboxyl and amidino groups; IV, V), and three
`modifications (modification of carboxyl, amidino, and
`phenyl hydroxyl groups; VI), Chart 1.
`Triple prodrug (VI) was found to be more orally
`active (lower ID50) in mice than double prodrugs (IV,
`V), which in turn showed higher oral activity than
`single prodrug (II, III). In a recent review Wang et
`al.77 discussed various prodrug-based strategies to
`improve bioavailability of peptidomimetic RGD (Arg-
`Gly-Asp) analogues.
`The prodrug approach can also be used to intercept
`with the P-glycoprotein-mediated efflux of PP drugs.
`However, to modify PP drugs to reduce or prevent
`their substrate activity for efflux systems it is im-
`portant to know the structural features that influence
`efflux-mediated transport for PP drugs.78 Broad
`substrate specificities shown by efflux systems make
`it difficult to identify the suitable chemical modifica-
`tions for altering susceptibility characteristics of PP
`drugs toward efflux systems. Instead, use of P-
`glycoprotein inhibitors, such as the R-isomer of
`verapamil, nonimmunosuppressive analogues of cy-
`closporin D (SDZ PSC833) and LY335979 as adju-
`vants appears to be a more realistic approach to help
`improve oral absorption of PP drugs that are sub-
`strates for intestinal efflux systems.58
`One of the most important features of a prodrug is
`the ability to be converted quantitatively to the
`parent peptide in vivo by a spontaneous or unspeci-
`fied plasma enzyme-catalyzed reaction after their
`absorption.79 Modified peptides that lack biorevers-
`ibility are considered to be new peptides rather than
`prodrugs, and the approach is known as an analogue
`approach. Chemical modification of proteins by suc-
`cinylation, acylation, guanidation, modification of
`amide bonds, and deamination conjugation with
`polymers such as dextran, albumin, DL-poly(amino
`acid), poly(vinylpyrrolidone), and poly(ethylene gly-
`col) have been tried to increase the blood circulating
`life and/or reduce immunogenicity.80-82 Toth and co-
`
`workers reported modification of N- and C-termini
`of TT-232, a tumor-selective somatostatin analogue,
`to improve its stability and bioavailability. They
`prepared lipoamino acid and liposaccharide conju-
`gates of TT-232, which resulted in amphipathic
`surfactant molecules with retained activity and im-
`proved transport across Caco-2 cell monolayers.83 In
`an attempt to use the lymphatic absorption pathway
`and thereby bypass hepatic first pass metabolism,
`Delie et al.84,85 prepared the diglyceride prodrug of a
`pentapeptide rennin inhibitor SR 42128. Conjugation
`of pentapeptide drug to 2-position of 1,3-diglyceride
`resulted in a prodrug of increased lipophilicity and
`better stability to degradation by proteases and
`peptidases (intestinal juice and R-chymotrypsin).
`However, lymphatic uptake of prodrug on oral ad-
`ministration to rats could not be established. Various
`approaches for derivatization of PPs and recently
`published studies highlighting use of the prodrug
`strategy to improve peroral bioavailability of PP
`drugs are summarized in Tables 3 and 4. In addition
`to altering the physicochemical properties of PP
`drugs to improve their transmembrane passive per-
`meability and stability to enzymatic degradation, the
`prodrug approach has been used to enhance substrate
`property of PP drugs to carrier-mediated active
`transport mechanisms, which is discussed later in the
`review.
`1. Pegnology
`Therapeutic proteins have been coupled to various
`polymers so as to reduce their immunogenic response,
`increase resistance to enzymatic degradation, and
`prolong their half-life. Oral absorption of PP drugs
`has been achieved by chemically changing the protein
`or peptide by covalent addition of the polymers
`composed of water- and fat-soluble elements. Poly-
`mers such as poly(ethylene glycol) (PEG), dextran,
`albumin, and poly(vinylpyrrolidone) have been stud-
`ied as protein carriers.3 Modification of proteins with
`PEG is known as pegnology or pegylation and has
`been shown to improve biopharmaceutical and clini-
`cal properties (including enhanced solubility, sus-
`tained absorption, reduced immunogenicity and pro-
`
`Bausch Health Ireland Exhibit 2039, Page 7 of 29
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`3282 Chemical Reviews, 2001, Vol. 101, No. 11
`
`Sood and Panchagnula
`
`Table 4. Some of the Prodrugs/Analogues of Proteins and Peptides Screened for Peroral Bioavailabilitya
`biologically active species
`prodrug/analogue
`results
`DDAVP
`pivalate, n-hexanoyl and n-octanoyl
`sterically hindered pivalate
`esters of the tyrosine phenolic group
`ester was more stable to
`in dDAVP
`enzymatic degradation
`Palins-1
`increased plasma radioactivity
`Palins-2
`on administration in polyoxyethylene
`hydrogenated castor oil (HCO60)
`provide affinity to transport carriers
`prodrugs were found to be stable
`to angiotensin-converting enzymes
`and aminopeptidases N enzymes that
`are responsible for degradation of Leu-
`enkephalin at the BBB and in plasma
`prodrugs were found to be more lipophilic,
`more stable against peptidase metabolism,
`and many fold better permeating across
`Caco-2 cell monolayers than
`their respective linear opioids;
`chemical stability studies
`revealed stoichiometric conversion
`of prodrugs to the corresponding
`peptides; however, for aycloxyalkoxy-
`based prodrugs, apical to basolateral
`permeability was lower than that of
`DADLE and also lower than their
`permeability in basolateral to apical
`direction due to polarized efflux system
`completely inert toward aminopeptidases
`and (cid:181)-chymotrypsin, decomposes at pH
`7.4 and 37 °C with half-lives of 30, 10.9,
`and 3.1 h, respectively, lipophilicity of
`prodrugs was increased; however, it could
`be easily degraded by carboxypeptidase A
`MTP-PE has immunostimulant effects
`similar to those of natural muramyl
`dipeptide and has a longer half-life in
`plasma and lower toxicity
`cyclic prodrugs degraded to linear
`hexapeptide in various biological media
`due to esterase activity; cyclic prodrugs
`were more stable to peptidase metabolism
`and more permeable when applied to apical
`side of Caco-2 cell monolayers
`derivatives were found to be completely
`resistant to hydrolysis by R-chymotrypsin
`prodrugs of RGD analogues showed
`enhanced membrane interaction potentials
`(determined from their partitioning between
`10 mM phosphate buffer, pH 7.4/acetonitrile
`as various concentrations, and an
`immobilized artificial membrane) and
`intrinsic membrane permeabilities
`(determined using Caco-2 cell
`monolayers); prodrugs were found to
`undergo esterase-catalyzed release of
`RGD analogues in the presence of
`porcine lever esterase; prodrug of
`compound MK-383 showed significant
`and prolonged antiplatelet activity
`(determined ex vivo after oral administration
`to a dog) in contrast t