Indian Journal of Experimental Biology Vol . 40, October 2002, pp. 1097- 1 109

Review Article

Lymphatic transport of orally administered drugs

L Hari Vardhan Reddy & R S R Murthy*

Department of Pharmaceuti cs, Faculty of Technology and Engineering, Kalabhavan, M.S. University of Baroda, Vadoda ra 39000 1, India

Severa l therapeutic mo lecules such as li pophili c drugs and pept ides suffe r from the problems of low oral bioavail abi l­ity. Improvement of the ir bioavail ab il ity and simultaneous prevent ion of the oral degradation of the prone molecules appears to be a chall enge. Ly mphatic system, which is responsible fo r the maintenance of nui d balance, im munity and metastati c spread of cancers, is also fo und to playa major role in the oral absorption of lipids and li pophil ic drugs from intestine. The spec ialized structure of gut associated lymphoid ti ssue can be util ized as a gateway for the de li very of part icu late systems containing drugs. Eventhough a large gap has ex isted in the fie ld of lymphatic drug deli very, the introduction of a large number of lipophilic drugs and peptides has brought a renewed interest of research in th is area. In thi s review, the mecha­nisms of intestinal lymphatic drug transport, approaches taken for the de li very of macromolecu les, lipophilic and peptide drugs, biochemical barri ers in volved in in testinal drug absorption, and animal models used in the studies of intestinal lym­phatic drug transport has bee n discussed.

The drug uptake and transport into the body through intestinal lymphatics has recently received consider­able attenti on I. The speciali zed structure and func­tions of the lymphatic system all ows the site-specifi c deli very to lymph and lymphoid tissue (8- and T­lymphocytes), of cytokines and immunomodul ators and in the treatment of viral diseases li ke AIDS2

-4, the sites associated with low density lipoprotein recep­tors5 and delivery to systemic circulation. The intesti­nal lymphatic drug transport prevents the first-pass metaboli sm of several prone drugs and improve the oral bioavailbility of highl y lipophilic drugs6

.9

.

Physiologically, a majority of the orally admini stered drugs di ffuse into the enterocytes and get absorbed into the portal vein and after processed in the li ver, enter the systemic circul ation. But, the highl y lipo­philic moeities may gain access to the intestinal lym­phatics and enter the systemi c circulation via the tho­racic lymph duct. The process time of the lymphatic absorption and transport is slower than that of the por­tal blood, because of a sequence of complex events invol ved in the lymphatic transport, and hence the plasma profiles of drug absorption by both processes vary 10.

The orally admi nistered lipids are digested and ab­sorbed into the intestinal lymphatics, and the particu­late systems may gain access to the intestinal lymph th rough the gut associated lymphoid ti ssue (GALT).

*Correspondent author E-mail : murthyrsr@satyam.net.in Fax: (0265) 4 18927

Several therapeutic substances like macromolecules including peptides and peptidomimetics, highly lipo­philic drugs and their prod rugs have been attempted fo r intestinal lymphatic deli very, and their physico­chemical requirements fo r better lymphatic absorption have been studied. Deli very systems containing lipids and biodegradable polymers have been developed, and the effect of various lipids and polymers in the lymphatic transport has been described. In thi s re­view, the aspects regarding anatomy and physiology of the lymphatic system, mechani sms of drug access to lymphatics from intestine, various attempts done to enhance the intestinal lymphatic drug absorption and animal models used in the assessment of lymphatic drug concentrations has been discussed.

Physiological anatomy and functions of the lymphatic system

The lymphatic system consists of lymph ducts, lymph nodes and lymphoid ti ssues distributed in all areas of the vascular tissues of the body, which func­tion to absorb the excess fluid and cellular elements like proteins and lipids, not reabsorbed by the blood capill aries . The lymphati c system do not fo rm a circu­lar system li ke blood vascul ar system, and has a uni­directional fl ow of lymph, collecting lymph from pe­ripheral tissues and emptying into the vascular sys­tem l l

. The terminal lymphatic capillaries in the pe­riphery collect lymph and unite to fo rm collecting (afferent) lymph ducts that transport lymph to the re­gional lymph nodes. Post nodal (efferent) lymph ducts

1098 INDIAN J EXP BIOL, OCTOBER 2002

drain the lymph between successive sets of lymph nodes and ducts into the collecting reservoir known as cysterna chyli, which also receive lymph from the intestinal, hepatic and lumbar regions. Ascending part of the cysterna chyli is continued into thoracic lymph duct (major lymphatic vessel), collecting lymph also from mediastinum and cranial parts of the body ex­cept the right quadrant and empties into the venous circulation at the junction between left internal jugular and left subclavian vein. Lymph from the upper right quadrant of the body may drain into the right thoracic lymph duct that empties into the venous circulation at the junction of the right internal jugular and right sub­clavian vein.

