COMMENTARY
`
`Anatomical, Physiological, and Experimental Factors Affecting the
`Bioavailability of sc-Administered Large Biotherapeutics
`
`ANAS M. FATHALLAH, SATHY V. BALU-IYER
`
`Department of Pharmaceutical Sciences, University of Buffalo, Buffalo, New York
`
`Received 15 August 2014; revised 27 October 2014; accepted 29 October 2014
`
`Published online 19 November 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24277
`
`ABSTRACT: Subcutaneous route of administration is highly desirable for protein therapeutics. It improves patient compliance and quality
`of life (McDonald TA, Zepeda ML, Tomlinson MJ, Bee WH, Ivens IA. 2010. Curr Opin Mol Ther 12(4):461–470; Dychter SS, Gold DA,
`Haller MF. 2012. J Infus Nurs 35(3):154–160), while reducing healthcare cost (Dychter SS, Gold DA, Haller MF. 2012. J Infus Nurs
`35(3):154–160). Recent evidence also suggests that sc administration of protein therapeutics can increase tolerability to some treatments
`such as intravenous immunoglobulin therapy by administering it subcutaneously (subcutaneous immunoglobulin therapy SCIG), which will
`reduce fluctuation in plasma drug concentration (Kobrynski L. 2012. Biologics 6:277–287). Furthermore, sc administration may reduce the
`risk of systemic infections associated with i.v. infusion (McDonald TA, Zepeda ML, Tomlinson MJ, Bee WH, Ivens IA. 2010. Curr Opin Mol
`Ther 12(4):461–470; Dychter SS, Gold DA, Haller MF. 2012. J Infus Nurs 35(3):154–160). This route, however, has its challenges, especially
`for large multidomain proteins. Poor bioavailability and poor scalability from preclinical models are often cited. This commentary will
`discuss barriers to sc absorption as well as physiological and experimental factors that could affect pharmacokinetics of subcutaneously
`administered large protein therapeutics in preclinical models. A mechanistic pharmacokinetic model is proposed as a potential tool to
`address the issue of scalability of sc pharmacokinetic from preclinical models to humans. C(cid:2) 2014 Wiley Periodicals, Inc. and the American
`Pharmacists Association J Pharm Sci 104:301–306, 2015
`Keywords: bioavailability; proteins; preclinical pharmacokinetics; absorption; biotechnology
`
`INTRODUCTION
`Protein therapeutics are classified based on their pharmaco-
`logical function into (1) proteins with enzymatic/regulatory
`function or (2) proteins with targeting function (monoclonal
`antibodies).1 The first class contains proteins ranging in size
`from small peptide hormones such as insulin and erythropoi-
`etin to the large multidomain proteins such as FVIII and acid
`alpha-glucosidase (GAA). These therapeutics are designed to:
`(1) replace lacking or aberrantly formed endogenous counter-
`parts to ameliorate disease conditions such as the use of insulin
`in diabetes; (2) augment existing pathways such as the use of
`human follicle-stimulating hormone for infertility; and (3) pro-
`vide a novel function such as hyaluronidase.2,3 The second class
`contains monoclonal antibodies (mAbs) and their derivatives.
`This class of protein therapeutics is characterized by unique
`pharmacokinetics because of their high-target-binding affinity
`and the presence of the Fc fragment (in the case of mAb), which
`imparts the prolonged half-life of this class of biologics.
