The Impact of Lymphatic Transport on the Systemic Disposition of Lipophilic Drugs

The Impact of Lymphatic Transport on the Systemic Disposition of Lipophilic Drugs ¨ SUZANNE M. CALIPH, ENYUAN CAO, JURGEN B. BULITTA, LUOJUAN HU, SIFEI HAN, CHRISTOPHER J. H. PORTER, NATALIE L. TREVASKIS Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences Monash University (Parkville Campus), Parkville, Victoria 3052, Australia Received 10 December 2012; revised 15 April 2013; accepted 17 April 2013 Published online 20 May 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23597 ABSTRACT: This work investigates the influence of drug absorption route (intestinal lymphatics vs. blood supply) on drug pharmacokinetics and tissue distribution. To achieve this aim, the pharmacokinetics and tissue distribution of model compounds [1,1-bis(4-chlorophenyl)-2,2,2trichloroethane, DDT; halofantrine] and lipids were assessed following intravenous delivery in lymph lipoproteins or plasma, and were found to differ significantly. For DDT, the clearance (CL) and volume of distribution (Vd ) were higher, whereas for halofantrine, CL and Vd were lower, after entry in lymph versus plasma due, in particular, to differences in adipose tissue and liver uptake. In a recent study, halofantrine CL and Vd were similar following entry in lymph or entry in plasma into the systemic circulation of animals predosed with lymph, whereas in the current study, predosing lymph did not influence DDT CL and Vd . For compounds such as DDT, changes to the route of absorption may thus directly impact on pharmacokinetics and tissue distribution, whereas for halofantrine factors that influence lymphatic transport may, by altering systemic lipoprotein concentrations, indirectly impact pharmacokinetics and tissue distribution. Ultimately, careful control of dosing conditions (formulation, prandial state), and thus the extent of lymphatic transport, may be important in assuring reproducible efficacy and toxicity for lymphatically transported drugs. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2395–2408, 2013 Keywords: absorption; clearance; disposition; distribution; lipids; lipoproteins; lymphatic transport; oral absorption; pharmacokinetics; intravenous

INTRODUCTION Dietary lipids may be transported from the intestine to the systemic circulation via either the intestinal blood capillaries and portal vein, or the intestinal lymphatic system.1–4 In the case of medium-chain triglycerides, digestion by pancreatic lipase in the small intestine lumen yields medium-chain fatty acids (and monoglycerides) that are absorbed into enterocytes and predominantly transported from the intestine to the systemic circulation via the blood capillaries.5 In contrast, the long-chain fatty acids and monoglycAbbreviations used: CE, cholesteryl ester; DDT, 1,1-bis(4chlorophenyl)-2,2,2-trichloroethane; HDL, high-density lipoprotein; LDL, low-density lipoprotein; VLDL, very low-density lipoprotein. Correspondence to: Natalie L. Trevaskis (Telephone: +613-9903-9138; Fax: +61-3-9903-9853; E-mail: Natalie.Trevaskis @monash.edu) Journal of Pharmaceutical Sciences, Vol. 102, 2395–2408 (2013) © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association

erides that are produced by triglyceride digestion in the intestinal lumen are preferentially incorporated into triglyceride resynthetic pathways within the enterocyte and assembled into intestinal lipoproteins [mostly chylomicrons and very low-density lipoprotein (VLDL)]. Intestinal lipoproteins are primarily secreted into the intestinal lymphatics as their large size precludes vascular access across the less permeable endothelium of blood capillaries.6 However, a significant proportion of ingested long-chain fatty acids are also recovered in the portal vein (∼40% of dose) in the form of free fatty acids and esterified to glycerides.7–9 The intestinal lymphatic system is also an important route for the absorption of some highly lipophilic drugs.3,4 Entry into the lymphatic system is achieved via drug association with intestinal lipoproteins during drug transit across the enterocyte.3,10 As such, lymphatic transport is typically only significant for drugs with very high lipophilicity (log P > 5 and

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 7, JULY 2013

2395

2396

CALIPH ET AL.

long-chain triglyceride solubility > 50 mg/g11 ) and/or affinity for lipoproteins.12–14 Furthermore, lymphatic transport is minimal in the fasted state or following drug administration with medium-chain lipids but enhanced significantly upon administration with a long-chain lipid source (food or formulation derived) that stimulates intestinal lipoprotein formation.3,15 When transported via the lymphatic system, drugs pass through the mesenteric and thoracic lymph ducts before emptying into the systemic circulation at the junction of the left subclavian and internal jugular veins. Drugs transported via the intestinal lymphatics therefore enter the systemic circulation in association with intestinal lipoproteins such as chylomicrons, VLDL and to a lesser extent, high-density lipoprotein (HDL).3,10 In contrast, drugs that are absorbed and transported via the portal vein are transported directly to the liver, where metabolism may limit bioavailability,15 and may be present in blood in free solution or in associated with red blood cells, plasma proteins (e.g., albumin) or plasma lipoproteins such as VLDL, low-density lipoprotein (LDL), and HDL.16 Differential binding to plasma proteins or lipoproteins under conditions where systemic protein or lipoprotein concentrations are altered can impact on the clearance (CL), volume of distribution, and tissue distribution of lipophilic drugs.16–18 This has led several authors to speculate that transport into the systemic circulation via the mesenteric lymph rather than the portal vein blood may impact on drug CL and tissue deposition.17,18 Importantly, this could have therapeutic consequences if, for example, the route of absorption of a highly lipophilic drug is inadvertently altered through changes in formulation components (e.g., the inclusion of long vs. medium-chain lipids) or following dosing in the fasted state when compared with the fed state.15 Promoting the lymphatic transport of lipophilic drugs may also be utilized as a strategy to positively influence drug pharmacokinetics and disposition to achieve targeted delivery to the site of action and to reduce toxicity due to distribution to off-target tissues.19 In a recent paper, we explored the impact of drug entry into the systemic circulation within lymph versus plasma on the pharmacokinetics (specifically the CL and volume of distribution) of a model lipophilic drug, halofantrine. In these studies, halofantrine distribution and CL were significantly lower following delivery into the systemic circulation of fasted rats within lymph or a lipid emulsion when compared with entry within plasma or an aqueous-based formulation.20 Interestingly, however, CL was similar following delivery of halofantrine in lymph or following delivery in plasma into the systemic circulation of animals predosed with lymph. This suggests that although the pharmacokinetics of halofantrine differs following administration in lymph or plasma alone, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 7, JULY 2013

where lymph and plasma enter the systemic circulation simultaneously, halofantrine rapidly equilibrates across the lipoproteins present in blood. As such, regardless of the route of entry into the systemic circulation, the systemic CL of halofantrine is likely dictated by systemic lipoprotein levels rather than the route of absorption per se, although long-chain lipid formulations and dosing conditions (fed state),20 which stimulate lymph lipoprotein and drug transport are likely to delay the CL and distribution of halofantrine. The aim of the current study was thus to investigate the impact of changes to the route of absorption (mesenteric lymph vs. portal vein blood) on the pharmacokinetics of a second highly lipophilic and lymphatically transported model compound, DDT,21–23 to determine whether the results for halofantrine were applicable to other lymphatically transported drugs. Importantly, the impact of drug entry into the systemic circulation within lymph or blood on the tissue deposition of halofantrine and DDT was examined to determine whether conditions that alter the extent of lymphatic transport might also lead to differences in drug disposition to sites of activity versus toxicity. Entry into the systemic circulation via the lymph or portal blood was simulated by intravenous (i.v.) administration of drug solubilized in either lymph or plasma. The CL and tissue deposition of lymph lipoprotein associated lipids when compared with lipids solubilized in plasma was also examined for comparison with the drug data. The pharmacokinetics and tissue deposition patterns of DDT, halofantrine, and lipids were significantly different after entry to the systemic circulation in lymph and plasma. This has significant implications for the development of lipophilic compounds as it reemphasizes the importance of exploring lymphatic transport as a potential absorption route and also indicates the potential impact that changes to formulation and dosing conditions (fed vs. fasted) which influence the extent of lymphatic transport, may have on the CL, distribution, safety, and efficacy of highly lipophilic drugs.

