Lymphatic transport of proteins after subcutaneous administration

MINI-REVIEW

Lymphatic Transport of Proteins AfterSubcutaneous Administration

CHRISTOPHER J. H. PORTER, SUSAN A. CHARMAN*

Department of Pharmaceutics, Victorian College of Pharmacy Monash University, 381 Royal Parade,Parkville, Victoria, 3052, Australia

Received 2 September 1999; revised 15 November 1999; accepted 18 November 1999

ABSTRACT: This mini-review summarizes the relevant literature regarding the lym-phatic transport of proteins after subcutaneous administration. A review of the physi-ology of the lymphatics and inherent anatomical differences between blood and lymphcapillaries is presented followed by a brief overview of the general characteristics ofprotein absorption and bioavailability following S.C. injection. A description of factorsknown to directly affect the lymphatic uptake of macromolecules follows and is sup-ported by representative data from this laboratory. A brief perspective on the impor-tance of lymphatic uptake and transport in understanding the biopharmaceutical prop-erties of protein drugs and potentially targeting the lymphatics is presented. © 2000Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89: 297–310, 2000

INTRODUCTION

Subcutaneous (S.C.) administration continues torepresent the primary route of delivery for pro-tein-based drugs despite significant efforts to de-velop non-parenteral delivery systems for theseagents. The reasons for this are many-fold andinclude the low and variable systemic bioavail-ability with many non-parenteral routes, the rela-tive ease and speed of development of parenteraldosage forms compared with more complicated,non-parenteral delivery systems (e.g., inhaledsystems), and stability considerations upon stor-age and administration. Even though S.C. deliv-ery has been utilized extensively for a number ofyears, very little is known about the processes

that govern the absorption of macromoleculesfrom the interstitial space and the resulting im-pact of these processes on bioavailability andpharmacokinetic profiles. Proteins larger thanabout 16–20 kDa are generally thought to betaken up primarily by the lymphatic system,1–7

but very few studies have experimentally inves-tigated this process. Furthermore, the effects ofvarious external factors, including the site andmethod of administration and even “simple” for-mulation variables (e.g., pH and ionic strength)on the lymphatic transport of subcutaneously-injected proteins are still largely undefined.

This mini-review summarizes the relevant lit-erature regarding the lymphatic transport of pro-teins after S.C. administration. A review of thephysiology of the lymphatics is presented, whichfocuses on the inherent anatomical differences be-tween blood and lymph capillaries which controltheir respective permeabilities. This is followed

*Correspondence to: S. A. Charman (E-mail: [email protected])Journal of Pharmaceutical Sciences, Vol. 89, 297–310 (2000)© 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association

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by a brief overview of the general characteristicsof protein absorption and bioavailability followingS.C. injection to provide a background of indirectevidence for potential transport via the lymphat-ics. Factors known to directly affect the lymphaticuptake of macromolecules from the injection sitewill then be discussed using recent data from thislaboratory to illustrate the role of lymphatic up-take and transport in the bioavailability of hu-man growth hormone (hGH) after S.C. adminis-tration. The final section will provide a brief per-spective on the importance of lymphatic transportfrom the standpoint of fully understanding thebiopharmaceutical properties of protein drugsand potentially targeting the lymphatics for thetreatment of various lymph-resident diseases andconditions. It is not within the scope of this com-munication to review the literature pertaining tolymphatic transport of particulates or micellar-and colloidal-based delivery systems for smallmolecular weight drugs. The reader is referred toother excellent reviews7–11 for further informa-tion on these topics.