Lymph is derived from intestinal fluid and contains most of the components of the plasma, but in lower concentrations l2

, and the electrolyte, non-electrolyte and iron levels donot differ much 13

. Lymph enters the lymph nodes by afferent lymphatic ducts and flow through medullary sinuses lined with macrophages that are responsible for phagocytosis of cellular and particulate matter, and exits through hilus into an ef­ferent lymphatic. The mechanisms of exchange of various materials between blood and lymph are poorly understood.

The lymphatic capillaries of small intestine (Iac­teals) are located in the central part of each intestinal villi, and are bigger in size than that of the lacteals of the large intestine. These capillaries from both small and large intestine unite with capillaries of mucosa and submucosa and form larger collecting lymphatics. The collecting lymphatics from small intestine and the ascending and transverse colon unite to form the su­perior mesenteric duct, which runs into the cysterna chyli, and through thoracic duct, opens into the vascu­lar system. The permeability of lymphatic capillaries to large molecules is more than that of blood capillar­ies. The fluid flow rate of portal blood is around 500times greater than that through lymph and hence, most of the orally administered drugs are preferen­tially absorbed into the portal circulation 14. Neverthe­less, the specialized structure of the lymphatic capil­laries may allow macromolecules and colloids for the lymphatic transport.

Mechanisms of drug access to lymphatics from in­testine

The drug access to intestinal lymphatics can be ex­plained by three mechanisms. 1. Macromolecular compounds are often favoured

for intestinal lymphatic transport because of high

molecular size. Small polar molecules could also be converted into macromolecular conjugates or their prodrugs can be prepared, for improved in­testinal lymphatic transport.

2. Lipid digestion and absorption pathway in the in­testine, could form an important route of the in­testinal lymphatic absorption and transport of highly lipophilic drugs with high pKa values. The drugs incorporated into the lipids, associate with them all throughout their digestion and absorption process, and transported to the lymphatic system.

3. Drugs incorporated into the colloidal systems, undergo intestinal uptake by the lymphoid folli­cles and Peyer's patches (PP) of the GALT, and transported to lymph directly or by phagocytosis effect of macrophages.

Lymphatic transport of macromolecules from intestine

The higher molecular size of macromolecules, avoids their absorption into the portal circulation and promotes uptake by intestinal lymphatics. Intestinal lymphatic transport of several macromolecules like enteric endotoxin 15, enterostaphylococcal endotoxin 16,

clostridium botulinum toxin 17, horse radish peroxi­dase l8

, albumin 19 and eiastase20 has been reported. However, the extent of macromolecular absorption is very limited, and is restricted by the barrier function of the intestine. The improvement of intestinal absorption of macromolecules could be achieved by the use of penetration enhancers21

• Yoshikawa et at. studied the effect of penetration enhancer on lymphatic transport of fluorescent labeled dextrans. These dextrans having molecular weights 10, 20, 40 and 70kD were dissolved in penetration enhancer solution (a mixed micellar solution of linoleic acid and polyoxyethylene lauryl ether) and administered to small and large intestine. From both the small and large intestine, the comparatively high molecular weight dextrans (HMD) (40 and 70kD) showed good affinity to intestinal lymphatics than that of the other dextrans in the study22, which may be due to the decreased permeability of blood capillaries to HMD.

Mixed micellar systems (MMS) have also been utilized for the absorption enhancement studies of a­and f)-interferon from the large intestine23

. MMS con­taining human fibroblast f)-interferon also showed more affinity to intestinal lymphatics resulting in very low blood concentration24

. Nevertheless, the penetra­tion enhancers should be used with caution, because of their toxicity related problems.