MATERIALS AND METHODS Chemicals Halofantrine HCl (1,3-dichloro-alpha-[2-(dibutylamino)ethyl]-6-(trifluoromethyl)-9-phenanthrenemethanol), 14 C-halofantrine HCl [a kind gift from Walter Reed Army Institute of Research (WRAIR)], 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT), 4,4-DDT-Ring-UL-14 C DDT, oleic acid, cholesterol, and sodium chloride (Sigma-Aldrich, Sydney, Australia), Tween 80 (BDH Chemicals, Darmstadt, Germany), 14 C-cholesterol and 14 Coleic acid (PerkinElmer Life Sciences, Waltham, Massachusetts), and normal saline for injection DOI 10.1002/jps

THE IMPACT OF LYMPHATIC TRANSPORT ON THE SYSTEMIC DISPOSITION OF LIPOPHILIC DRUGS

(Baxter Healthcare, Old Toongabbie, Australia) were obtained from listed suppliers. Water was obtained from a Milli-Q (Millipore, Milford, Massachusetts) purification system. Ketamine, Xylazine (Ilium R R and Xylazil ; Troy Laboratories, GlendenKetamil ning, Australia) and acepromazine (Delvet, Asquith, R Australia) were used for anesthesia. Irgasafe plus (Packard Bioscience, Meriden, Connecticut) liquid scintillation cocktail was used for liquid scintillation counting of radioactivity levels. Polyethylene (PE) and polyvinyl chloride cannulas with 0.96 and 0.58 mm and 0.8 and 0.5 mm external and internal diameters were obtained from Microtube Extrusions, Eastwood, Australia. All other chemicals were analytical reagent grade. Animal Experiments All surgical and experimental procedures were approved by the local institutional animal ethics committee. The studies were performed in 280–320 g male Sprague–Dawley rats. The rats were anesthetized, and anesthesia was maintained, using a previously described combination of ketamine, xylazine, and acepromazine.24 While anesthetized, the body temperature of the rats was maintained by placing them on a heated pad at 37◦ C (Ratek, Australia). At the conclusion of experiments, rats were killed via a lethal intraperitoneal or i.v. dose of 1 mL of sodium pentobarbitone (100 mg/mL). Preparation of Lymph Containing Radiolabeled DDT, Halofantrine, or Lipids Lymph was collected from donor rats on the day before dosing to recipient rats. Donor rats were anesthetized throughout the surgery and lymph collection period. The mesenteric lymph duct and duodenum were cannulated as described previously.5,25 The animals were rehydrated with an intraduodenal infusion of 2.8 mL/h normal saline for 30 min following surgery. After the rehydration period, a lipid emulsion was infused into the duodenum of the animals over 2 h. The emulsion consisted of 200 mg oleic acid dispersed in 5.6 mL of 0.5% Tween 80 in normal saline.25 In experiments to examine the pharmacokinetics of DDT after i.v. dosing in lymph, formulations also contained 1 mg DDT and 5 :Ci 14 C-DDT. In experiments to determine the CL or tissue deposition of lymph lipoprotein (chylomicron)-associated lipids after i.v. dosing, the formulation also included 2 mg cholesterol and 5 :Ci 14 C-cholesterol, or 15 :Ci 14 C-oleic acid, respectively. After completion of the lipid emulsion infusion, 2.8 mL/h normal saline was infused into the duodenum for the remainder of the experiment. Lymph was collected continuously for 6 h into a 10 mL PE tube R ) containing EDTA. (BD Vacutainer DOI 10.1002/jps

2397

The lymph from rats administered 14 C-DDT, 14 Ccholesterol, or 14 C-oleic acid was not further prepared before administration to recipient rats. The dose of 14 C-DDT, 14 C-cholesterol (which was likely present in the lymph as 14 C-cholesterol ester (CE) as well as 14 C-cholesterol as cholesterol is esterified in the intestine before incorporation into lipoproteins) or 14 Coleic acid (which was likely present in the lymph as 14 C-triglyceride) administered to recipient rats was determined by scintillation counting of the radiolabel as described below. The data for DDT and cholesterol per mL lymph was dose normalized to 0.5 mg/kg rat. To prepare the lymph containing radiolabeled DDT or halofantrine for tissue distribution studies, stock solutions containing radiolabeled and nonlabeled DDT or halofantrine dissolved in ACN were added to a 10 mL PE tube and the solvent evaporated to dryness under a steam of high purity nitrogen using a N-EVAP 112 evaporator (Organomation, Berlin, Massachusetts). The appropriate volume of blank lymph was subsequently added to the tube such that the final lymph concentrations were 150 :g/mL DDT with 0.5 :Ci/mL 14 C-DDT or 200 :g/mL halofantrine HCl with 0.5 :Ci/mL 14 C-halofantrine HCl. To ensure that all DDT or halofantrine was solubilized in lymph lipids, the tubes were placed in a shaking water bath at 37◦ C overnight and the following morning the lymph was centrifuged at 3000g for 3 min to precipitate any undissolved drug. This preparation method was validated by spiking blank lymph samples with a range of concentrations of DDT or halofantrine HCl to determine the maximum mass of each compound that could be solubilized in lymph. Additionally, the concentration of radiolabeled DDT or halofantrine contained within the lymph dosed to recipient animals was confirmed on the day of dosing by scintillation counting as described below. Preparation of Plasma Containing Radiolabeled DDT, Halofantrine, or Lipids Radiolabeled DDT, halofantrine and oleic acid were dissolved in plasma using the same method as described above for lymph such that the final concentrations were: 150 :g/mL DDT with 0.5 :Ci/mL 14 CDDT, 200 :g/mL halofantrine·HCl with 0.5 :Ci/mL 14 C-halofantrine·HCl or 0.7 :Ci/mL 14 C-oleic acid in plasma. The preparation method was similarly validated by spiking blank plasma samples with a range of concentrations of DDT, halofantrine·HCl or oleic acid to ensure that the mass of DDT, halofantrine and oleic acid added was below the maximum solubility of each compound in plasma. Additionally, the concentration of radiolabeled DDT, halofantrine or oleic acid contained within the plasma dosed to recipient animals was confirmed on the day of dosing by scintillation counting. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 7, JULY 2013

2398

CALIPH ET AL.

Surgery, Dosing, and Sample Collection from Recipient Rats The jugular vein and carotid artery of recipient rats were cannulated under anesthesia as described previously.5,10 The cannulas were exteriorized to the back of the neck and the rats were placed in a harness, spring and swivel system, which enabled dosing and blood sampling from the cannulas to be performed from outside of the cage as described previously.5,26 The rats were allowed to partially regain consciousness while maintained on the heated pad at 37◦ C (Ratek), after which time they were placed in metabolic cages. The rats were fasted overnight with free access to water and then infused with model compounds in lymph or plasma, as described below, via the jugular vein cannula. To ensure that the entire dose of lymph or plasma was received by the animals, 0.5 mL of heparin saline solution (10 IU/ mL) was infused through the cannula immediately after dosing. Serial blood samples or organ and tissue samples were subsequently taken from the animals. In experiments to compare the pharmacokinetics of DDT after entry into the systemic circulation in lymph versus plasma, rats were intravenously dosed with 1 mL of lymph containing 14 C-DDT over 5 or 60 min, 1 mL of plasma containing 14 C-DDT over 5 min, or 1 mL of plasma containing 14 C-DDT over 5 min immediately following bolus i.v. administration of 1 mL of blank lymph. To enable comparison between the CL of lipids, DDT and halofantrine following systemic entry in lymph lipoproteins, a group of rats also received, via i.v. infusion over 5 min, 1 mL of rat lymph collected from donor animals administered 14 C-cholesterol. After i.v. administration of lymph or plasma containing 14 C-DDT or cholesterol, blood samples (200 :L) were taken from the carotid artery cannula at intervals of −5, 5, 10, and 30 min and 1, 2, 4, 6, 7, or 8, 24, and 28 h after initiation of i.v. dosing. The blood samples were placed in eppendorfs containing 10 IU of heparin and plasma was separated from the blood via centrifugation at 3000g for 5 min. Radiolabel concentrations in plasma were measured by scintillation counting as described below. In the pharmacokinetic studies, rats were administered 14 C-DDT in lymph over 5 or 60 min as the typical flow rate of lymph in rats is 1–3 mL/h27 suggest-