OVERVIEW OF THE LYMPHATIC SYSTEM

Most of the constituents of plasma move freelythrough capillary walls to form interstitial fluid,however, more fluid leaves the blood capillariesthan is returned by absorption with the excessfluid (about 3 L/day) draining into the lymphaticsto form lymph. The lymphatics draining the in-terstitial spaces are responsible for the absorp-tion of excess fluid, protein, and cellular elementswhich are not reabsorbed by the blood capillaries.However, unlike the blood circulation, the flow oflymph is unidirectional, recovering fluid from theperiphery and returning it to the vasculature.12

The initial or terminal lymphatics collectlymph from the periphery and unite to formprenodal collecting vessels or afferent lymphaticswhich transport lymph to the regional lymphnodes. Postnodal (efferent) lymph vessels carrylymph between successive sets of nodes or tolarger lymphatic collecting vessels which thendrain the lymph from the final set of lymph nodesinto the principle lymph ducts. Lymph from theintestinal, hepatic, and lumbar areas finallydrains into the cisterna chyli, which acts as a col-lecting reservoir at the distal end of the thoraciclymph duct. The major lymphatic vessel, the tho-racic lymph duct, ascends from the cisterna chyli,receiving lymph from the mediastinum and even-

tually, from all parts of the body (except in somecases where the upper right quadrant is drainedby a “right” thoracic lymph duct), and then emp-ties directly into the venous blood at the junctionof the left internal jugular vein and the left sub-clavian vein.12

All lymph passes through at least one set oflymph nodes (and in many cases several sets ofnodes) on its passage from the periphery back tothe systemic circulation.13 Lymph enters thelymph nodes via one, or many, afferent lymphducts, flows through the medullary sinuses, andexits through the hilus into a single efferent lym-phatic. Exchange of various materials betweenthe blood and the lymph may occur in the lymphnodes, however, the mechanism of this exchangeis poorly understood. Large numbers of macro-phages line the medullary sinuses and are re-sponsible for the phagocytosis of cellular and par-ticulate material from the lymph. The lymphnodes are also a center for lymphocyte prolifera-tion with B-lymphocytes, T-lymphocytes, and B-lymphocyte-derived antibodies entering thelymph via the lymph nodes.14

Physiology of the Interstitial Space

The structural characteristics of the interstitialspace are similar in all tissues, consisting of afibrous collagen framework supporting a gelphase made up of glycosaminoglycans, salts, andplasma-derived proteins. The glycosaminoglycansare polyanionic polysaccharides that are fullycharged at physiological pH and which, with theexception of hyaluronan, are bound covalently toa protein backbone to form proteoglycans whichare immobilized in the interstitium. Hyaluronan,is not immobilized and may be removed from theinterstitium via the lymph in a flow-dependentmanner.15–17

The proteins present in the interstitial spaceare qualitatively the same as those in the plasma,although quantitatively, they are present in lowerconcentrations most likely due to restricted ex-travasation by transcapillary flux.12,18,19 One ofthe major functions of interstitial proteins is tomaintain extravascular colloid osmotic pressureand in this regard, protein concentrations in theinterstitium are approximately 50% of those inplasma resulting in the interstitial colloid osmoticpressure (COPI) being less than that in plasma.20

In comparison, the fluid interstitial hydrostaticpressure (Pi) is small and slightly negative.

Since the capillary endothelial barrier is rela-

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tively poorly permeable to large hydrophilic mac-romolecules, endogenous proteins are primarilycleared from the interstitium via the lymph and,at least in the initial lymphatics, the lymphaticprotein concentrations reflect those in the inter-stitial space.20 Endogenous protein removal fromthe interstitium has been estimated using albu-min to be approximately 2–2.5%/h and is sensi-tive to both physical and pathophysiologicalchanges, increasing 3–4-fold during muscle activ-ity or edema.21,22

Diffusion of macromolecules within the inter-stitium may be physically retarded by the fibrouscollagen network and the gel structure of the pro-teoglycans as well as by electrostatic interactionwith charged components of the interstitial archi-tecture. Although contradictory reports exist,23

the prevailing view is that the negative chargessupplied by hyaluronan and the proteoglycansseem likely to provide an overall net negativecharge in the interstitium.20,24 The interstitiumalso displays a high degree of structural hetero-geneity and it is thought that aqueous pores orchannels are present which provide a “free-fluid”phase in addition to the presence of a “colloid-rich” gel phase.25–29 Electron microscopic investi-gations suggest channel diameters of 50–100nm,28 whereas application of pore theory to thediffusivity of proteins across the interstitium sug-gests pore sizes some five times smaller (120–200Å).26 Other studies have suggested that the effec-tive interstitial distribution volumes are dictatednot only by the presence of a free-fluid phase anda gel phase, but also by the presence of endog-enous macromolecules within the gel phasethereby restricting “access” to the available vol-ume. This network is thought to effectively reducethe distribution volume such that the intersti-tium acts in a size exclusion manner, excludingvery large molecules and thereby promoting theirinterstitial transport.19,27,30–32