`The wide range in the size and properties of protein ther-
`apeutics makes it difficult to treat them as a single class of
`therapeutics, especially when discussing sc absorption. Fur-
`thermore, the classification of protein therapeutics based on
`pharmacological function may be irrelevant when discussing
`absorption from the subcutaneous space. This necessitates a
`different categorization system based on the size rather than
`the function of these therapeutics. The following sections dis-
`cuss the physical barriers to sc absorption of protein thera-
`peutics, which should help in classifying protein therapeutics,
`
`Correspondence to: Sathy V. Balu-Iyer (Telephone: +716-645-4836; Fax: +716-
`645-3693; E-mail: svb@buffalo.edu)
`Journal of Pharmaceutical Sciences, Vol. 104, 301–306 (2015)
`C(cid:2) 2014 Wiley Periodicals, Inc. and the American Pharmacists Association
`
`based on size, into (1) small proteins of less than 10 nm in diam-
`eter, (2) large proteins of greater than 10 nm in diameter, and
`(3) mAbs. Next, we discuss presystemic degradation as a con-
`tributing factor to incomplete bioavailability before presenting
`possible experimental artifacts in preclinical models that can
`further contribute to poor scalability to humans.
`
`BARRIER TO sc ABSORPTION OF PROTEIN
`THERAPEUTICS
`
`Physical Barriers
`
`After a drug is deposited in the sc space, it must traverse the
`extracellular matrix to reach an entry point into systemic cir-
`culation. Entry can be directly into the blood stream or by tran-
`siting through the lymphatics.4
`
`Direct Uptake into Blood
`
`Uptake into blood requires entry at the postcapillary bed or
`by traversing the basal membrane of blood vesicles, both of
`which are size limiting. The postcapillary bed is involved in
`blood/tissue fluid exchange, it is also the primary site of leuko-
`cytes and plasma protein leakage.5 These capillaries preferen-
`tially reabsorb particles up to 10 nm.6 Alternatively, the drug
`enters systemic circulation by crossing the basal membrane of
`blood vessels via the paracellular or transcellular pathway. The
`former is limited by the size of the fenestrations in the basal
`membrane reported to be 6–12 nm for most nonsinusoidal blood
`capillaries.7
`The transcellular pathway may not be a major player in pro-
`tein uptake. Indeed, large proteins have been shown to have
`poor transcellular trafficking.8 Those therapeutic proteins are
`generally hydrophilic, which prevents them from traversing the
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`COMMENTARY
`
`cell membrane. Protein entering through pinocytosis or phago-
`cytosis will likely be degraded leading to the loss of protein.
`One exception is monoclonal antibodies. Transcellular trans-
`port of mAbs has been recognized since the early 1970s.8 This
`is mediated by FcRn receptors on the surface of endothelia cells.
`FcRn not only facilitates the bidirectional transport of mAbs,9,10
`but it also protects the antibody during fluid phase pinocytosis
`by binding the antibody and sorting it away from the lysoso-
`mal pathways.11–13 FcRn-mediated transport explains the high
`bioavailability and the saturable nature14 of mAb uptake from
`sc.
`
`Physicochemical properties of antibodies that can potentially
`affect transcellular trafficking of mAbs such as isotype, FcRn-
`binding affinity, charge, hydrophobicity, and solubility have
`been investigated by a number of researchers in the field with
`conflicting results. For example, Khawli et al.15 showed no ef-
`fect of charge variants of IgG1 on their pharmacokinetics after
`sc administration in rats. This, however, is in contrast to other
`reports showing that alteration to the isoelectric point of mAbs
`altered the bioavailability after sc administration in mice.16 In-
`terestingly, both groups reported no change in FcRn-binding
`affinity as a function of changes in pI of the protein.15,16
`The role of pI and protein surface charge in sc uptake
`could be explained by charge–charge interaction during fluid
`phase pinocytosis. It has been shown that IgGs with higher
`pI have higher cellular uptake.17,18 This suggests that a posi-
`tively charged IgG interacts more favorably with the negatively
`charged cell surface allowing for more uptake of mAb during
`fluid phase pinocytosis16–18; the IgG will then bind to FcRn,
`which will protect it from degradation. Negatively charged IgG,
`on the contrary, will have lower uptake because of the repulsion
`between the negative protein and the negative cell surface.16
`However, in the context of the sc space, the repulsive forces be-
`tween the negative protein and the negative extracellular ma-
`trix could enhance convective movement of the protein through
`the extracellular matrix and improve lymphatic trafficking, as
`we will discuss below.