ing that infusion over 60 min represents entry over a physiologically relevant time period. However, the pharmacokinetics of DDT (Table 1) and halofantrine (as described previously28 ) were not significantly different after delivery in lymph over 5 versus 60 min such that an infusion time of 5 min was chosen for subsequent experiments to examine organ and tissue deposition profiles after systemic entry of halofantrine, DDT and lipids in lymph versus plasma. In these experiments, rats were intravenously infused with 1 mL plasma or lymph containing 14 C-oleic acid, 14 C-DDT or 14 C-halofantrine over 5 min. The rats were subsequently killed 4 h after dosing and the stomach, upper half of the small intestine, lower half of the small intestine, liver, kidney, heart, spleen, lungs, thymus, testes, bladder, brain, pancreas, cervical lymph nodes, and approximately 1 g portions of thigh muscle and (retroperitoneal, perirenal and/or flank) fat tissues were subsequently collected. Each organ or tissue was weighed and the entire organ or tissue (if the mass was less than 300 mg) or two to three 200–300 mg samples of the organ or tissue transferred into 20 mL glass scintillation vials and prepared for scintillation counting as described below. Scintillation Counting Lymph and plasma samples were prepared for scintillation counting via the addition of 10–20 :L (of the lymph or plasma dosing solutions) or 60 :L (of plasma samples from pharmacokinetic studies) into scintillaR scintillation tion vials with 3 mL of Irgasafe plus fluid. The samples were vortexed for 30 s before scintillation counting. The organ and tissue samples weighing 200– 300 mg were solubilized in preparation for scintillaR , brief tion counting via addition of 4 mL of Solvable ◦ vortexing and incubation at 50 C for 6 h. The samples were then clarified via addition of 300–400 :L of 30% hydrogen peroxide (adding 100 :L at a time), followed by a further vortex mix for 10–20 s and incubation for R scin2 h at 50◦ C. Sixteen milliliters of Ultima Gold tillation fluid was subsequently added, the samples vortexed for 30 s and allowed to light and temperature adjust at 4◦ C for a minimum of 3 days. Samples were subsequently analyzed on a scintillation counter at 4◦ C.

Table 1. Noncompartmental Analysis Pharmacokinetic Parameters (Mean ± SEM; n = 4) of DDT Following Intravenous Administration of a Normalized Dose of 0.5 mg/kg DDT Delivered in 1 mL of Rat Lymph or Rat Plasma

DDT AUC0–∞ (ng h/mL) CL [mL/(h kg)] Vss (L/kg) t1/2 (h) ∗

Lymph 5 Min Infusion

Lymph 60 Min Infusion

Plasma 5 Min Infusion

Plasma 5 Min Infusion (Predosed with Blank Lymph)

912 ± 137∗ 583 ± 78∗ 9.2 ± 2.2∗ 13.0 ± 1.7

907 ± 96∗ 569 ± 58∗ 4.5 ± 0.6∗ 10.0 ± 0.4

2314 ± 481 242 ± 43 3.6 ± 0.2 13.1 ± 2.0

3018 ± 236 169 ± 13 2.2 ± 0.2 10.4 ± 1.3

Significantly different (p < 0.05) to DDT delivered in plasma-dosed groups.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 7, JULY 2013

DOI 10.1002/jps

THE IMPACT OF LYMPHATIC TRANSPORT ON THE SYSTEMIC DISPOSITION OF LIPOPHILIC DRUGS

2399

To validate the scintillation counting methods blank plasma, lymph, each organ/tissue sampled, and water were spiked in triplicate with three different known quantities of 14 C cholesterol (plasma and lymph samples only), halofantrine (tissue and organ samples only), DDT, and oleic acid. The efficiencies of counting ranged between 75% and 110% and the standard curves were precise and reproducible (±10%). To additionally ensure the accuracy of measurements, a standard curve consisting of blank plasma, organs, or tissues spiked with known quantities of 14 C cholesterol, halofantrine, or DDT was processed with each set of samples to generate a standard curve, to determine background counts and evaluate the efficiency of counting for each set of samples. Pharmacokinetic Analysis Both a compartmental and noncompartmental pharmacokinetic analyses of the DDT plasma concentration versus time data were performed. WinNonlin Professional version 5.2.1 was used to calculate noncompartmental values for the area under the plasma concentration versus time profiles from time zero to last sampling time point (AUC0–tz ) and to infinity (AUC0–∞ ), CL, elimination half-life (t1/2 ), and volume of distribution (Vss ). A population pharmacokinetic modeling analysis was also conducted using the importance sampling estimation algorithm in S-ADAPT (pmethod = 4, version 1.57) and facilitated by SADAPT-TRAN.29,30 Linear one-, two-, three-, and four-compartment models with a time-delimited zeroorder input were considered. Log-normal distributions were used for all parameters to describe the between subject variability. The residual error was modeled by a joint additive and proportional model. The Beal M3 method was used to account for observations below the quantification limit.31 Model discrimination was performed as described previously.32 Statistical Analysis Results were analyzed using one-way analysis of variance for multiple comparisons and t-tests for two group comparisons using SPSS v19 for Windows (SPSS Inc., Chicago, Illinois). A p value of less than 0.05 was considered to be a significant difference.

RESULTS Systemic CL of DDT and CE Delivered in Intestinal Lymph and Plasma According to the noncompartmental calculation of pharmacokinetic parameters, the CL and volume of distribution of DDT were significantly higher (p < 0.05) after delivery to the systemic circulation in

DOI 10.1002/jps

Figure 1. Plasma DDT concentration versus time profiles following intravenous (i.v.) administration of 0.5 mg/kg of DDT in 1 mL of rat plasma infused over 5 min (䊊), 1 mL rat lymph infused over 5 min (), or 60 min () or 1 mL rat plasma infused over 5 min immediately after an i.v. bolus dose of 1 mL of blank rat lymph (䊉). Data represent mean ± SEM for n = 4 rats. Dotted line indicates the mean of the individual fits from the three compartmental pharmacokinetic analysis for each group.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 7, JULY 2013

2400

CALIPH ET AL.

Table 2. Compartmental Pharmacokinetic Parameter Estimates for DDT (Arithmetic Mean ± SD; n = 4) Following Intravenous Administration of a Normalized dose of 0.5 mg/kg DDT Delivered in 1 mL of Rat Lymph or Rat Plasma, Using a Linear Three-Compartment Model for a Population Pharmacokinetic Analysis Lymph

DDT in CL (mL h/kg) Vss (L/kg) CLD1 (L h/kg) CLD2 (L h/kg) V1 (L/kg) V2 (L/kg) V3 (L/kg)

Plasma

5 min Infusion

60 min Infusion

5 min Infusion

5 min Infusion (Predosed with Blank Lymph)

635 ± 20∗ 9.34 ± 2.56∗ 7.90 ± 2.96 1.05 ± 0.007 1.15 ± 0.22 4.07 ± 2.43 4.12 ± 0.06

659 ± 25∗ 3.00 ± 0.64 6.71 ± 1.82 1.06 ± 0.002 0.96 ± 0.84 1.98 ± 1.15 0.062 ± 0.008

261 ± 101 3.72 ± 0.15 7.43 ± 2.99 1.06 ± 0.02 0.47 ± 0.11 0.67 ± 0.04 2.58 ± 0.01

228 ± 15 1.85 ± 0.20 6.75 ± 1.84 1.06 ± 0.02 0.14 ± 0.06 0.47 ± 0.08 1.24 ± 0.06

∗

Significantly different (p < 0.05) to DDT delivered in plasma-dosed groups. CLD1 , distribution clearance to shallow peripheral compartment; CLD2 , distribution clearance to deep peripheral compartment; V1 , volume of distribution of central compartment; V2 , volume of distribution of shallow peripheral compartment; V3 , volume of distribution of deep peripheral compartment.

intestinal lymph over 5 or 60 min when compared with delivery in plasma over 5 min (Fig. 1 and Table 1). The AUC0–∞ was therefore significantly lower (p < 0.05) in the groups administered DDT in lymph when compared with plasma. In addition, the pharmacokinetics of DDT was similar after administration in lymph over 5 versus 60 min with no significant difference in CL, volume of distribution, AUC or half-life. Intravenous delivery of blank lymph to the rats 5 min before i.v. delivery of DDT in plasma also did not significantly alter the CL and volume of distribution of DDT when compared with delivery of DDT in plasma into the systemic circulation of fasted rats (Fig. 1 and Table 1). The plasma half-life of DDT was similar for all lymph versus plasma dosing groups. The population pharmacokinetic modeling was generally in good agreement with the results of the noncompartmental pharmacokinetic analysis (Table 1 and 2). Although a one-compartment model was clearly inadequate to describe the data, the threecompartment model had a significantly better objective function (p = 0.00001, likelihood ratio test) than a two-compartment model. Including a fourth compartment neither improved the objective function nor the curve fits. The final three-compartment model yielded unbiased and precise curve fits (Fig. 1 dotted lines show the means of individual fits from the population pharmacokinetic modeling). The CL was estimated to be about 2.7-fold higher when DDT was administered in lymph (for both 5 and 60 min infusion groups) when compared with plasma (Table 2) and the CL values estimated from the compartmental modeling were within 10% of noncompartmental estimates. Vss estimates from population pharmacokinetic modeling were slightly lower than the noncompartmental analysis results, however, still within 10%–20% of noncompartmental analysis estimates. As DDT is transported in the lymph in association with core lipids of chylomicrons such as CE, plasma JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 7, JULY 2013