The process of macromolecular diffusion acrossthe interstitium is likely dictated by molecularsize, the presence of diffusional microdomains,and physical and electrostatic interactions withthe various components of the interstitium. Inthis regard, Bell and co-workers suggest thatsteric effects predominate,19 however, a clear pic-ture of these complex interactions is still elusive.The composition and structure of the interstitiumhas been extensively reviewed elsewhere,20,24,33

and the interested reader is directed to these re-views for further information.

Lymphatic Drainage of the Interstitial Space

The rate of filtration and reabsorption of fluidacross the vascular capillaries is high (20–40L/day) in comparison to the small quantities ofinterstitial fluid drained by the lymph (2–4L/day). Consequently, small molecules (<2,000molecular weight) which can access lymph andblood capillaries equally are predominantlycleared by the blood vessels, whereas particulatesand molecules of increasing molecular size appearto favor drainage into the more open capillaries ofthe lymph. Three different types of blood capillar-ies exist within the vasculature and may be clas-sified in terms of their endothelial structure aseither continuous, fenestrated or discontinuous.The blood capillaries supplying the subcutaneousspace are generally continuous in nature andcharacterized by tight interendothelial junctionsand an uninterrupted basement membrane.These blood capillaries are relatively permeableto the exchange of small, lipophilic molecules, andby virtue of capillary “pores” (probably a combi-nation of plasmalemmal vesicles, transendothe-lial channels, and endothelial junctions), somehydrophilic molecules such as water. In contrast,the endothelium of blood vessels constitutes a sig-nificant barrier to the transfer of large, hydro-philic molecules such as proteins.

The structure of the lymphatic capillaries dif-fers markedly from that of blood capillaries as itconsists of a single layer of overlapping endothe-lial cells with an incomplete basal lamina and anabsence of interendothelial tight junctions (Fig-ure 1). The lymphatic endothelium thus has amore “open” structure with larger intercellularjunctions than found in blood capillaries. Esti-mates of intercellular junctional distances varyfrom several microns34–36 to 15–20 nm.37,38 Thestructure of the lymphatic capillaries is main-tained by anchoring filaments which attach to thewall of the lymphatic capillary and to the collagenfibers of the interstitium.35,39 These anchoringfilaments, in association with the overlapping en-dothelial cells, provide an endothelial “flap,” orlymphatic endothelial microvalve, which allowsthe unidirectional flow of fluid into the initiallymphatics when interstitial fluid pressure israised. In the presence of an increase in intralu-minal pressure, the flap falls back against thelymph wall, preventing fluid flow back into theinterstitium.40 Transfer of various tracers such asferritin and horseradish peroxidase also occurs

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across the lymphatic endothelium in associationwith cytoplasmic vesicles.38,41–43

The driving force for fluid transfer from theinterstitium into the initial lymphatics has beenthe subject of significant debate. Clearly, a poten-tial energy difference in the fluid phase betweenthe interstitium and the lymphatics is requiredand may take the form of a chemical gradient,with the associated osmotic or oncotic pressuregradients, or a hydrostatic pressure differential.Casley-Smith has suggested that initial lym-phatic filling is facilitated by an osmotic gradientset up in the initial lymphatics by the filtration ofprotein-poor fluid from the lymphatics into theinterstitium.44 However, other investigators haveshown that the colloid osmotic pressures in theinterstitium and the initial lymphatics are simi-lar at steady state.45 More recent studies suggestthat lymphatic filling is facilitated via periodicchanges in interstitial pressure and volume

which are transmitted via the anchoring fila-ments to the lymphatics causing them to expandand contract.46–51 The periodic tissue pressuresinherent in this explanation include mechanismsprevalent in “inactive” subjects such as arterialpressure pulsations, respiration, and contractionsof the heart,49,52 in addition to larger variationsstimulated by skeletal muscle contraction,51 skinrubbing,50,51 and external compression such asthat experienced during running or walking. Theadditional lymphatic filling pressures provided byphysical movement appear to explain the en-hanced ability of unanesthetized animals to com-bat edema via an increase in lymph flow.53–55