`
`Uptake by the Lymphatics
`
`Uptake by lymphatics is less restrictive. The initial lym-
`phatics, where interstitial fluid enters the lymphatic system
`and becomes lymph fluid, do not have a continuous basal
`membrane.6,19 Rather, the endothelial cells of the initial lym-
`phatic overlap while being anchored by collagen VII to the ex-
`tracellular matrix.6,19 The lack of the basal membrane allows
`for large proteins as well as small cells, bacteria, and viruses to
`enter the lymphatics.19 Anchorage to the extracellular matrix,
`on the other hand, allows for the transmission of mechanical
`forces from the extracellular matrix to the lymphatic lumen.19
`This can allow the initial lymphatics to open up in response
`to mechanical movement; this can explain the improved lymph
`flow in response to massaging or movement.
`Despite the lax size limitation of lymphatic uptake, there are
`still a number of other impediments to absorption of biologics
`via this route. After injection, the protein must navigate the ex-
`tracellular matrix to reach a point of entry into the lymphatics.
`The density of the initial lymphatics at the injection site4 will
`affect the proficiency of lymphatic uptake of protein from the
`injection site. This process can also be affected by the size and
`charge of the proteins.6 Larger proteins are selectively taken up
`by the lymphatics; however, the larger the protein, the slower
`
`the uptake6 because of the increased resistance to convective
`and/or diffusive movement. Also, electrostatic interaction with
`glycosaminoglycanes, the negatively charged component of the
`glycocalyx matrix,5,6 can hide or promote the movement of the
`protein through the extracellular matrix.6,20 Indeed, positively
`charged proteins have been reported to reach the lymphatics at
`a delayed time as compared with negatively charged protein of
`comparable size.21
`It is important to note that the above-mentioned uptake
`pathways are not mutually exclusive, and protein absorption
`can occur via one or more of the pathways discussed above. For
`example; small protein therapeutics can utilize the postcapil-
`lary bed as well as the fenestrations in the basal membrane
`of blood vessels; this explains their good bioavailability. mAbs
`can utilize FcRn receptors on the surface of endothelial cells as
`well as lymphatic uptake. Large protein therapeutics, however,
`must utilize lymphatic uptake.
`Strategies that can overcome one or more of the above-
`mentioned physical barriers could enhance bioavailability of
`protein therapeutics. For example, the use of hyaluronidase
`to “loosen” the extracellular matrix enhances the diffusion
`of the coadministered biologics.3 Another example is the
`use of albumin to manipulate the oncotic pressure, and by
`extension the interstitial fluid volume in sc space, to en-
`hance sc bioavailability.22,23 Other strategies to manipulate
`the environment in the sc space to improve overall bioavail-
`ability of protein therapeutics such as viscosity, osmolarity,
`and volume of injection have been recognized by a number
`of workers in the field and are discussed in a number of
`reviews.4,24
`In our own work, we found a relationship between increased
`buffer tonicity and improved bioavailability of subcutaneously
`administered rituximab in a mouse model.25 Furthermore, our
`data suggest that the effect of buffer hypertonicity on ritux-
`imab bioavailability is excipient specific. We found that manni-
`tol, a neutral excipient, performed better than the negatively
`charged O-phospho-L-serine at equal tonicities. The enhanced
`bioavailability was associated with enhanced lymph node up-
`take of rituximab.25 We propose that hypertonic buffers per-
`turb the isotonicity of the interstitial space altering the forma-
`tion and reabsorption of interstitial fluid at the postcapillary
`beds. The draining of excess interstitial fluid by the lymphatics
`enhances bulk movement of fluid through the sc space carry-
`ing with it the protein from the injection site through to the
`lymphatic.25
`
`Presystemic Degradation
`
`Another complicating factor for sc absorption of protein
`therapeutics is presystemic elimination.4,14 This could be
`due to degradation at the injection site by proteolytic en-
`zymes. The presence of such enzymes is supported by reports