REDDY & MURTHY : LYMPHATIC TRANSPORT OF ORALLY ADMINISTERED DRUGS 1099

Lymphatic transport of lipophilic molecules from intestine through lipid absorption pathway

Highly lipophilic drugs could be delivered to intes­tinal lymphatics by association with dietary lipids. The dietary lipids are digested in the intestine, ab­sorbed into the intestinal lymphatics and transported to the systemic circulation2s.26. In this case, the ab­sorption of drugs does not depend on their molecular size, but it is the extent of lipophilicity and chain length of lipids that determine the drug absorption. Better drug candidates for this route of absorption would be the molecules having high pKa value indi­cating high lipophilicity and good lipid solubility, which allows better association with lipoproteins formed during the re-esterification of triglycerides in the enterocyte. The digestion and absorption pathway of dietary lipids is shown in Fig. 1.

Digestion and absorption of dietary lipids The normal diet contains lipids predominantly in

the form of triglycerides (TGs). These TGs undergo hydrolysis by the action of lingual and gastric lipases, resulting in the formation of diglyceride (OG) and fatty acid (FA) in the stomach. The shear produced by the antral contractions aid in the formation of crude emulsion of the above amphiphilic lipid digestion products (LOPs) and their emptying into the duode­num. Lipid entry into the duodenum stimulates the release of pancreatic fluids, and bile salts and biliary lipids from gall bladder, which aid in the formation of a more stable emulsion having high surface area. The action of pancreatic lipase and co-lipase on the sur­face of emulsion droplets, result in the formation of one molecule of 2-monoglyceride (MG) and two fatty acid molecules (FA) for each molecule of TG27. These LOPs pinch off from the surface of emulsion and form liquid crystalline structures, which in the pres­ence of bile salts form both unilamellar and multi­lamellar vesicles. At sufficient bile salt concentra­tions, these vehicles are solubilised to form mixed micelles28.29, which greatly enhance the luminal solu­bility of LOPs (lOOO-fold) and provide a concentra­tion gradient for the diffusion of LOPs into the en-

3031 f d· . . f . d . II terocyte . , a ter ISSOCIatlOn rom mlxe mice es, due to a low pH environment at the intestinal absorp­tion site.

FAs are transported to the enterocyte by fatty acid binding protein (FABP) and fatty acid transporter (FAT)34.3s. Finally, the transport of lipids from en­terocyte depends upon their chain length. Short and medium chain lipids (less than 12-C atoms) directly

gain access to the portal circulation, where as the long chain lipids (greater than 12-C atoms), enter the en­doplasmic reticulum (ER) and undergo re­esterification to triglycerides by one of the two path­ways, i.e. monoacyl glycerol pathway (majod6 and phosphatidic pathway (minor). The re-esterified TG assemble into the intestinal lipoproteins, gets stabi­lized and fuse with basolateral cell membrane of en­terocyte and transported to the systemic circulation via thoracic lymph duct.

Formulation approaches for intestinal lymphatic transport of lipophilic drugs

The important pre-requisites of drug candidates for intestinal lymphatic delivery is to possess high log P (log partition coefficient) values above 5 (which im­plies about 50,000times greater affinity for lymph lipid than the portal blood) and good solubility (greater than 50mg/ml) in triglycerides (for better as­sociation during re-esterification in the ER of the en­terocyte)37. Examples of lipophilic drugs that were studied for intestinal lymphatic transport include naftifine38, mepitiostanes.39, ooro, benzo(a)pyrene41, probucol42, cyclosporine43.44, ontazolast6 and halofan­trine4s (Table 1).

Lipid-based vehicles are generally used for en­hanced delivery of drugs to intestinal lymphatics, in a view that they stimulate the lymph lipoprotein turn over through the enterocyte, and increase the lipopro­tein-based lipid sink into which the drugs can parti­tion46. Several lipid-based delivery systems of differ­ent compositions and functional properties have been attempted for the formulation of lipophilic drugs47.49 . The lymphatic transport of ontazolast, a highly lipo­philic and potent LTB4 inhibitor has been studied by administering as an aqueous-based suspension and four different lipid-based emulsion systems. The re­sults indicate that the emulsion systems provided higher lymphatic drug concentration in comparison to aqueous suspension44. In another study, similar results were observed by Hauss et at. in case of a lipophilic lipid regulating agentSo

.