The initial lymphatics drain into larger collect-ing lymphatics which, unlike the initial lymphat-ics, contain a layer of smooth muscle makingthem capable of spontaneous contraction. This ac-tion propels lymph through the rest of the lym-phatic system and the lymph nodes through to thethoracic duct and into the systemic circulation.Even though the concentration of albuminchanges on passage through lymph nodes in re-sponse to alterations in venous pressure andlymph colloid osmotic pressure,56–58 there ap-pears to be almost complete recovery of albuminon passage through the lymphatics except atmarkedly increased lymphatic outflow pres-sures.59,60

ABSORPTION OF PROTEINS AFTERS.C. ADMINISTRATION

After S.C. administration, the absolute bioavail-ability of proteins is generally variable and in-complete relative to an I.V. dose with values rang-ing from about 20% up to 100% (Table I). Thebasis for the reduced S.C. bioavailability is notcurrently known, although degradation or me-tabolism at the site of injection has been proposedas a general mechanism.3,4,61,62 Subcutaneousdegradation has been specifically suggested forseveral proteins including hGH,63,64 erythropoie-tin (EPO),3,65 and insulin,66–68 however, direct ex-perimental evidence for this putative mechanismis limited. For example, whilst aprotinin, a serineprotease inhibitor, has been reported to enhancethe rate and extent of absorption of parathyroidhormone,69 calcitonin,69 and insulin,67,70,71 Lindeet al. have also demonstrated a concomitant in-crease in subcutaneous blood flow with aprotininwhich may account for the change in the absorp-tion kinetics.71

Figure 1. Electron micrographs illustrating the over-lapping endothelial cells of lymphatic capillaries. Theintercellular cleft regions are denoted by (*). Magnifi-cation (a) ×76,500, (b) ×21,250, (c) ×31,150, (d) ×23,800.(Reproduced from J. Cell Biol., 1971, 50, 300–323, bycopyright permission of The Rockefeller UniversityPress.)



Rate of Protein Absorption AfterS.C. Administration

Subcutaneously administered proteins generallyexhibit a relatively slow rate of absorption as evi-denced by a prolonged terminal half-life in com-parison to that observed after I.V. administration.Maximum plasma concentrations after S.C. injec-tion occur from 2 to 20 h post-dosing (Table I)with the rate of absorption being dependent onvarious external factors including the site of in-jection, the subcutaneous blood flow, and the ap-plication of heat or massage.4

The influence of the site of injection on proteinabsorption has been demonstrated for insu-lin,70,72–74 hGH,75 and EPO.76 In these studies,the rate of absorption was increased with S.C.injection in the abdomen in comparison to moreperipheral extremities such as the thigh or theupper arm. While this effect is typically ascribedto differences in local blood flow, several authorshave also highlighted a possible link to regionalvariations in lymph flow.4,76 Although other ex-ternal factors such as exercise might be expected

to increase both blood and lymph flow, factorssuch as massage are also known to dramaticallyincrease lymph flow,20,49,50 without a measurableeffect on subcutaneous blood flow.77 Unfortu-nately, variations in blood and lymph flow at thedifferent anatomical sites utilized for S.C. injec-tions are not well defined.