`showing this proteolytic activity in the sc space to be sat-
`urable by administering high doses of the drug as well as
`inhibited by coadministering protease inhibitors.14,26,27 This
`proteolytic activity has been proposed as a reason for in-
`complete bioavailability of biologics. Another form of presys-
`temic elimination is the uptake and processing of protein
`therapeutics by professional antigen presenting cells in the
`skin. Upon sc administration, dermis and epidermis resi-
`dent dendritic cells migrate to the injection site to sam-
`ple the injected protein.28 This leads to maturation of these
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`cells, which is accompanied by the release of proinflamma-
`tory cytokine and chemokines.28–30 This can recruit more
`antigen presenting cells to the injection site for sustained
`sampling of the injected drug for later presentation in the
`lymph node.28–30 This sustained sampling of the drug can be
`a significant form of elimination at the injection site. Re-
`ducing the residence time of the drug in the subcutaneous
`space could reduce the effect of the aforementioned degrada-
`tion pathways. This could be achieved by enhancing lymphatic
`uptake, which will siphon the drug away from the injection
`site. Such a strategy could also prove particularly effective
`in situations where saturable uptake, such as FcRn uptake
`of mAbs, is in effect. Trafficking the drug effectively through
`the lymphatic may alleviate the load on uptake transporters
`and enhance overall bioavailability of injected therapeutic
`proteins.
`
`PRECLINICAL MODELS AND EXPERIMENTAL ARTIFACTS
`
`Anatomy and Physiology of Preclinical Models
`
`The complexity of absorption processes after sc administration,
`as discussed in the previous section, and the variety of path-
`ways that are involved in this process are key contributing fac-
`tors to the poor scalability and poor predictive power of preclin-
`ical species. This section will examine some physiological and
`experimental factors that can further contribute to this issue.
`The suitability of rodent models for predicting pharmacokinet-
`ics of subcutaneously administered biotherapeutic in humans
`has been questioned recently. Anatomical and physiological dif-
`ferences in rodent sc space viz a viz humans are often cited. For
`example, rodent models exhibits a wide lateral expansion of
`an sc-injected dose because of loose connective tissue in the
`sc space.4 This lateral expansion means a wider surface area
`for the drug to diffuse through and can result in better ab-
`sorption. Another commonly cited difference is the panniculus
`carnosus, a muscular layer embedded in the hypodermis under
`the superficial adipose tissue and above the membranous layer
`(Fig. 1a). This layer is less pronounced in larger nonfurred an-
`imals and almost lacking in humans.31
`Other preclinical models, such as swine models, have a fixed
`skin with a tight attachment to the subcutaneous tissues.32
`This may reduce the issue of wide lateral expansion after sc ad-
`ministration. In general, swine models are regarded suitable
`for experimental toxicologic, dermatologic, and wound-healing
`studies33 and are gaining popularity in pharmacokinetics stud-
`ies. This is mainly because of structural and biochemical simi-
`larities between human and pig skin.32–34 There are, however,
`potentially important differences that can affect the absorption
`of biotherapeutics from sc space in pigs as compared with hu-
`mans. Reduced vasculature in pig’s skin and a unique inverted
`architecture of swine lymph nodes32,35 may affect the uptake
`and trafficking of drug from sc space.
`Despite the anatomical similarities between swine models
`and human skin, the fact remains that there is no reliable pre-
`clinical model to predict pharmacokinetics of sc-administered
`protein therapeutics in humans.31 Indeed, a comparison of the
`bioavailability of 13 biotherapeutic in human versus monkeys,
`dogs, rats, mice, and pigs showed no strong correlation between
`results in humans and any one species.4 If anatomy and phys-
`iology, per se, cannot account for the differences in sc uptake
`
`COMMENTARY
`
`303
`
`and bioavailability, then one must examine some experimental
`artifacts that could help explain the observed differences.