Figure 2. Correlation between DDT versus CE plasma concentrations at different time points following intravenous administration of 0.5 mg/kg of DDT or CE in 1 mL of rat lymph over 5 min to fasted rats. Data represent mean ± SEM for n = 4 rats in each group.

concentrations of DDT delivered in the lymph over 5 min were also compared with plasma concentrations of radiolabeled CE after delivery in lymph lipoproteins over 5 min. Fig. 2 shows that a strong correlation was evident between DDT and CE concentrations in the systemic circulation at different time points, reflecting the similar plasma half-life for CE (15.4 ± 3.7) and DDT (13.0 ± 1.7) dosed in lymph. Systemic Disposition of Lipid Delivered in the Intestinal Lymph and Plasma The organ deposition profiles of lipid, as percent dose recovered in the whole organ and percent dose per gram of tissue, after i.v. administration of 14 C-oleic acid containing triglyceride in the lymph versus 14 Coleic acid in plasma are shown in Table 3 and Fig. 3. A significant proportion of the dose (7%–9%) was recovered in the liver after delivery in plasma or lymph. Distribution to tissues including the liver, gastrointestinal tract (upper and lower small intestine and stomach), lungs, testes, heart, thymus and bladder DOI 10.1002/jps

THE IMPACT OF LYMPHATIC TRANSPORT ON THE SYSTEMIC DISPOSITION OF LIPOPHILIC DRUGS

2401

Table 3. Lipid Biodistribution Profile in Rats after Intravenous Administration of 1 mL Lymph Collected from Animals Administered 14 C-Oleic Acid or 1 mL Plasma to which 14 C-Oleic Acid was Added Percent Dose Recovered in Entire Tissue

Liver Lower small intestine Upper small intestine Stomach Kidneys Lungs Testes Pancreas Heart Brain Bladder Thymus Spleen Musclea Adipose tissuea Total

Percent Dose Recovered Per Gram

Plasma

Lymph

Plasma

Lymph

6.98 ± 1.29 0.51 ± 0.02 0.76 ± 0.07 0.42 ± 0.05 0.81 ± 0.03∗ 0.60 ± 0.10 0.14 ± 0.01 0.66 ± 0.09∗ 0.25 ± 0.05 0.1 ± 0.02 0.01 ± 0.003 0.17 ± 0.04 0.26 ± 0.03∗ 26 3.4–5.2 41–43

8.66 ± 0.98 0.53 ± 0.03 0.75 ± 0.03 0.35 ± 0.11 0.52 ± 0.05 0.47 ± 0.03 0.13 ± 0.01 0.24 ± 0.03 0.19 ± 0.02 0.06 ± 0.01 0.03 ± 0.007 0.10 ± 0.03 0.14 ± 0.02 12 6.1–9.2 31–34

0.59 ± 0.06 0.21 ± 0.02 0.27 ± 0.02 0.24 ± 0.02 0.27 ± 0.03∗ 0.30 ± 0.13 0.04 ± 0.001 0.29 ± 0.06 0.20 ± 0.06 0.04 ± 0.002 0.11 ± 0.03 0.37 ± 0.03 0.28 ± 0.06∗ 0.17 ± 0.07 0.10 ± 0.01

0.63 ± 0.07 0.19 ± 0.01 0.26 ± 0.01 0.18 ± 0.03 0.18 ± 0.02 0.15 ± 0.04 0.03 ± 0.001 0.24 ± 0.04 0.11 ± 0.01 0.04 ± 0.001 0.48 ± 0.21 0.26 ± 0.07 0.15 ± 0.01 0.08 ± 0.005 0.18 ± 0.04∗

Tissue samples were removed 4 h postadministration. Lipid recovery is presented as % dose recovered in each tissue sampled, or as % dose recovered per gram of tissue sampled. Data represent mean ± SEM; n = 3 rats. ∗ Represents a significant difference (t-test, p < 0.05) in lipid recovery between the lymph versus plasma-dosed groups. a Total recovery in adipose tissue and muscle was calculated using the assumption that 10%–15%, and 45% of the rat body weight, respectively, consists of adipose tissue and muscle.

was not significantly different after delivery in lymph versus plasma. In contrast, uptake into the kidneys, pancreas, and spleen was significantly higher after administration in plasma when compared with lymph. The largest differences in uptake, however, were seen in adipose tissue and muscle. Uptake into adipose was significantly greater after administration in lymph lipoproteins, whereas uptake into muscle was significantly greater after administration as a free fatty acid in plasma, consistent with previous reports.33–35 As adipose and muscle tissue account for approximately 10%–15% and 45% of the body weight of a 300 g rat36–38 the total mass of oleic acid deposited in adipose tissue may represent 3%–5% and 6%–9% of the dose, and in muscle may represent 26% and 12% of the dose, when delivered in the plasma and lymph, respectively, if it is assumed that the radiolabel was distributed evenly across adipose and muscle depots. Overall, the total recovery of radiolabel in all tissues represented 31%–43% of the dose after administration in either plasma or lymph. A minor proportion of the radiolabel dose was likely present in plasma as previous studies by Bragdon et al.35 demonstrated that ∼1% of an oleic acid dose remains in plasma 200 min following i.v. injection as unesterified or chylomicron associated 14 C-fatty acid. A significant proportion of the radiolabel was therefore likely present in tissues that were not analyzed, consistent with previous studies in which radiolabel was recovered in the rat carcass following i.v. entry of unesterified and chylomicron associated 14 C fatty acids and removal of organs and tissues.33–35 Additionally, up to 20% of unesterified or chylomicron associated DOI 10.1002/jps

14

C fatty acid is oxidized and expired as 14 C labeled CO2 within 10 min of administration39,40 suggesting that a proportion of the radiolabel administered in the current study was removed via metabolism, oxidation and/or expiration. Indeed, the 14 C-label in the current study was present in the one position of the 14 C-oleic acid backbone and liable to loss via oxidation and expiration. Systemic Disposition of DDT Delivered in Intestinal Lymph and Plasma The organ deposition profiles of DDT calculated as percent dose recovered per whole organ and percent dose recovered per gram of tissue, after i.v. delivery of 14 C-DDT in lymph versus plasma are shown in Table 4 and Fig. 4. A large proportion of the DDT dose was recovered in the liver (15%–16%) and muscle (14%–16%) after delivery in lymph and plasma and there was no significant difference in uptake between the two groups. A significantly greater mass (p < 0.05) of DDT was, however, recovered in adipose tissue after delivery in lymph when compared with plasma, consistent with the data seen for lipid deposition. Estimated total recoveries in adipose tissue were 48%–72% and 18%–27% of the dose when delivered in the lymph and plasma, respectively. A large proportion of the DDT dose was also recovered in the gastrointestinal tract (the stomach, upper and lower small intestine) in groups administered DDT in both lymph and plasma, and DDT recovery was greater than threefold higher in the gastrointestinal tract when it was delivered in plasma. Significant JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 7, JULY 2013

2402

CALIPH ET AL.

sistent with the higher volume of distribution of DDT seen in the pharmacokinetic studies (Table 1 and 2, Fig. 1). Systemic Disposition of Halofantrine Delivered in the Intestinal Lymph and Plasma

Figure 3. (a and b) Lipid biodistribution profile in rats after intravenous administration of 1 mL lymph (white bars) from rats intraduodenally administered 14 C-oleic acid or 1 mL plasma to which 14 C-oleic acid was added in vitro (black bars). Tissue samples were removed 4 h postadministration. Lipid recovery is presented as the per cent of the dose of radiolabel recovered in the organ or tissue sampled. Data represent mean ± SEM; n = 4 rats. LSI, lower small intestine; USI, upper small intestine. For fat, total recovery is calculated using the assumption that 15% of the rat body weight consists of fat (adipose) tissue. ∗ Represents a significant difference (t-test, p < 0.05) in lipid recovery between the lymph versus plasma-dosed group.

differences (p < 0.05) in DDT recovery were also seen in the thymus, spleen, bladder and heart tissues after delivery in lymph when compared with plasma. The greatest difference in DDT deposition between lymph and plasma groups was seen in a lymphoid organ, the thymus, where >fivefold increase in deposition was seen when delivered in lymph. Overall, the total recovery of DDT in all organs was greater after delivery in lymph when compared with plasma (Table 4), conJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 7, JULY 2013