The effect of blood flow on the rate of proteinabsorption from the interstitial space is fre-quently inferred; however, only a few studieshave simultaneously measured subcutaneousblood flow (SBF) and the rate of protein absorp-tion, and most of these studies have been con-ducted with insulin. Insulin absorption is typi-cally studied by monitoring the disappearance of125I-labeled material from the injection site withthe assumption that the resulting disappearancerate equals the rate of appearance in blood. In aseries of studies examining the disposition of in-sulin,78 an increased rate of absorption in re-sponse to alterations in injection site, skinfoldthickness, exercise, orthostatic changes, ambienttemperature, and other external factors were pri-

Table I. Representative Bioavailability Values and Times to Reach MaximumPlasma Concentrations Following S.C. Administration of Various Protein Drugs*

Protein

ApproximateMonomericMW (×103) Species

Absolute BA(%)

TMAX(h) Reference

Insulina 5.6 Humans 84 1–2 114–116IGF-1 7.6 Humans 100 7 117, 118IL-3 13.2 Monkeys 40 2–4 119IL-2 15.5 Humans 30–80 2–4 120, 121IL-10b 18.7 Humans 42 4–6 88IL-11 19 Humans 65 2–3 122TNF-a 17.4 Humans c 2 123, 124GM-CSFd 15.5–19.5 Humans 50 3 125G-CSFd 18–22 Monkeys 40–50 2–3 126hGH 22 Humans 50 5–6 127–129IFN-a 19.5 Humans >80 6–8 130, 131IFN-b 23 Rabbits 12 1 105IFN-gd 20–25 Humans 30–70 6–13 131, 132FSHb,d 36 Humans 66 0.5 133, 134EPOd 34–39 Humans 20–36 13–18 65, 135Factor IX 56 Dogs 63.5 19 136

* Abbreviations: insulin-like growth factor I (IGF-I), interleukin (IL), tumor necrosis factor(TNF), granulocyte macrophage-colony stimulating factor (GM-CSF), granulocyte-colony stimu-lating factor (G-CSF), human growth hormone (hGH), interferon (IFN), follicle stimulating factor(FSH), erythropoietin (EPO).

a Normally present as a hexamer.b Normally present as a dimer.c Too low to be assessed.d May be glycosylated.

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marily attributed to increased SBF measured bydisappearance of 133Xe from the subcutaneous tis-sue. A curvilinear relationship between SBF andthe rate of disappearance of radiolabeled insulinwas found and as a result, factors other thanblood flow (i.e., depolymerization, interstitial dif-fusion, capillary permeability) were proposed asbeing rate limiting at high SBF. The authors wenton to describe the capillary diffusion capacity, de-fined as the amount of tracer passing the capil-lary membrane per 100 g of tissue per min, asbeing 5–10-fold lower for insulin than for inulin,which is of a similar monomeric molecular weightas insulin. On this basis, it was proposed thatinsulin must be transported within the intersti-tium in a polymeric form, yet lymphatic transportwas suggested to represent only a minor compo-nent of absorption. Further studies74,79–82 havealso shown an inverse relationship between therate of disappearance of radiolabeled insulin fromthe subcutaneous injection site and the averageeffective size of the insulin unit, although lym-phatic transport has not typically been thought tocontribute significantly to insulin absorption.Studies presented by Binder in 196983 supportthis hypothesis and suggest that lymphatic up-take does not play a significant role in the absorp-tion of porcine (hexameric) insulin, although morerecent results by Supersaxo et al.2 have shownthat lymphatic transport accounts for approxi-mately 20% of the absorption of subcutaneouslyadministered inulin, which is of a similar size tothe insulin monomer. Given the size of mono-meric insulin and of the insulin hexamer, it ispossible that multiple mechanisms control the ab-sorption and further studies are necessary tomore clearly define these processes.

Mathematical Models to CharacterizeProtein Absorption

Several mathematical models have been proposedto account for the slow absorption kinetics of pro-teins following S.C. administration. Initial phar-macokinetic models used to describe insulin ab-sorption assumed a split-pool model, whereby in-sulin absorption takes place from an undefinedpool secondary to the injection pool.84 More recentreports have utilized a combined diffusion–dissociation model to account for the absorptionkinetics and the differences in concentration andvolume effects for monomeric and hexameric in-sulin.85 While these models adequately describethe kinetic profiles for S.C. insulin and are in

agreement with clinical data,86 they do not pre-clude a potential lymphatic component for insulinabsorption, even though the contribution may beonly minor.