However, on the other hand, some interesting ob­servations were made, indicating high lymphatic transport of drugs from non-lipid based (lipid-free) vehicles. In case of a 5a-reductase inhibitor, the ad­ministration of aqueous suspension formulation showed increased lymphatic transport, when com­pared to other lipid-based formulations

s,. Similarly,

an aqueous polysorbate 80 micellar solution contain­ing retinyl palmitate showed a two-fold increase in

1100 IND IAN J EXP BIOl. OCTOBER 2002

lymphatic absorption, than the lipid-based systems52.

However, in some studi es, not much difference was found in lymphatic transport of drugs, when lipid and non-lipid based systems were used5

) . The enhance­ment of lymphatic transport of drugs from non-lipid based vehicles may be due to increased solubilization and altered membrane permeability due to surfactants.

such as lipid class, fatty acid chain length and degree of unsaturation were also found to affect the intestinal lymphatic drug transport54

-57

. Indi vidual lipids di ffe r in their rate of absorption and biochemical metabo­lism. The greater the degree of un saturati on of the fatty ac id, the rapid the onset of chylomicron sy nthe­sis, and rapid transport to the mesenteric lymph5s

.

Hence, proper selection of lipids is necessary for pro­ducing a formulation with better lymphatic drug ab-

Apart from the drug characteristics, the choice and properties of co-administered or drug-carrier li pids

Mouth

Stomach

-----j~~ Triglycerides (dietary lipids)

~ Lingual lipase

Diglycerides+Fatly acid

-----l~~ lO""k I;p'" Mi XiI of lipids by pylori c contracti ons

Intestine ----~~ Bile salts and biliary lipids

Stable emulsion havi ng very high surface area

~pancreatic lipase and Co-lipase

2-Monoglyceride+2 Fatly ac id molecules

• u nilan~e ll a r and multilamcllar vesicles

Mixedinicelles

/nintoe~

Absorbed into the portal vein Enter the endoplasmic reticulum

~MOnOaCY I glycerol pathway

~PhoSPhatidic ac id pathway

Re-esterification to triglycerides and

association to fo rm lipoproteins

~ Mesenteric lymph ---'~~ Cysterna chyli

• Systemic circulation .-- Thoracic lymph

Fi g. I - Schematic representation of diges ti on and absorption pattern of dietary lipids

REDDY & MURTHY : LYMPHATIC TRANSPORT OF ORALLY ADMINISTERED DRUGS 1101

sorption capabilities. For further details regarding lipid selection and formulation related issues in the development of oral lipid-based delivery systems, the interested readers are referred to a recent review by Wasan59.

The in vitro determination of digestion profiles and phase behavior of the lipids used in the lipid drug formulations, may be useful in predicting the fate of drug during the phase of lipid digestion. (i.e. the pre­absorptive phase). The rate and extent of digestion and phase behavior of the lipolytic products depend much on the fatty acid chain length . The extent and rate of digestion and aqueous phase distribution of medium chain lipolytic products is greater than the corresponding long chain lipids and is independent of bile salt concentration60

, to produce monoglyceride and fatty acid. This is due to the partial ionization of medium chain fatty acids to a greater extent than the long chain fatty acids61 at physiological pH resulting in the formation of sodium or calcium soaps or incor­poration into bile salt micellar systems to form aque­ous dispersions . The phase behavior of the dispersion systems in the intestine is important in directing the dietary lipids to the absorptive surface and their pene­tration across the enterocyte62. Prod rug approach was also attempted for lipophilic drugs to enhance the lymphatic absorption and, increased intestinal lymph concentrations of prodrug were observed in compari­son to the free drug63.64

.

Biochemical and pharmacological considerations in intestinal lymphatic drug delivery

Biochemical barriers such as enterocyte-based cy­tochrome P450 3A (CYP 3A)65 and P-glycoprotein (P-g) are one of the factors that affect the drug ab-

Table I-List of drugs studied for the oral lymphatic transport

Drug Reference

Naftifine 38

Mepitiostane 39,53

DDT 40,55

Benzo(a)pyrene 41

Probucol 42

Ontazolast 44

Halofantrine 45

CI-976 (a lipid regulating agent) 50

MK-386 (5-a reductase inhibitor) 51

Retinyl Palmitate 52

Naproxen 64 Nicotinic acid 64

Verapamil 73

sorption from the intestine. CYP 3A enzymes present on the ER of the enterocyte are involved in phase I drug metabolism, and enterocyte P-g, an energy­dependent efflux transporter present in the brush bor­der region of the intestine, limits the drug transport by retrograde efflux into the lumen66. These two proc­esses could act in an inter-relative manner in inhibit­ing the drug absorption67 . The oral bioavailability of several lipophilic drugs such as digoxin68