`
`Experimental Artifacts Affecting Pharmacokinetics of
`sc-Administered Protein Therapeutics
`
`One might argue that the experimental procedure may sig-
`nificantly contribute to the poor scalability of bioavailability
`estimates obtained in rodents or large animals to humans. One
`such artifact is the volume of injection used in preclinical mod-
`els. Those volumes could create superphysiological pressure in
`the sc space that will not translate into humans. For example,
`a 100-:L sc injection into a 20-g mouse, corresponds to a vol-
`ume of 350 mL for an average 70 kg human if scaled based
`on body weight. Or 23 mL if scaled up based on surface area.
`Both of which are unrealistic superphysiological volumes. This
`indicates that rodent models are being injected with massive
`volumes creating ultraphysiological pressure in the interstitial
`space. As the volume spreads laterally in the sc space, it may
`cause shear stress. This is known to cause alteration in the
`glycocalyx structure and the release of nitric oxide,5 a potent
`intracellular-signaling molecule, altering the permeability of
`the endothelial barrier.5
`The effect of large injection volumes (relative to body size)
`could be a major contributor to the altered rate of uptake in
`small animals and is not usually accounted for in typical phar-
`macokinetics models. Furthermore, this effect is species inde-
`pendent as it depends on the size of the animals. One could
`argue that this artifact could plague data from other preclini-
`cal models with more relevant sc anatomy and physiology but
`small body size and surface area such as minipigs. This is a
`very important question to investigate, especially with the in-
`creased interest in using minipigs as preclinical models for sc
`administration of protein-based therapeutics.36 Swine models
`not only have similar sc anatomy and physiology compared with
`humans, but their surface area is also comparable to that of an
`adult human.32 This makes them a good candidate for a pre-
`clinical model of sc absorption. However, because of difficulty
`in handling, housing, and experimenting with typical “farm”
`pigs, miniature breeds called minipigs were developed out of
`necessity by selective breeding.33 Those breeds, however, are
`one-fifth to one-tenth the size of their “farm” or domestic coun-
`terparts depending on the breed (Hanford or Yucatan vs. Sin-
`clair or Go(cid:2)ttingen).33 This means that minipigs have a smaller
`body surface area and as such could be susceptible to the ef-
`fects of large volume of injection (compared with body surface
`area) as other “small” preclinical models. Indeed, a study com-
`paring the bioavailability of nine mAbs in Go(cid:2)ttingen minip-
`igs (which can grow to about 10 kg) showed poor correlation
`between bioavailability and absorption rate obtained in those
`pigs and humans.36 Values obtained from minipigs were higher
`for the majority of the tested mAbs.36 This is despite good corre-
`lation in clearance and similar FcRn-binding properties in both
`species.
`Injection technique is also recognized as a determining factor
`in the bioavailability of sc-administered biologics.31 When we
`examined the pinch method in mouse models, one of the more
`common methods of sc injection into mice, we observed that
`this method results in separation of the membranous layer of
`the sc space in mice (Figs. 1a and 1b). This indicates that the
`injected dose is being deposited much deeper in rodents as op-
`posed to humans. In humans, the dose is expected to deposit in
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`(a-I) 5× magnification of skin and abdomen samples. On the left, Trichrom staining of the abdominal intact wall-skin samples
`Figure 1.
`obtained from HA mice. On the right is the skin sample obtained after a simulated sc injection using the “pinch” method. This figure shows
`that the “pinch” method is separating the skin at the membranous layer, which results in a much deeper deposit of the sc dose than expected
`in humans. Arrows are pointing at: (I) dermis, (II) epidermis, (III) superficial adipose tissue, (IV) deep adipose tissue, (V) membranous layer.
`(b) 40× magnification of skin and abdomen samples. On the left, Trichrom staining of the abdominal wall-skin samples obtained from HA mice
`(see the panniculus carnosus in red intermittent muscular layer). On the right is the skin sample obtained from the “pinch” method. Here, we
`can see the membranous layer (blue) appearing thinner when compared with the image to the right.
`
`the superficial adipose tissue and not the membranous layer.