Other models have been proposed to describethe plasma pharmacokinetics of proteins afterS.C. administration assuming at least partial up-take via the lymphatics. Bocci et al.87 proposed athree-compartment model with parallel zero- andfirst-order input to describe the plasma concen-tration versus time profiles of interferon-a2 (IFN-a, MW 19,000) after S.C. administration in com-bination with albumin. The prolonged absorptionin the presence of albumin was attributed to en-hanced lymphatic transport due to an increase inthe colloid osmotic pressure of the interstitialfluid. Studies by Radwanski et al. published in199888 utilized a similar model to describe theplasma pharmacokinetics for subcutaneously ad-ministered interleukin-10 (IL-10, non-covalentdimer with monomeric MW 18,700). In thesestudies, the rapid appearance of the protein inplasma was attributed to direct delivery to theblood via a zero-order process, whereas the lagphase and the associated prolonged plasma con-centrations were ascribed to gradual uptake andtransport via the lymph as described by a first-order process presumed to be controlled primarilyby lymph flow rate. Based on the fitted absorptionparameters, the first-order lymphatic componentwas found to represent 95% of the overall absorp-tion process. These authors highlighted that thepresence of proteolytic enzymes in S.C. or lymphcompartments may partially account for the re-duced bioavailability (42% in the case of IL-10relative to an I.V. dose) with this delivery route.

It is apparent that the rate and extent of ab-sorption of proteins directly into the systemic cir-culation after S.C. administration is significantlylower than that commonly seen for small mol-ecules. Even though many studies have alluded tothe possibility of lymphatic transport of thesemolecules, confirmatory studies which define theextent of lymphatic transport and the contribu-tion of this pathway to the absorption kineticsand bioavailability are limited.

Factors Affecting Lymphatic Transport ofProteins after S.C. Administration

The lymphatic transport of exogenous macromol-ecules was first reported in the 1950s after intra-muscular and intrapleural administration of high



molecular weight salts of antibiotic bases such asstreptomycin and neomycin.89 Transport of mono-clonal antibodies and radiolabeled macromol-ecules including dextran, albumin, and hydroxy-ethyl starch via the lymphatics has also been ex-tensively examined in relation to imaging agentsin lymphoscintigraphic studies, although the ab-solute extent of lymphatic transport has rarelybeen quantified.90–95 Muranishi and co-workers96

also utilized the lymph-directing qualities of dex-tran macromolecules by forming a dextran (MW500,000) prodrug of bleomycin (BLM) to enhancedelivery to the regional lymph nodes after injec-tion into the stomach wall of the rat. Takakuraand co-workers97–99 further confirmed the utilityof macromolecular prodrugs to enhance lym-phatic transport in studies using a series of dif-ferent sized (MW range of 10,000–500,000) posi-tively and negatively charged dextran prodrugs ofthe antitumor antibiotic, mitomycin C (MMC) af-ter intramuscular (I.M.) injection. In the latterstudies, macromolecules of increased size andpositive charge displayed lower rates of absorp-tion from the interstitial injection site, relativelyhigher affinity for local lymph nodes, and reducedappearance in the thoracic lymph, compared withsmaller, negatively charged prodrugs. Furtherevidence of the lymphotropic nature of macromo-lecular prodrugs was supplied by the studies ofMaeda and co-workers,100–102 who showed thataccumulation of a conjugate of the anti-tumoragent, neocarzinostatin (NCS) with a poly(maleicacid)/styrene oligomer in the regional lymphnodes was up to 10-fold higher after S.C. admin-istration than after I.V. administration.