, cyc-10sporin69, terfenadine70 and ritonavir71 is affected by one or both the above mechanisms. Studies were per­formed to understand the inter-relative nature of P-g and CYP 3A72.73 . Administration of cyclosporine oral formulation (Sandimmune®) with and without water­soluble vitamin E (TPGS, tocopheryl polyethylene glycol 1000 succinate) in healthy volunteers, showed an increase in cyclosporine AUC in formulation con­taining TPGS72

. TPGS donot have any effect on CYP 3A metabolism, but acts as an inhibitor of p_g74, which might resulted in improved drug transport through the enterocyte and hence the reduced meta­bolic effect by CYP 3A.

The effect of P-g efflux and the metabolism of verapami I in the intestine during its transport is stud­ied by Johnson etal73. The studies were performed in animals, by isolating the rat jejunum and mounting it in side-by-side diffusion chambers containing buffer. The drug flux and metabolism are observed in both mucosal to serosal (m to s) and serosal to mucosal (s to m) directions. The s to m flux of verapamil is found high in comparison to m to s flux, indicating the P-g efflux process, due to the reduced availability of drug to CYP 3A metabolism. Also the extent of drug metabolism was found more in the m to s direc­tion, as a result of the prolonged residence time of the drug in enterocyte because of P-g efflux resulting in increased metabolism by CYP 3A.

In vitro cell cultures were widely used for studying the role of P-g and its inter-relationship with CYP 3A75.77

. However, a strong supportive human clinical data are still required to confirm, whether the intesti­nal P-g is a significant limiting barrier for drug ab­sorption at clinically relevant doses78. The membrane permeability of the drugs should also be considered while studying their susceptibility to P-g, as in some cases, the high passive permeability of drugs may compensate for P-g mediated efflux, resulting in good oral bioavailbili ty79.

The excipients used in the formulation of lipid­based dosage forms may sometimes affect the above

1102 INDIAN J EXP BIOL, OCTOBER 2002

mentioned drug absorption limiting mechanisms, es­pecially some surfactantsRO and TPGS74 (D-a­tocopheryl polyethylene glycol lOOO succinate) are found to inhibit the P-g mediated efflux in invitro studies. Hence, consideration of all the above factors is essential in order to establish a proper correlation and uti lize such interactions in modifying the drug absorption barriers and achieving improved oral bioavailability of drugs.

On the other hand, the binding of lipophilic drugs to plasma lipoproteins (chylomicrons, VLDL, LDL and HDL) was found to vary the pharmacokinetic and pharmacodynamic parameters of the drugssi . Exa-:1-pies of drugs that bind to the plasma lipoproteins in­clude cyclosporin, halofantrine, amphotericin and an­tiarrhythmic drugs. Lipoprotein binding has been shown to alter the plasma free drug concentration and hence the activity of the cyclosporin82

-83

. Similar ef­fect is observed in case of halofantrine84 after post­prandial administration, as a result of which, signifi­cant decrease in volume of distribution and clearance is seen . Sometimes, this interaction could also be positively utilized in site-specific delivery, wherein the drug-plasma lipoprotein bound fraction can be targeted to the lipoprotein receptors present in the body. After binding to receptors, they may undergo enzymatic degradation and release the drug, or enter the cell and facilitate drug release inside the ce1l81

.

Lymphatic transport of drugs via gut associated lymphoid tissue (GALT)

Drugs incorporated into the colloidal carriers such as microparticles could be delivered orally to lym­phatic system via gut associated lymphoid tissue (GALT). The specialized structure of GALT facili­tates the uptake of particulate systems from the intes­tine especially by the Peyer's patches. The lymphoid tissue is divided into two components. GALT and gut associated lamina propia compose the central lym­phoid tissue, and the lymph nodes form the peripheral lymphoid tissue. The GALT consists of lymphoid fol­licles (e.g. Peyer's patches), plasma cells and lym­phocytes. The Peyer's patches playa major role in particulate uptake. Generally, the Peyer's patches are oval in shape and are found usually on the opposite side to the mesenteric wall of the intestine. Light mi­croscopy and electron microscopic studies showed that the Peyer's patch is divided into four zones, a) the germinal center, b) the small lymphocytic areas, c) the inter follicular area, and d) the sub-epithelial zone.