The organ deposition profiles of halofantrine, as percent dose recovered in the whole organ and percent dose recovered per gram of tissue, after i.v. delivery of 14 C-halofantrine in the lymph versus plasma are shown in Table 5 and Fig. 5. Similar to DDT, a substantial proportion of the dose (18%–27%) of halofantrine was recovered in the liver after delivery in plasma or lymph. However, for halofantrine, significantly more was recovered in the liver after delivery in plasma (p < 0.05). Halofantrine was also recovered in reasonable quantities in the gastrointestinal tract (stomach, upper and lower small intestine) when delivered in the lymph and plasma. Unlike DDT, however, there was no significant difference in halofantrine recovery in the gastrointestinal tract after delivery in lymph versus plasma. Halofantrine recovery in adipose tissue and muscle was similar between groups and the mass recovered in adipose tissue was substantially lower than that recovered after administration of DDT. Significant differences (p < 0.05) in halofantrine recovery were also seen in the lungs, bladder, brain, and testes with higher deposition of halofantrine in these organs when delivered in plasma. Overall, the recovery of halofantrine in all organs and tissues was higher after delivery in plasma when compared with lymph (Table 5), consistent with previous studies that show a higher volume of distribution of halofantrine after delivery in plasma when compared with lymph,20 but in contrast to the data obtained with DDT.

DISCUSSION Conditions that alter the extent of drug binding to plasma proteins or lipoproteins (for example, changes to systemic lipoprotein levels in the fed vs. fasted state or in normolipidemia vs. hyperlipidemia), influence the CL and distribution of lipophilic drugs.16–18,41,42 This has led to suggestions that the route of drug absorption following oral delivery (lymph vs. portal vein), and therefore the route of drug entry into the systemic circulation, may influence CL and tissue deposition, and in doing so impact on the efficacy and toxicity of lipophilic drugs.20,43 Consistent with this suggestion we have recently shown that the CL and distribution of a lipophilic drug, halofantrine, are significantly lower after drug entry into the systemic circulation in association with lymph lipoproteins when compared plasma.20 However, in this case, the concentration of lymph lipoproteins in the systemic circulation, rather than the route of entry, was the more DOI 10.1002/jps

THE IMPACT OF LYMPHATIC TRANSPORT ON THE SYSTEMIC DISPOSITION OF LIPOPHILIC DRUGS

Table 4.

2403

DDT Biodistribution Profile in Rats after Intravenous Administration of 1 mL radiolabeled 14 C-DDT in Lymph or Plasma

Liver Lower small intestine Upper small intestine Stomach Kidneys Lungs Testes Pancreas Heart Brain Bladder Thymus Spleen Musclea Adipose tissuea Total

Percent Dose Recovered in Entire Tissue

Percent Dose Recovered Per Gram

Plasma

Lymph

Plasma

Lymph

15.8 ± 1.72 16.3 ± 1.53∗ 2.46 ± 0.23∗ 1.69 ± 0.26∗ 1.66 ± 0.28 0.83 ± 0.46 0.32 ± 0.02 0.19 ± 0.06 0.15 ± 0.03 0.16 ± 0.03 0.13 ± 0.06∗ 0.1 ± 0.01 0.1 ± 0.01∗ 14 18–27 71–81

15.2 ± 1.53 3.91 ± 1.38 0.83 ± 0.05 0.62 ± 0.12 1.06 ± 0.28 0.88 ± 0.48 0.56 ± 0.27 0.26 ± 0.12 0.19 ± 0.02∗ 0.31 ± 0.10 0.03 ± 0.01 0.51 ± 0.06∗ 0.04 ± 0.02 16 48–72 88–112

1.05 ± 0.06 5.09 ± 0.59∗ 0.79 ± 0.04∗ 0.97 ± 0.16∗ 0.49 ± 0.07 0.33 ± 0.14 0.09 ± 0.01 0.22 ± 0.06 0.12 ± 0.01 0.09 ± 0.08 0.34 ± 0.09∗ 0.21 ± 0.03 0.093 ± 0.01∗ 0.10 ± 0.02 0.64 ± 0.18

1.08 ± 0.14 1.54 ± 0.27 0.30 ± 0.05 0.33 ± 0.08 0.30 ± 0.14 0.36 ± 0.20 0.17 ± 0.09 0.34 ± 0.17 0.16 ± 0.01∗ 0.18 ± 0.06 0.16 ± 0.03 0.55 ± 0.16∗ 0.033 ± 0.02 0.12 ± 0.02 1.27 ± 0.31∗

Tissue samples were removed 4 h postadministration. DDT recovery is presented as % dose recovered in each tissue sampled, or as % dose recovered per gram of tissue sampled. Data represent mean ± SEM; n = 4 rats. ∗ Represents a significant difference (t-test, p < 0.05) in DDT recovery between the lymph versus plasma-dosed groups. a Total recovery in adipose tissue and muscle was calculated using the assumption that 10%–15%, and 45% of the rat body weight, respectively, consists of adipose tissue and muscle.

important determinant because CL and volume of distribution of halofantrine were also reduced when animals were predosed lymph lipoproteins before drug delivery to the circulation in plasma. The current study has expanded this investigation to examine the impact of absorption route (lymph vs. portal vein) on the CL and distribution of a second (and more lipophilic) compound, DDT, and on the tissue deposition profile of lipids, halofantrine and DDT. The results show that for DDT, the route of absorption into the systemic circulation directly influences CL, distribution, and tissue deposition, regardless of the systemic concentration of lymph lipoproteins, and that for DDT, halofantrine and dietary lipid, entry into the systemic circulation within lymph lipoproteins when compared with plasma significantly alters patterns of tissue deposition. The CL and volume of distribution of DDT were significantly lower after entry to the systemic circulation in plasma over 5 min when compared with lymph over 5 or 60 min (Fig. 1, Table 1). In lymph, DDT is associated almost entirely (∼97%) with lipid-rich lipoproteins (chylomicrons and VLDL).44 In contrast, when incubated with rat plasma, Mohammed et al.45 found that ∼18% of the mass of DDT is associated with albumin and 65%–82% with plasma lipoproteins (HDL, LDL, and VLDL). In the latter study, DDT distribution across plasma lipoprotein fractions was in direct proportion to the cholesterol content in each lipoprotein.45 After administration in lymph versus plasma, DDT was therefore likely to have been associated, at least initially, with different plasma fractions (more in chylomicrons and VLDL after administration in lymph and more in smaller lipoproteins DOI 10.1002/jps

and lipoprotein free fractions after administration in plasma). Differences in the rate of drug desorption from different binding species may therefore have led to the subsequent changes in CL and distribution of DDT after delivery in lymph versus plasma. A number of lines of evidence suggest that DDT may transfer less rapidly from plasma proteins to tissues when compared with lymph lipoproteins and that this may lead to more rapid CL and distribution of DDT after delivery in lymph when compared with plasma. For example, Mohammed et al.45 found that following entry of DDT into the systemic circulation, DDT concentrations in association with plasma lipoproteins declined more rapidly than concentrations associated with plasma albumin, suggesting that DDT more readily dissociates from lipoproteins than from albumin and may subsequently be transferred to plasma albumin, cleared or taken up into tissues. Furthermore, DDT is very rapidly cleared from the systemic circulation in the first 10–30 min following delivery in intestinal lymph chylomicrons.21,46 During this 30 min postdose period, DDT is even more rapidly cleared than chylomicron core lipids (triglycerides, CEs) and Vost and MacLean suggested DDT is removed from chylomicrons via two mechanisms: passive diffusion (independent of release of lipoprotein lipids), and diffusion with the assistance of lipoprotein lipase mediated hydrolysis of core lipoprotein lipids.44 The initial rapid disappearance of DDT from chylomicrons in vivo was further associated with rapid uptake into the liver, with 30% of the dose of DDT recovered in the liver 14 min after systemic delivery in chylomicrons.44 A detailed analysis of DDT uptake into other tissues, including adipose, was JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 7, JULY 2013

2404

CALIPH ET AL.