Interest in the lymphatic disposition of thera-peutic proteins after S.C. or I.M. injection has in-creased relatively recently due primarily to theincreasing number of proteins in various stages ofdevelopment and the overwhelming prevalence ofparenteral administration for these drugs. In thisregard, Supersaxo and co-workers published a se-ries of seminal articles which quantified, for thefirst time, the extent of uptake of selected macro-molecules into the peripheral lymphatics after in-terstitial injection.2,103 Using a sheep model inwhich the efferent lymphatic duct of the popliteallymph node was cannulated, the studies demon-strated that the primary route of initial absorp-tion of recombinant interferon-a2a (IFN-a) afterintradermal or S.C. administration into the lowerhind leg was the regional lymphatics with ap-proximately 59% of the dose being recovered inthe popliteal lymph. Further studies examined

the lymphatic transfer of a series of compoundswith increasing molecular weights (5-fluoro-28-deoxyuridine, MW 246.2; inulin, MW 5,200; cyto-chrome c, MW 12,300; and IFN-a, MW 19,000)and showed a direct correlation between molecu-lar weight and the extent of recovery in the pop-liteal lymph (Figure 2).2 The correlation sug-gested that more than 50% of an administereddose would be absorbed by the regional lymphat-ics after S.C. administration for molecules withmolecular weights exceeding approximately16,000.

The lymphatic transport of IFN-a and glyco-sylated IFN-b into the central (thoracic) lymphhas also been widely studied.87,104–109 In contrastto the large extent of peripheral lymphatic trans-port of IFN-a reported by Supersaxo et al.,103 theamount of IFN-a recovered in the thoracic lymphof rabbits after a single S.C. injection was low(<0.1% of dose) but increased 4-fold when injectedat multiple sites. In the case of IFN-b, the extentof lymphatic transport was reported as the tho-racic lymph/plasma concentration ratio andranged from 1.2 after S.C. injection107 to 3.8107

and 1–2110 after I.M. injection. By way of com-parison, the lymph/plasma concentration ratiosestimated for thoracic lymph and IFN-a were ap-proximately 20–30,104 and the concentration ra-tios for IFN-a in peripheral lymph relative to sys-temic plasma were in the range of 2,000–8,000.103

Figure 2. Relationship between molecular weightand cumulative % dose recovered in popliteal lymphfollowing S.C. administration into the lower part of thehind leg of sheep (mean ± SD, n 4 3–4). Data fromSupersaxo et al. (●) and Charman et al. (s).

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Subsequent studies examined the effect of al-bumin inclusion in the formulation, speculatingthat albumin would increase interstitial oncoticpressure thereby stimulating lymph flow. Bocci etal. showed a 2-fold increase in thoracic lymphatictransport of both IFN-a in the presence of 4% al-bumin104 and IFN-b in the presence of 13% albu-min107 after S.C. administration relative to albu-min-free formulations. Conversely, 13% albuminreduced the lymphatic transport of IFN-b afterI.M. injection, and other studies have shown thatintradermal administration of IFN-a with either0.5% or 13% albumin had no effect on the extentof IFN transport into the politeal (peripheral)lymph.103 Bocci et al. have also demonstrated in-creased lymphatic transport after inclusion of hy-aluronidase in formulations in an effort to stimu-late interstitial edema104 and increased lymphand blood transport with the inclusion of hista-mine or bradykinin as alternative edematogenicagents.109 Again, the extent of lymphatic trans-port in both cases was low in mass terms. Thetransport of human tumor necrosis factor (TNF,MW 20,000–40,000) into thoracic lymph afterS.C. injection to rats was also found to be ex-tremely low (0.02–0.03% of dose), although lym-phatic transport was considerably enhanced afterinjection into the stomach wall or gut wall.111

The reasons for the marked differences in theextent of protein transport into the peripheraland central lymph have not been fully defined butmay be related to the different animal models andmore importantly, to the different sites of lymphcannulation. Studies recently conducted in thislaboratory have extended the sheep model origi-nally described by Hall and Morris112 and laterused by Supersaxo et al.,1,2,103 to allow for simul-taneous sampling of blood and collection of eitherperipheral or central lymph in the same species(Figure 3).113 A parallel group study was con-ducted using human growth hormone (hGH, MW22,000) as a model protein with one I.V. and threeS.C. treatment groups. For I.V. and S.C. controlgroups, only blood was sampled for the estimationof systemic availability. For the peripherallymph-cannulated group, blood was sampled andlymph collected continuously from the popliteallymph duct and for the central lymph-cannulatedgroup, blood was sampled and lymph was col-lected from the thoracic lymph duct. Approxi-mately 61.7% of a S.C. dose was collected in pe-ripheral lymph draining the injection sitewhereas only 8.6% was collected in central (tho-racic) lymph (Figure 4). These data are consistent