Overlying the Peyer's patches is the membranous epithelial cell layer known as follicle associated epi­thelium (FAE), which is composed of absorptive cells, goblet cells, M cells and enteroendocrine cells85

.

FAE possess ability to transport macromolecules and also minimize their degradation during passage and along with GALT, it also performs the function of uptake of bacteria, viruses and pathogens from the . . 86 M II 1I1test1l1e. ce s endocytose and transport the lu-minal antigens to the basolateral cell membrane, and release into the extracellular space.

The particulate systems are taken up by the Peyer's patches and the extent of uptake depends on their size and surface characteristics. The particulate internali­zation studies showed that the particulate systems in the intestine, adhere to surface of the M cells, and the M cell pseudopods facilitate vesicular transfer of these particulates to the underlying lymphocytes and antigen presenting cells in the central hollow region of M cells87

-88

. M cells are deficient in Iysosomes, and hence the destruction of particulates is avoided during their transport through follicle associated epithelium (FAE). Majority of the particulates are taken up by the Peyer's patches in comparison to other lymphoid follicles, and the particulate uptake across the intes­tine was found to be 0.01 % of the dose of 170 to 250nm latex particles and only 0.0055% of the carbon particles. This was later confirmed by quantitising the intestinal uptake of fluorescent microspheres through flow cytometric techniques89

.9o

. Though the quantity of particulate uptake is low, the rate of uptake is

·d90 H rapl . owever, such low particulate uptake would not be advantageous for the delivery of most of the drugs, except the highly potent drugs. The findings were not similar in all the cases and some studies re­ported the uptake of large quantities of particulates j' h · . 9 1-92 I d 9? • rom t e 1I1test1l1e . n one stu y -, the partIculate uptake as high as 33% of the dose for very small par­ticles (50nm) and less than 5% of the larger particles (I )lm) has been observed. In another studl3

, the ab­sorption of 40% of the dose of l.l)lm and 13% of the dose of 3.1)lm polystyrene particles has been re­ported.

As mentioned earlier, the particle size and surface properties of the particulate systems are important considerations, which also decide the extent of ab­sorption. The extent of transport of small hydrophobic particles is more than the large and hydrophilic parti­cles94

. The penetration of the particulates into Peyer's patches is limited by their larger size, resulting in

REDDY & MURTHY : LYMPHATIC TRANSPORT OF ORALLY ADMINISTERED DRUGS 1103

reduced uptake, because of getting trapped in the sub­mucosal layer of the Peyer's patches. Reduced uptake is observed when the particulates are coated with hy­drophilic polymers such as poloxamers, and increased particulate uptake, when coated with immunoglobu­lins (SigA)950r specific antibodies (anti-M-cell anti­body)96. These principles were also applied in the development of oral vaccines for immunization, using particulate delivery systems97.98

, as even the absorp­tion of about 1 % of the particulate systems is sufficient for antigen delivery . Hence it becomes ap­parent that, a careful selection of delivery vehicles is important in attempting the drug delivery via GALT. Recently, effective targeting of HIV in intestinal mu­cosa by 3'-Azido 3'-deoxythymidine (AZT) en­trapped into poly(isohexyl cyanoacrylate) nano­spheres, has been described by Oembri et a199

.

Intestinal lymphatic transport of peptides Because of the large molecular size and hydro­

philicity, most of the peptides suffer from the problem of low oral bioavailability, and are often degraded by proteolytic enzymes in the mucosa and various tis­sues. Hence, the successful oral delivery of peptides always remained a challenge to the drug delivery field. The peptides can be protected from degradation by chemical conjugation or incorporation into the col­loidal carriers. A successful strategy in oral peptide delivery would be the proper selection of carrier and approaches to protect against degradation by various

Table 2-List of macromolecules and peptides studied for the oral lymphatic transport

Bioactive material

Macromolecules:

Enteric endotoxin

Enterostaphylococcal endotoxin

Clostridium botulinum toxin

Horse radish peroxidase

Albumin

Elastase

Dextrans

Human fibroblast B-interferon

Peptides and peptidomimetics:

Tetragastrin

Muramyl tripeptide

Cyclosporin A

Calcitonin

Insulin

Vasopressin

Reference

15

16

17

18

19

20 22

24

102

103

43, 104

108

108, liD, III

112

enzymesIOO,IOI. Protection of some peptides from enzymatic degradation has been attempted with success 102-104 (Table 2).