Figure 4. (a and b) DDT biodistribution profile in rats after intravenous administration of 1 mL radiolabeled 14 CDDT containing lymph (white bars) or plasma (black bars). Tissue samples were removed 4 h postadministration. DDT recovery is presented as the per cent of the dose recovered in the organ or tissue sampled. Data represent mean ± SEM; n = 4 rats. LSI, lower small intestine; USI, upper small intestine. For fat, total recovery is calculated using the assumption that 15% of the rat body weight consists of fat (adipose) tissue. ∗ Represents a significant difference (ttest, p < 0.05) in DDT recovery between the lymph versus plasma-dosed group.

not performed in the study by Vost and MacLean. However, in a more recent study Kohan et al.47 found that DDT associated with chylomicrons is more rapidly taken up into adipocytes than chylomicron core lipids and that this uptake is independent of lipoprotein lipase mediated hydrolysis of chylomicron core lipids suggesting the likelihood that rapid uptake into adipose tissue contributes to the initial rapid disappearance of DDT from chylomicrons in vivo. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 7, JULY 2013

DDT may thus have a very rapid initial rate of transfer from lymph lipoproteins and uptake into tissues such as the liver and/or adipose tissue, which is partly responsible for the rapid CL and higher volume of distribution observed here after delivery in lymph when compared with plasma. In the current study, however, DDT plasma concentrations were well correlated with plasma concentrations of CE from 30 min to 24 h following entry of DDT or CE into the systemic circulation within lymph lipoproteins (Fig. 2) suggesting that during this later time period DDT transfer from lymph lipoproteins may be more dependent on the CL and distribution of lipoprotein core lipids. In contrast to the tissue deposition data seen by Vost and MacLean44 14 min after delivery in chylomicrons, 4 h after delivery of DDT in lymph or plasma, a greater proportion of the dose was recovered in adipose tissue after delivery in lymph lipoproteins (∼48%–72% of dose) when compared with plasma (∼18%–27% of dose) (Table 4). A significant proportion of the dose was also recovered in the liver 4 h after administration (15%–16% of the dose of DDT), but recovery was similar regardless of the route of entry (lymph vs. plasma). The significantly higher CL and volume of distribution of DDT after delivery in lymph lipoproteins may thus have resulted from avid uptake into adipose tissue, and potentially initial rapid uptake into the liver. The high recovery of DDT in adipose after delivery in lymph is also consistent with higher recovery of lipids in adipose after delivery in lymph lipoproteins as triglyceride associated fatty acids, when compared with delivery as free fatty acids in plasma, according to results from the current study (Fig. 3, Table 3) and previous studies.35 This again suggests that DDT transfer from lymph lipoproteins to tissues is dependent on the CL and distribution of lipoprotein lipids. The lower CL and volume of distribution of DDT after delivery in plasma when compared with lymph lipoproteins may, in contrast, reflect a particularly high affinity to, and a slower rate of dissociation from, a component of plasma (e.g., a plasma protein or lipoprotein), possibly albumin because the data of Mohammed et al.45 suggest that DDT concentrations in plasma lipoproteins decline more rapidly than concentrations associated with albumin. The data in Fig. 1 and Table 1 further show that DDT CL and volume of distribution are the same, irrespective of dosing to fasted animals or to animals predosed with lymph lipoproteins, again suggesting that DDT has a high affinity for a component of plasma, and does not readily transfer from plasma proteins to lymph lipoproteins. In contrast, DDT has been found to rapidly distribute from chylomicrons to more dense lipoprotein fractions (HDL, LDL) and to plasma proteins both in vitro and in vivo, suggesting that DDT is more readily able to dissociate from lymph DOI 10.1002/jps

THE IMPACT OF LYMPHATIC TRANSPORT ON THE SYSTEMIC DISPOSITION OF LIPOPHILIC DRUGS

Table 5. Plasma

2405

Halofantrine (Hf) Biodistribution Profile in Rats after Intravenous Administration of 1 mL Radiolabeled 14 C-Hf in Lymph or

Percentage Dose Recovered in Entire Tissue

Liver Lungs Lower small intestine Upper small intestine Stomach Kidneys Spleen Pancreas Heart Thymus Testes Brain Bladder Musclea Adipose tissuea Total

Percentage Dose Recovered Per Gram

Plasma

Lymph

Plasma

Lymph

26.5 ± 1.5∗ 8.5 ± 1.9∗ 8.4 ± 1.4 1.1 ± 0.3 0.7 ± 0.1 2.6 ± 0.6 1.4 ± 0.3 0.5 ± 0.2 0.5 ± 0.1 0.22 ± 0.08 0.27 ± 0.1∗ 0.14 ± 0.08∗ 0.05 ± 0.02∗ 21 16–24 88–96

18.0 ± 1.8 4.0 ± 0.1 10.3 ± 1.4 1.9 ± 0.2 0.6 ± 0.1 1.7 ± 0.1 1.0 ± 0.1 0.8 ± 0.2 0.4 ± 0.02 0.3 ± 0.09 0.09 ± 0.04 0.02 ± 0.01 0.02 ± 0.01 14 13–19 66–72

1.93 ± 0.06∗ 3.38 ± 0.57∗ 2.64 ± 0.44 0.71 ± 0.15 0.40 ± 0.06 0.77 ± 0.19 1.14 ± 0.12 0.63 ± 0.19 0.46 ± 0.12 0.83 ± 0.30 0.10 ± 0.04∗ 0.08 ± 0.05∗ 0.22 ± 0.11∗ 0.15 ± 0.07 0.53 ± 0.13

1.19 ± 0.13 1.95 ± 0.27 3.58 ± 0.68 0.53 ± 0.02 0.34 ± 0.04 0.57 ± 0.02 0.96 ± 0.10 0.99 ± 0.21 0.29 ± 0.03 0.28 ± 0.12 0.03 ± 0.01 0.011 ± 0.01 0.12 ± 0.04 0.10 ± 0.03 0.43 ± 0.06

Tissue samples were removed 4 h postadministration. Hf recovery is presented as % dose recovered in each tissue sampled, or as % dose recovered per gram of tissue sampled. Data represent mean ± SEM; n = 4 rats. ∗ Represents a significant difference (t-test, p < 0.05) in Hf recovery between the lymph versus plasma-dosed groups. a Total recovery in adipose tissue and muscle was calculated using the assumption that 10%–15%, and 45% of the rat body weight, respectively, consists of adipose tissue and muscle.

lipoproteins when compared with plasma proteins.21,44,45,48 Differences in affinity and the rate of dissociation of DDT from lymph lipoproteins versus plasma components such as albumin may thus influence the CL and distribution of DDT after dosing in lymph versus plasma. In contrast to the data for DDT, the CL and distribution of halofantrine were delayed following delivery to the systemic circulation in lymph lipoproteins, and were higher after delivery in plasma.20 Like DDT, halofantrine is expected to be associated with the apolar lipid core of lipid-rich lipoproteins (chylomicrons and VLDL) when delivered in lymph as 96%–98% of halofantrine recovered in lymph is also present in lipid-rich lipoproteins.10 In plasma, halofantrine is also expected to distribute between a lipoprotein-free fraction and plasma lipoproteins (HDL, LDL, VLDL). For halofantrine, 28%–45% of the dose was recovered in the lipoprotein-free fraction in rat plasma upon in vitro incubation.49 However, the remainder was found within plasma lipoproteins (HDL, LDL, VLDL) with the amount in each fraction directly proportional to apolar lipid content49 rather than cholesterol (i.e., surface) lipid content as was the case for DDT.45 There are therefore differences in the distribution patterns of halofantrine and DDT amongst different protein and lipoprotein fractions in lymph and plasma, and perhaps in the patterns of association with lipoprotein components (core vs. surface lipids) which may also influence CL and distribution. More importantly, predosing lymph led to significant reductions in the CL and distribution of halofantrine delivered in plasma suggesting that halofantrine readily transDOI 10.1002/jps

fers from plasma proteins and lipoproteins to lymph lipoproteins. In the opposite manner to DDT, halofantrine may thus have a higher affinity for, and slower rate of dissociation from, lymph lipoproteins when compared with plasma proteins/lipoproteins, and thus slower uptake into metabolic pathways and tissues after delivery in lymph lipoproteins. Indeed, total recovery of halofantrine in tissues was lower after delivery in lymph (Table 5). In particular, less halofantrine was recovered in the liver (18% vs. 26.5% of dose), lungs, testes, brain, and bladder and unlike DDT, the recovery of halofantrine in adipose tissue and muscle was not significantly different after delivery in lymph versus plasma (Table 5, Fig. 5). The total recovery of halofantrine in adipose tissue in all groups was also significantly lower than for DDT (Table 4), consistent with the lower lipophilicity (log P) of halofantrine when compared with DDT. It is possible, therefore, that DDT transfers from lymph lipoproteins to tissues particularly rapidly, and more rapidly than halofantrine, because of its high lipophilicity and because lipoproteins tend to interact with lipid storage tissues such as the liver and adipose tissue. Delivery of both halofantrine and DDT into the systemic circulation in lymph or plasma thus influenced their plasma CL and volume of distribution. For halofantrine, the data are consistent with previously reported data for halofantrine42,50,51 and other drugs16 showing that CL is delayed following administration in conditions where systemic lipoprotein levels are raised (postprandially or in the setting of hyperlipidemia). For halofantrine, CL is believed to be delayed, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 7, JULY 2013

2406

CALIPH ET AL.