with previously reported results with respect tothe extent of transport into peripheral lymph (seeFigure 2) and the dramatic difference between theamounts of protein recovered in peripheral andcentral lymph. In non-lymph-cannulated sheep(i.e., the S.C. control group), the systemic avail-ability was approximately 58% whereas absorp-tion into the blood decreased to 32–40% when ei-ther peripheral or central lymph was collected.When the total recovery was calculated as thesum of the systemic availability and the cumula-tive amount recovered in lymph, approximately93% of the dose was accounted for in the periph-eral lymph-cannulated group in comparison toonly 49% for the central lymph-cannulated group(Figure 4). These data indicated that loss at theinjection site was minimal and that the apparent“clearance” within the lymphatics was the likelybasis for the reduced bioavailability of hGH inthis animal model. While the initial transfer fromthe interstitium to the lymphatic capillariesmay be dictated primarily by molecular size, it islikely that clearance mechanisms within the lym-phatics will be protein-specific. Further studiesare required to more clearly characterize theseprocesses.

SUMMARY AND PERSPECTIVES

In spite of the considerable effort directed towardthe design of non-parenteral delivery systems fortherapeutic proteins, the bioavailability limita-tions associated with non-parenteral routes still

Figure 3. Schematic representation of the sites ofS.C. injection, blood sampling, and peripheral and cen-tral lymph sampling in the sheep model used by Char-man et al.



dictate that the majority of proteins are currentlyadministered parenterally with the S.C. routepredominating. For the most part, the mecha-nisms underlying protein drainage from the injec-tion site and transport to the systemic circulationhave been only partially defined. It is apparentthat the lymphatic system plays a significant rolein the distribution of S.C. administered proteins,although the available data are complicated byvariations in the animals models used and thesites of lymphatic cannulation. Review of the lit-erature suggests that proteins drain from the in-jection site into both the peripheral lymph andthe blood capillaries and that uptake into the lym-phatics increases with molecular size in an ap-proximately linear manner. As such, greater than50% of a S.C. dose might be expected to drain intothe peripheral lymph for molecules with molecu-lar weights exceeding approximately 16,000. Fur-ther preliminary results from this laboratory sug-gest that protein “clearance” upon passagethrough the lymphatics may be significant lead-

ing to much lower concentrations in thoraciclymph than in peripheral lymph and possibly ac-counting, in part, for the reduced systemic avail-ability of these molecules.

A further understanding of the inherentlymph-directing properties of proteins after S.C.administration and the possibility of increasedresidence or uptake upon passage through thelymphatics has significant ramifications in termsof the activity and toxicity of these agents. Thelymphatics present an appealing target for im-munomodulatory agents, vaccines, and anti-metastatic compounds, and optimization of lym-phatic transport may prove beneficial for suchtherapeutic agents. Conversely, if lymphaticclearance leads to a reduction in systemic bio-availability, opportunities to reduce lymphatictransport may improve the availability and activ-ity of molecules for which the active site residesoutside of the lymphatics. Application of the para-digm which dictates that assessment of the “ex-posure” of patients to S.C. administered proteinsshould be via plasma or serum bioavailability es-timations may be inappropriate.

Realizing the importance of the potential roleof the lymphatics in the absorption and distribu-tion of therapeutic proteins after S.C. administra-tion, future studies might usefully address the po-tential effect of protein charge or protein modifi-cation on lymphatic uptake and/or clearance, aswell the importance of formulation variables suchas ionic strength, pH, and viscosity on the effec-tive size of proteins within the interstitium andthe resulting effect on lymphatic drainage.Greater insight into these and other factors mayindicate useful approaches to maximize the po-tential benefit of this route of absorption.

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Lymphatic transport of proteins after subcutaneous administration