Administration of lipid micellar solutions contain­ing cyclosporin A (CA), a highly lipophilic peptide with seven N-methyl groups, by intra-gastric route showed much higher levels in lymph, compared to the rectal route 105. The total amount of lymphatic trans­port of CA in rats is found to be only 2-5%. Such low concentrations, inspite of having good lipophilicity, may be due to insufficient 10gP (2.99 octanollbuffer) of the drug 106. Peptides incorporated into colloidal systems, access the lymphatic system via the peyer's patches in the intestinal mucosa lO7 . The particle size and surface characteristics of the carrier systems play an important role in the effective lymphatic absorp­tion 108. Several peptides were attempted for oral de­livery by incorporation into polymersl09-113, and a de­tailed review on this aspect is presented by Alle'mann et a1 114

•

Enhanced intestinal lymphatic absorption of pep­tides can also be achieved by administering as prod­rugs. Yoshikawa et af. 115 studied the intestinal absorp­tion of bleomycin conjugated with an anionic high molecular weight dextran (500kO), incorporated in MMS, and compared with free bleomycin in MMS. In large intestine, the conjugated bleomycin in MMS showed significantly higher lymph concentrations than the unconjugated drug in MMS 116. However, the lymph selectivity of conjugated drug is reduced, when administered to the small intestine, which may be due to decreased stability of the conjugate in that envi­ronment l17 . Studies on similar lines have been per­formed on peplomycin, which is used in the treatment of esophageal cancerI18.119.

Animal models in the assessment of intestinal lym­phatic drug transport

The literature describes a number of animal models for the assessment of intestinal lymphatic drug trans­port. Apart from several factors affecting the lym­phatic drug transport from intestine, the selection of animal models and methodologies adopted for lymph collection can have a significant influence on the data of lymphatic drug absorption and its overall bioavail­ability. Generally, two types of animal models such as small animal models and large animal models are used. In both of these models, depending on the ad­vantages and disadvantages offered, at times the con­scious and unconscious models were used.

1104 INDIAN J EXP BIOL, OCTOBER 2002

Small animal models Rat model s are extensively used in the category of

small an imal models, because of the ease of handling and ex perimentation in compari son to other sma ll animals. Both unconsc ious45 and conscious 120-121 models have been used in various studies.

In unconscious rat model, the studies are performed by cannulating the mesenteric lymph duct for the col­lection of intestinal lymph , the carotid artery for the collection of blood and the duodenum fo r the admini­stration of rehydration solution. Collection of all the lymph draining the small intestine can be ensured by mesenteric lymph duct cannulation. The absolute quantity of drug transported is calculated by multiply­ing the concentration in lymph by the respective lymph volume produced during the peri od of each collection 122 . However, in order to determine the complete oral bioavailability of drug, the absorption into portal circul ation is also to be considered. The unconsc ious rat model possess the advantage of avoidance of problems related to an imal movement during experimentation, which are generally observed in conscious models. The rate of lymph flow varies with the conscious state of animal, and is 0.1-0.6mllhr in anaesthetized animal, and may increase up to 1-3ml/hr in case of unanaesthetized animal, and such difference may be due to variation in the gastric mo­tility, capillary permeability, interstiti al fluid forma­tion and venous return in both states in which the rate of above processes gets reduced in anaestheti zed ani­mals. In unconscious model, the effective shear caused and gastric emptying process differs and their effect on lipid digesti on is largely neglected, and maintenance of animal in unaesthetised state for a long time may lead to hi gh mortality rates .