`This deeper deposit in rodent models can have two effects: (1)
`the drug has to traverse a shorter distance to reach an uptake
`site and (2) the drug may be taken up by initial lymphatics
`of different compliance and elasticity affecting its propulsion.
`Negrini and Moriondo19 highlight the effects of the local sur-
`roundings of initial lymphatics on lymph drainage, retention,
`
`and propulsion. Their model predicts that vessels surrounded
`by loose compliant tissue are themselves compliant and as such
`act as reservoirs of drained lymphatics, but have a slower lym-
`phatic propulsion as compared with those surrounded by stiff
`tissue.19
`Another experimental artifact that can account for the dif-
`ferences between sc data obtained in rodent models versus
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`
`Table 1. FVIII Recovery from Plasma After 3 h of sc Administration
`of 5 IU/g of FVIII into the Lower Dorsal Region or the Abdominal
`Region of HA Mice
`
`Plasma (IU/mL)
`
`0.25
`
`00
`
`2.20
`0.28
`1.18
`
`Injection Site
`
`Dorsal side
`
`Abdominal
`
`humans, is the site of injection. Active areas in the body or those
`with cyclical compression and expansion, such as the thoracic
`region, tend to have higher lymph flow because of high mechan-
`ical stress imposed on the lumen of the initial lymphatics.19 In
`our laboratory, we observed marked differences in the amount
`of FVIII recovered in plasma after 3 h of injecting FVIII sc
`in the ventral (abdominal skin) versus the dorsal side (scruff)
`of HA mice (Table 1), highlighting the effects of injection site
`on absorption in small animals. The rapid breathing of small
`rodents, such as mice, results in rapid cyclical motion in the
`thoracic and abdominal region of the animal. As humans have
`slower breathing rate and less pronounced movement of the
`abdominal region with every breath, we do not expect marked
`difference in bioavailability between abdominal injection and
`injections in the thigh, for example. Another common experi-
`mental practice in preclinical settings is the injection into the
`paw of the hind leg. This is carried out for ease of collection
`of lymph data. This practice is also seen when larger animals
`are used, such as sheep and dogs. Those anatomical sites have
`no equivalent in humans; this practice may also result in an
`exaggerated response not seen in humans.
`Limitations, notwithstanding, we argue that small preclini-
`cal models, such as rodent models, are suitable for mechanis-
`tic studies and can help shed light on factors affecting uptake
`from sc space. Also, we must acknowledge that because of logis-
`tic and practical considerations, especially in academic setting,
`rodent models will not be replaced anytime soon by larger ani-
`mals. However, by understanding their limitations, we can cau-
`tiously extrapolate into higher species and humans. Minipigs
`are also emerging as a possible practical model for sc absorp-
`tion; however, caution must be practiced to account for some
`physiological and experimental effects as mentioned above.
`We further propose that a mechanistic pharmacokinetic
`model (such as the one proposed in Fig. 2) for sc uptake that
`takes into account the effects of different barriers, limitation,
`and experimental artifacts on sc absorption should help us bet-
`ter predict sc bioavailability in humans from preclinical data.
`In the model presented in Figure 2, the skin in perfused with
`arterial blood at a rate “Q,” and drained by lymph flow and
`venular blood flow at rates QL and Q−QL, respectively. Within
`the skin, the blood and interstitial compartments are presented
`separately; interstitial fluid is formed and reabsorbed based on
`known physiological parameters at steady state. Excess inter-
`stitial fluid is drained by the lymph flow. Such a model will
`allow for physiological parameter to change as we extrapolate
`from one species to another. Depositing a dose in the interstitial
`space will perturb steady-state conditions. This will result in
`changes in permeability, interstitial volume, and flow. The mag-
`nitude and extent of such changes will differ from one species
`
`Figure 2. A proposed physiological model for sc absorption (see text
`for discussion and details).
`
`to another. This could be determined from in vitro and in vivo
`studies, and then could be incorporated into the model.