Figure 5. (a and b) Halofantrine (Hf) biodistribution profile in rats after intravenous administration of 1 mL radiolabeled 14 C-Hf containing lymph (white bars) or plasma (black bars). Tissue samples were removed 4 h postadministration. Hf recovery is presented as the per cent of the dose recovered in the organ or tissue sampled. Data represent mean ± SEM; n = 4 rats. LSI, lower small intestine; USI, upper small intestine. For fat, total recovery is calculated using the assumption that 15% of the rat body weight consists of fat (adipose) tissue. ∗ Represents a significant difference (t-test, p < 0.05) in Hf recovery between the lymph versus plasma-dosed group.

as it is a low extraction ratio drug that it primarily cleared by the liver, and increases in systemic lipoprotein levels reduce the free plasma concentrations and thus concentration available for metabolism. Similarly, uptake into tissues is believed to be reduced due to a reduction in the free fraction in plasma.50 Previous data examining the impact of raised systemic lipoprotein levels on the CL and volume of distribution of DDT have also found reduced CL of DDT in the setting of hyperlipoproteinemia,17,18 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 7, JULY 2013

whereas in the current study DDT was more rapidly cleared after delivery in lymph lipoproteins when compared with plasma. This variability is consistent with previous data for cyclosporine, for which the area under the plasma concentration versus time profile has been found to increase, decrease or not change depending on the model of hyperlipidemia used16 suggesting that the precise types and concentrations of lipoproteins present in the systemic circulation may markedly influence the CL and distribution of lipophilic drugs. The changes to the tissue distribution of lipids, halofantrine, and DDT after delivery in lymph versus plasma seen here are also somewhat consistent with previous studies. Thus, chylomicron associated triglycerides have been shown to be preferentially taken up into adipose tissue, whereas plasma fatty acids are more specifically taken up into tissues such as muscle, where they are more readily utilized as an energy source.35 This is believed to be related to the need to store excess energy in the adipose tissue postprandially, whereas the utilization of fatty acids as an energy source by tissues such as muscle is required during periods of fasting when fatty acids are released into the systemic circulation following adipose tissue lipolysis of triglyceride. For halofantrine, tissue distribution is different after administration to normo- versus hyperlipidemic animals consistent with the current study where changes to systemic lipoprotein concentrations via administration in lymph lipoproteins versus plasma altered tissue deposition. In particular, hyperlipidemia increased halofantrine uptake into the spleen and decreased uptake into lung and adipose tissue, whereas concentrations were relatively similar in the liver and brain.50 In the current study, delivery in lymph also appeared to reduce uptake into the lungs, liver, and brain when compared with delivery in fasted plasma. For DDT, hyperlipidemia has previously been found to reduce uptake into the brain and the onset of related pharmacodynamic endpoints (facial muscle tremor resulting from neurotoxicity). In terms of a direct impact of lymphatic drug transport on tissue distribution, Hauss et al. have also shown that the uptake of a lipophilic lipid regulator, CI-976, into perirenal adipose was significantly greater after oral delivery in a lipid emulsion which promoted increased lymphatic transport when compared with a suspension formulation.43 In summary, delivery into the systemic circulation within lymph versus plasma had a significant impact on the CL, volume of distribution, and tissue deposition of halofantrine, DDT, and lipid. For DDT, CL, and distribution were altered regardless of the lipoprotein lipid concentrations in the systemic circulation. This suggests that changes to the route of absorption (i.e., stimulation of lymph versus portal vein absorption) of compounds such as DDT have the potential DOI 10.1002/jps

THE IMPACT OF LYMPHATIC TRANSPORT ON THE SYSTEMIC DISPOSITION OF LIPOPHILIC DRUGS

to directly impact on drug CL, distribution, and tissue deposition and ultimately on pharmacodynamic profiles. For halofantrine, CL and volume of distribution is more dependent on systemic lipoprotein lipid concentrations (with CL delayed when lipoprotein concentrations are increased) because halofantrine redistributes from plasma to lymph lipoproteins rapidly. Conditions that enhance the lymphatic transport of halofantrine, such as coadministration with food or formulation lipids, are therefore likely to influence CL, distribution, and tissue deposition, but to do so by altering systemic lipoprotein lipid levels. Collectively, the data suggest that changes to the extent of lymphatic transport as a result of formulation or prandial state changes may significantly influence the CL and tissue deposition profiles of highly lipophilic, lymphatically transported drugs, but that the patterns of these changes are highly drug (and lipoproteins binding pattern) dependent. For lymphatically transported drugs, therefore, careful control of dosing conditions may be important in assuring reproducible efficacy and toxicity.

ACKNOWLEDGMENTS The authors would like to acknowledge Walter Reed Army Institute of Research (WRAIR) for their kind gift of 14 C-halofantrine HCl.

REFERENCES 1. Mansbach CM, Dowell RF, Pritchett D. 1991. Portal transport of absorbed lipids in rats. Am J Physiol 261(3):G530–G538. 2. Nordskog BK, Phan CT, Nutting DF, Tso P. 2001. An examination of the factors affecting intestinal lymphatic transport of dietary lipids. Adv Drug Deliv Rev 50(1–2):21–44. 3. Trevaskis NL, Charman WN, Porter CJ. 2008. Lipid-based delivery systems and intestinal lymphatic drug transport: A mechanistic update. Adv Drug Deliv Rev 60(6):702–716. 4. Williams HD, Trevaskis NL, Charman SA, Shanker RM, Charman WN, Pouton CW, Porter CJ. 2013. Strategies to address low drug solubility in discovery and development. Pharmacol Rev 65(1):315–499. 5. Caliph SM, Charman WN, Porter CJ. 2000. Effect of short-, medium-, and long-chain fatty acid-based vehicles on the absolute oral bioavailability and intestinal lymphatic transport of halofantrine and assessment of mass balance in lymph-cannulated and non-cannulated rats. J Pharm Sci 89(8):1073–1084. 6. Dixon JB. 2010. Mechanisms of chylomicron uptake into lacteals. Ann N Y Acad Sci 1207 Suppl 1:E52–E57. 7. Chaikoff IL, Bloom B, Stevens BP, Reinhardt WO, Dauben WG. 1951. Pentadecanoic acid-5-C14; its absorption and lymphatic transport. J Biol Chem 190(2):431–435. 8. Bloom B, Chaikoff IL, Reinhardt. 1951. Intestinal lymph as pathway for transport of absorbed fatty acids of different chain lengths. Am J Physiol 166(2):451–455. 9. Kiyasu JY, Bloom B, Chaikoff IL. 1952. The portal transport of absorbed fatty acids. J Biol Chem 199(1):415–419. 10. Porter CJ, Charman SA, Charman WN. 1996. Lymphatic transport of halofantrine in the triple-cannulated anesthetized

DOI 10.1002/jps

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21. 22.

23.

24.

25.

26.

27.