The draw backs associated with unconscious mod­els cou ld be reduced by employing consc ious rat models. The method used for lymph collection is similar to that of the unconscious model, except that the cannulas of mesenteric lymph duct and jugular vein are set to travel from the bottom side of the skin, and exit at the backside of the neck , and are connected to a saddle-type arrangement to allow continuos infu­sion and sampling. The surgical procedures invol ved in cannulation and sample collection are well ex­plained by Edwards et 0[122. In conclusion, the rat models are simple, easy to handle and inexpensi ve. Apart from the above advantages, they also possess the di sadvantage that the lymphatic transport data could not be confidently extrapolated to other clinical situations, and also the administration of large dosage forms is not possible.

Large animal models The limitations associated with the rat model could

be overcome by employ ing the large animal models such as pig l23 and dog '24 , which fac ilitate accurate assessment of drug absorption. An anaesthetized pig model l23 was developed, which allows for simultane­ous sampling of lymph and blood. Before cannulat ion, a li pophilic dye (sudan black) is orally admini stered to aid in better visuali zati on of the mesenteric lymph duct. However, the model did not found much appli ­cation, because it allows onl y periodical sampling.

Several dog models were described for lymph col­lection , which in volved the cannulation of thoracic ductI 25. 126. But in these models the need to perform thoracotomy fo r gaining access to the duct, compli­cates the process and necess itates intensive care of the animals. Rajpal et al. 127 described a dog model fo r collection of thoracic lymph avoiding thoracotomy. More recentl y, Khoo et al. 12R described a simpler method for direct cannulation of thoracic lymph duct to study the intestinal transport of drugs. However, in this model, the lymph cannot be collected from mes­enteric lymph duct, which arises the chances of over­estimation of actual intestinal lymphatic transport be­cause the thoracic lymph duct is also suppli ed by pe­ripheral sources43

. Hence, the cannulation site is also important in drug transport studi es . Several other ex­perimental facto rs that influence the intestinal lym­phatic drug absorption studies include, fasting status of the animal, extent of rehydrati on and presence or absence of lipid feeding prior to surgery. In fusion of rehydrati on so lutions and lipids were found to in­crease the lymph fl ow and extent of lymph lipoprotein transport l29.

Finally, several factors affec t the accurate assess­ment of intestinal lymphatic drug transport. Hence a careful consideration and examinati on of all the as­pects, starting fro m model selection to the method­ologies of lymph collection and drug estimation IS

very important in proper interpretation of the data.

Conclusion Intestinal lymphatic transport of therapeutic mole­

cules offers several advantages like avoidance of first­pass metaboli sm and oral degradation associated with several peptides. The hi gh molecular size, and the formulation rel ated approaches taken to improve the selectivity to intestinal lymphatic transport, has led to the delivery of a large number of macromolecu les. The problem of low oral bioavailability associated with a large number of highl y lipophilic drugs, has

REDDY & MURTHY : LYMPHATIC TRANSPORT OF ORALLY ADMINISTERED DRUGS 1105

been rcduced by administering into lipid-based vehi­cles. However, the considerations related to the lipid vehicles, and other formulation related parameters are important for the effective delivery of these drugs. On the other hand , the biochemical barriers affecting the drug absorption mechanisms should be carefully con­sidered and the excipient related interactions of the efflux mechanisms could be utili zed in modifying the drug absorption barriers and an improved oral bioavailability could be achieved. The prod rug ap­proach for lipophilic drugs resulted in improved asso­ciation with lipid digestion products and better ab­sorption into the intestinal lymphatic system. The specialized structure and functions of the GALT al­lowed the delivery of several peptides and antigens by incorporation into particulate carriers. Though the particulate uptake is low, such low concentrations may be sufficient for stimulating the mucosal immune system. The intestinal ti ssue and the associated im­munocompetent cells acts as a target for the potential antiviral agents like azidodeoxythymidine (AZT), as the HIY is considered to reside in the intestinal tissue in HIY infection.

A large number of animal models were utilized for estimating the intestinal lymphatic drug transport. Every model has its own advantages like ease of han­dling and relative simplicity of small animals such as rats, and more accurate reflectance of human physiol­ogy by large animals. Finally , the selection of animal models for the assessment of intestinal lymphatic drug transport is very critical and necessitates the consid­eration of a number of factors such as conscious status of the animal, site of cannulation and methodologies used for lymph collection, for better and effective comparison of data and reaching to definite conclu­sions.

Acknowledgement The authors are thankful to UGC, New Delhi for

funding this work.

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