`For example, studying the effect of skin biopsy homogenates
`on protein therapeutic stability in vitro can shed light on the
`rate of protein degradation after sc injection. This rate could
`differ depending on the preclinical model being used. Another
`useful parameter to determine from in vitro analysis of skin
`biopsy is the glycosaminoglycan content of the sc space in dif-
`ferent animal models and how it related to humans sc space.
`Swabb et al.37 showed a relationship between glycosaminogly-
`can content in different tissue and the dominance of convective
`versus diffusive forces on macromolecule movement in those
`tissue. Indeed, the disruption of the glycosaminoglycan ma-
`trix by enzymes such as hyaluronidase can alter the disper-
`sion of coinjected therapeutic proteins as well as reduce inter-
`stitial pressure,2,38 further highlighting the importance of the
`glycosaminoglycan matrix in macromolecule transport and ab-
`sorption from the sc space. The rate of interstitial fluid flow in
`the proposed mechanistic model can be modulated to account
`for interspecies differences in glycosaminoglycan content. The
`binding affinity of the therapeutic protein to possible ligand in
`the sc space could also be determined from in vitro studies. If
`such a ligand exists in the sc space, it can hinder the migra-
`tion and absorption of the therapeutic protein by trapping it in
`the skin. This is especially important in the case of monoclonal
`antibodies. mAbs are designed to bind their target with high
`affinity, if the target is abundant in the sc space; this can hin-
`der the absorption of mAbs. Furthermore, FcRn binding and
`uptake is key in mAb absorption from sc space.14 Thus, deter-
`mining the affinity and abundance of mAb ligands in different
`preclinical models as well as mAb/FcRn binding affinity as com-
`pared with humans, can further improve the predictive power
`of preclinical data.
`Similar all-encompassing models for oral absorption exist
`with successful commercial software applications capable of in-
`corporating a wide range of information such as drug proper-
`ties, in vitro–in vivo correlation of transport and metabolism
`data as well as physiological data. Similar efforts are needed
`to develop such models that can better predict the absorption
`and bioavailability of this important class of therapeutics after
`sc administration.
`
`ACKNOWLEDGMENTS
`
`The authors would like to acknowledge Krithika Shetty for
`her input and help during the revision of this manuscript. The
`
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`COMMENTARY
`
`authors are grateful for the financial support from National
`Heart, Lung and Blood Institute, National Institute of Health,
`HL-70227 to Dr. Sathy Balu-Iyer.
`The authors declare that there is no financial conflict of in-
`terest.
`
`REFERENCES
`1. Leader B, Baca QJ, Golan DE. 2008. Protein therapeutics: A sum-
`mary and pharmacological classification. Nat Rev Drug Discov 7(1):21–
`39.
`2. Bookbinder LH, Hofer A, Haller MF, Zepeda ML, Keller GA, Lim JE,
`Edgington TS, Shepard HM, Patton JS, Frost GI. 2006. A recombinant
`human enzyme for enhanced interstitial transport of therapeutics. J
`Control Release 114(2):230–241.
`3. Frost GI. 2007. Recombinant human hyaluronidase (rHuPH20): An
`enabling platform for subcutaneous drug and fluid administration. Ex-
`pert Opin Drug Deliv 4(4):427–440.
`4. Richter WF, Bhansali SG, Morris ME. 2012. Mechanistic determi-
`nants of biotherapeutics absorption following SC administration. AAPS
`J 14(3):559–570.
`5. Sarah Y. Yuan and Robert R. Rigor. 2010. Structure and function
`of exchange microvessels. Regulation of Endothelial Barrier Function.
`San Rafael (CA): Morgan & Claypool Life Sciences.
`6. Swartz MA. 2001. The physiology of the lymphatic system. Adv Drug
`Deliv Rev 50(1, ¨A`I2):3–20.
`7. Sarin H. 2010. Physiologic upper limits of pore size of different blood
`capillary types and another perspective on the dual pore theory of mi-
`crovascular permeability. J Angiogenes Res 2(14):14.