2407

rat model: Effect of lipid vehicle dispersion. J Pharm Sci 85(4):351–356. Charman WN, Stella VJ. 1986. Estimating the maximum potential for intestinal lymphatic transport of lipophilic drug molecules. Int J Pharm 34:175–178. Gershkovich P, Fanous J, Qadri B, Yacovan A, Amselem S, Hoffman A. 2009. The role of molecular physicochemical properties and apolipoproteins in association of drugs with triglyceride-rich lipoproteins: In-silico prediction of uptake by chylomicrons. J Pharm Pharmacol 61(1):31–39. Trevaskis NL, Shanker RM, Charman WN, Porter CJ. 2010. The mechanism of lymphatic access of two cholesteryl ester transfer protein inhibitors (CP524,515 and CP532,623) and evaluation of their impact on lymph lipoprotein profiles. Pharm Res 27(9):1949–1954. Trevaskis NL, McEvoy CL, McIntosh MP, Edwards GA, Shanker RM, Charman WN, Porter CJ. 2010. The role of the intestinal lymphatics in the absorption of two highly lipophilic cholesterol ester transfer protein inhibitors (CP524,515 and CP532,623). Pharm Res 27(5):878–893. Porter CJ, Trevaskis NL, Charman WN. 2007. Lipids and lipid-based formulations: Optimizing the oral delivery of lipophilic drugs. Nat Rev Drug Discov 6(3):231– 248. Wasan KM, Brocks DR, Lee SD, Sachs-Barrable K, Thornton SJ. 2008. Impact of lipoproteins on the biological activity and disposition of hydrophobic drugs: Implications for drug discovery. Nat Rev Drug Discov 7(1):84–99. Gershkovich P, Hoffman A. 2007. Effect of a high-fat meal on absorption and disposition of lipophilic compounds: The importance of degree of association with triglyceride-rich lipoproteins. Eur J Pharm Sci 32(1):24–32. Gershkovich P, Shtainer D, Hoffman A. 2007. The effect of a high-fat meal on the pharmacodynamics of a model lipophilic compound that binds extensively to triglyceride-rich lipoproteins. Int J Pharm 333(1–2):1–4. Trevaskis NL, Charman WN, Porter CJ. 2010. Targeted drug delivery to lymphocytes: A route to site-specific immunomodulation? Mol Pharm 7(6):2297–2309. Caliph SM, Trevaskis NL, Charman WN, Porter CJ. 2012. Intravenous dosing conditions may affect systemic clearance for highly lipophilic drugs: Implications for lymphatic transport and absolute bioavailability studies. J Pharm Sci 101:3540–3546. Pocock DE, Vost A. 1974. DDT absorption and chylomicron transport in rat. Lipids 9(6):374–381. Noguchi T, Charman WNA, Stella VJ. 1985. Lymphatic appearance of DDT in thoracic or mesenteric lymph duct cannulated rats. Int J Pharm 24(2–3):185–192. Gershkovich P, Hoffman A. 2005. Uptake of lipophilic drugs by plasma derived isolated chylomicrons: Linear correlation with intestinal lymphatic bioavailability. Eur J Pharm Sci 26(5):394–404. Johnson BM, Chen W, Borchardt RT, Charman WN, Porter CJ. 2003. A kinetic evaluation of the absorption, efflux, and metabolism of verapamil in the autoperfused rat jejunum. J Pharmacol Exp Ther 305(1):151–158. Trevaskis NL, Porter CJ, Charman WN. 2005. Bile increases intestinal lymphatic drug transport in the fasted rat. Pharm Res 22(11):1863–1870. Edwards GA, Porter CJ, Caliph SM, Khoo SM, Charman WN. 2001. Animal models for the study of intestinal lymphatic drug transport. Adv Drug Deliv Rev 50(1–2):45–60. Trevaskis NL, Caliph SM, Nguyen G, Tso P, Charman WN, Porter CJ. 2013. A mouse model to evaluate the impact of species, sex, and lipid load on lymphatic drug transport. Pharm Res.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 7, JULY 2013

2408

CALIPH ET AL.

28. Caliph SM, Trevaskis NL, Charman WN, Porter CJ. 2012. Intravenous dosing conditions may affect systemic clearance for highly lipophilic drugs: Implications for lymphatic transport and absolute bioavailability studies. J Pharm Sci 101(9):3540–3546. 29. Bauer RJ, Guzy S, Ng C. 2007. A survey of population analysis methods and software for complex pharmacokinetic and pharmacodynamic models with examples. AAPS J 9(1):E60–E83. 30. Bulitta JB, Bingolbali A, Shin BS, Landersdorfer CB. 2011. Development of a new pre- and post-processing tool (SADAPTTRAN) for nonlinear mixed-effects modeling in S-ADAPT. AAPS J 13(2):201–211. 31. Beal SL. 2001. Ways to fit a PK model with some data below the quantification limit. J Pharm Pharmacodyn 28(5):481–504. 32. Bulitta JB, Okusanya OO, Forrest A, Bhavnani SM, Clark K, Still JG, Fernandes P, Ambrose PG. 2013. Population pharmacokinetics of fusidic acid: Rationale for front-loaded dosing regimens due to autoinhibition of clearance. Antimicrob Agents Chemother 57(1):498–507. 33. Belfrage P, Borgstr¨om B, Olivecrona T. 1963. The tissue distribution of radioactivity following the injection of varying levels of fatty acid labeled chylomicrons in the rat. Acta Physiol Scand 58(2–3):111–123. 34. Olivecrona T. 1962. The metabolism of 1-C14-palmitic acid labeled chylomicrons in rats. Acta Physiol Scand 55(2–3):170–176. 35. Bragdon JH, Gordon RS, Jr. 1958. Tissue distribution of C14 after the intravenous injection of labeled chylomicrons and unesterified fatty acids in the rat. J Clin Invest 37(4):574–578. 36. Schemmel R, Mickelsen O, Gill JL. 1970. Dietary obesity in rats: Body weight and body fat accretion in seven strains of rats. J Nutr 100(9):1041–1048. 37. Stimpson SA, Turner SM, Clifton LG, Poole JC, Mohammed HA, Shearer TW, Waitt GM, Hagerty LL, Remlinger KS, Hellerstein MK, Evans WJ. 2012. Total-body creatine pool size and skeletal muscle mass determination by creatine-(methyld3) dilution in rats. J Appl Physiol 112(11):1940–1948. 38. Arola L, Herrera E, Alemany M. 1979. A method for the estimation of striated muscle mass in small laboratory animals. Revista Espanola de Fisiologia 35(2):215–218. 39. Bragdon JH. 1958. C1402 excretion after the intravenous administration of labeled chylomicrons in the rat. Arch Biochem Biophys 75(2):528–533. 40. Mc CC, Gates HS, Jr., Gordon RS, Jr. 1957. C1402 Excretion after the intravenous administration of albuminbound palmitate-1-C14 to intact rats. Arch Biochem Biophys 71(2):346–351.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 7, JULY 2013

41. Wasan KM, Pritchard PH, Ramaswamy M, Wong W, Donnachie EM, Brunner LJ. 1997. Differences in lipoprotein lipid concentration and composition modify the plasma distribution of cyclosporine. Pharm Res 14(11):1613– 1620. 42. Humberstone AJ, Porter CJ, Edwards GA, Charman WN. 1998. Association of halofantrine with postprandially derived plasma lipoproteins decreases its clearance relative to administration in the fasted state. J Pharm Sci 87(8):936– 942. 43. Hauss DJ, Mehta SC, Radebauch GW. 1994. Targeted lymphatic transport and modified systemic distribution of CI-976, a lipophilic lipid-regulator drug, via a formulation approach. Int J Pharm 108(2):85–93. 44. Vost A, Maclean N. 1984. Hydrocarbon transport in chylomicrons and high-density lipoproteins in rat. Lipids 19(6):423–435. 45. Mohammed A, Eklund A, Ostlund-Lindqvist AM, Slanina P. 1990. Distribution of toxaphene, DDT, and PCB among lipoprotein fractions in rat and human plasma. Arch Toxicol 64(7):567–571. 46. Trevaskis NL, Tso P, Rider T, Charman WN, Porter CJ, Jandacek R. 2006. Tissue uptake of DDT is independent of chylomicron metabolism. Arch Toxicol 80(4):196– 200. 47. Kohan AB, Vandersall AE, Yang Q, Xu M, Jandacek RJ, Tso P. 2013. The transport of DDT from chylomicrons to adipocytes does not mimic triacylglycerol transport. Biochim Biophys Acta 1831(2):300–305. 48. Maliwal BP, Guthrie FE. 1982. In vitro uptake and transfer of chlorinated hydrocarbons among human lipoproteins. J Lipid Res 23(3):474–479. 49. McIntosh MP, Porter CJ, Wasan KM, Ramaswamy M, Charman WN. 1999. Differences in the lipoprotein binding profile of halofantrine in fed and fasted human or beagle plasma are dictated by the respective masses of core apolar lipoprotein lipid. J Pharm Sci 88(3):378– 384. 50. Patel JP, Fleischer JG, Wasan KM, Brocks DR. 2009. The effect of experimental hyperlipidemia on the stereoselective tissue distribution, lipoprotein association and microsomal metabolism of (+/−)-halofantrine. J Pharm Sci 98(7):2516–2528. 51. Brocks DR, Wasan KM. 2002. The influence of lipids on stereoselective pharmacokinetics of halofantrine: Important implications in food-effect studies involving drugs that bind to lipoproteins. J Pharm Sci 91(8):1817–1826.

DOI 10.1002/jps