`8. Jones EA, Waldmann TA. 1972. The mechanism of intestinal uptake
`and transcellular transport of IgG in the neonatal rat. J Clin Invest
`51(11):2916–2927.
`9. Dickinson BL, Badizadegan K, Wu Z, Ahouse JC, Zhu X, Simister
`NE, Blumberg RS, Lencer WI. 1999. Bidirectional FcRn-dependent IgG
`transport in a polarized human intestinal epithelial cell line. J Clin
`Invest 104(7):903–911.
`10. Claypool SM, Dickinson BL, Wagner JS, Johansen FE, Venu N,
`Borawski JA, Lencer WI, Blumberg RS. 2004. Bidirectional transep-
`ithelial IgG transport by a strongly polarized basolateral membrane
`Fcgamma-receptor. Mol Biol Cell 15(4):1746–1759.
`11. Ghetie V, Hubbard JG, Kim JK, Tsen MF, Lee Y, Ward ES. 1996.
`Abnormally short serum half-lives of IgG in beta 2-microglobulin-
`deficient mice. Eur J Immunol 26(3):690–696.
`12. Junghans RP, Anderson CL. 1996. The protection receptor for IgG
`catabolism is the beta2-microglobulin-containing neonatal intestinal
`transport receptor. Proc Natl Acad Sci USA 93(11):5512–5516.
`13. Tzaban S, Massol RH, Yen E, Hamman W, Frank SR, Lapierre
`LA, Hansen SH, Goldenring JR, Blumberg RS, Lencer WI. 2009.
`The recycling and transcytotic pathways for IgG transport by FcRn
`are distinct and display an inherent polarity. J Cell Biol 185(4):673–
`684.
`14. Kagan L, Turner MR, Balu-Iyer SV, Mager DE. 2012. Subcutaneous
`absorption of monoclonal antibodies: Role of dose, site of injection, and
`injection volume on rituximAb pharmacokinetics in rats. Pharm Res
`29:9.
`15. Khawli LA, Goswami S, Hutchinson R, Kwong ZW, Yang J, Wang
`X, Yao Z, Sreedhara A, Cano T, Tesar D, Nijem I, Allison DE, Wong
`PY, Kao YH, Quan C, Joshi A, Harris RJ, Motchnik P. 2010. Charge
`variants in IgG1: Isolation, characterization, in vitro binding properties
`and pharmacokinetics in rats. mAbs 2(6):613–624.
`16. Igawa T, Tsunoda H, Tachibana T, Maeda A, Mimoto F, Moriyama
`C, Nanami M, Sekimori Y, Nabuchi Y, Aso Y, Hattori K. 2010. Reduced
`elimination of IgG antibodies by engineering the variable region. Pro-
`tein Eng Des Sel 23(5):385–392.
`17. Devenny JJ, Wagner RC. 1985. Transport of immunoglobulin G
`by endothelial vesicles in isolated capillaries. Microcirc Endothelium
`Lymphatics 2(1):15–26.
`
`18. Hong G, Chappey O, Niel E, Scherrmann JM. 2000. Enhanced cel-
`lular uptake and transport of polyclonal immunoglobulin G and fab
`after their cationization. J Drug Target 8(2):67–77.
`19. Negrini D, Moriondo A. 2011. Lymphatic anatomy and biomechan-
`ics. J Physiol 589(Pt 12):2927–2934.
`20. Reddy ST, Berk DA, Jain RK, Swartz MA. 2006. A sensitive
`in vivo model for quantifying interstitial convective transport of in-
`jected macromolecules and nanoparticles. J Appl Physiol 101(4):1162–
`1169.
`21. Xie DD, Hale V. 1996. Factors affecting the lymphatic absorption
`of macromolecules following extravascular administration. Pharm Res
`13p S396
`22. Bocci V, Muscettola M, Grasso G, Magyar Z, Naldini A, Szabo G.
`1