N,N-Dimethyl Tertiary Amino Group Mediated Dual Pancreas- and

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N, N-Dimethyl tertiary amino group mediated dual pancreasand lung-targeting therapy against acute pancreatitis Shi Luo, Peiwen Li, Sha Li, Zhengwu Du, Xun Hu, Yao Fu, and Zhirong Zhang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00028 • Publication Date (Web): 01 Mar 2017 Downloaded from http://pubs.acs.org on March 2, 2017

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Molecular Pharmaceutics

N, N-Dimethyl tertiary amino group mediated dual pancreas- and lung-targeting therapy against acute pancreatitis Shi Luo, Peiwen Li, Sha Li, Zhengwu Du, Xun Hu, Yao Fu*, Zhirong Zhang* Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu 610041, China *Correspondence: Zhirong Zhang Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, Sichuan University No. 17, Block 3, Southern Renmin Road, Chengdu 610041 China Email: [email protected] Tel: +86-28-85501566; Fax: +86-28-85501615 Yao Fu Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, Sichuan University No. 17, Block 3, Southern Renmin Road, Chengdu, 610041, China. Email: [email protected] Tel:

+86-28-85503798

ACS Paragon Plus Environment

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ABSTRACT

Acute pancreatitis (AP) is a sudden inflammation of the pancreas with high-mortality rate worldwide. As a severe complication to AP, acute lung injury has been the major cause of death among patients with AP. Poor penetration across the blood pancreas barrier (BPB), and insufficient drug accumulation at the target site often result in poor therapeutic outcome. Our previous work successfully demonstrated a dual-specific targeting strategy to pancreas and lung using a phenolic propanediamine moiety. Inspired by this, a simplified ligand structure, N, Ndimethyl tertiary amino group, was covalently conjugated to celastrol (CLT) to afford tertiary amino conjugates via either an ester (CP) or an amide linkage (CTA). With sufficient plasma stability, CTA was subjected to the following studies. Compared to CLT, CTA exhibited excellent cellular uptake efficiency in both rat pancreatic acinar cell line (AR42J) and human pulmonary alveolar epithelial cell line (A549). Organic cation transporters were proven to be responsible for this active transport process. Given systemically, CTA specifically distributed to pancreases and lungs in rats thus resulting in a 2.59-fold and 3.31-fold increase in tissue-specific accumulation as compared to CLT. After CTA treatment, tissue lesions were greatly alleviated and the levels of pro-inflammatory cytokines were downregulated in rats with sodium taurocholate-induced AP. Furthermore, CTA demonstrated marginal adverse effect against major organs with reduced cardiac toxicity compared to CLT. Together, tertiary amine mediated dual pancreas- and lung-targeting therapy represents an efficient and safe strategy for AP management.

KEYWORDS: Tertiary amine, Dual-specific targeting, Acute pancreatitis, Celastrol, Antiinflammatory


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1. INTRODUCTION As a devastating inflammatory condition of pancreas, acute pancreatitis (AP) is characterized by both local and systemic symptoms. Although the pathogenesis of this disease is controversial, the autodigestion theory of pancreas was accepted to account for the initiation of pancreatitis1-4. NF-κB plays a critical role in the progression of AP in a number of different animal models5. In response to the acinar cell injury and the consequent release of intracellular content, local leukocytes migrated into pancreas, proinflammatory cytokines were rapidly generated and released into the blood circulation system to recruit leucocyte infiltration, leading to a life-threatening systemic multiple organ dysfunction syndrome (MODS)6, 7. Besides pancreatic injury, pulmonary dysfunction has been the most relevant manifestation of extrapancreatic organ failure in AP patients which accounts for the majority of AP-related death8. In spite of improvements in diagnosis and therapeutics, the overall population mortality rate for AP has remained exceedingly high over the last decade, which posed tremendous emotional, physical and financial burden on patients and society9, 10. Currently, therapeutic efforts for the management of AP are limited to antibiotic therapy, nutritional support, and fluid resuscitation11. However, intravenously administered prophylactic antibiotics failed to prevent infected necrosis12. Despite the promising results of continuous infusion of protease inhibitors, the clinic outcome had not been improved13. Owing to the poor penetration across the blood pancreas barrier (BPB), most therapeutic agents failed to achieve sufficient accumulation in the pancreas14. Additionally, insufficient therapeutic concentration in lungs contributes to failures in the management of AP15. Hence, delivery strategies that improve BPB permeability may be promising to achieve effective AP therapy.


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Based on the specific pathogenesis of AP, our group previously proposed a dual-specific targeting strategy to overcome the obstacles in the treatment of AP. HPDM-rhein, fabricated by coupling an analogue of phenolic propanedimine moiety with a linear tertiary amine residue to rhein, was proven to specifically accumulated in pancreases and lungs when systemically administered in rats16. Additionally, podophyllotoxin modified with a benzoic propanedimine moiety with a cyclic tertiary amine residue showed excellent targetability towards lungs17. Given that lung and pancreas are featured with acidic microenvironment and basic lipophilic moieties can be stored in acidic organelles, we hypothesized that the tertiary amine residues in the proposed ligands mediated the organ-specific accumulation in the pancreas and lung. In our study, we selected celastrol (CLT) as the model compound, which is a pentacyclic triterpenoid extracted from traditional Chinese medicine “God of Thunder Vine”18. The in vivo anti-inflammatory effect has been demonstrated in animal models such as collagen-induced arthritis19, Alzheimer disease20, asthma21, systemic lupus erythematosus22, and rheumatoid arthritis23, 24. CLT was validated to suppress the NF-κB activation by targeting the Cys-179 in the activation loop of IKKβ to exert the anti-inflammatory effect18, 25, 26. Specifically, N, N-dimethyl ethanolamine and N, N-dimethyl-1,2-diaminoethamine were conjugated to CLT via either an ester or an amide bond to afford CP (Fig. 1 A) and CTA (Fig. 1 B). Next, we characterized the stability of the two conjugates in vitro, and studied the biodistribution of CTA in vivo. The cytotoxicity, selective cell uptake efficiency and underlying mechanisms were investigated systematically. Also, the therapeutic effect against sodium taurocholate induced AP in rats was further evaluated in vivo.


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Figure 1. The chemical structure of CP (A) and CTA (B).

2. EXPERIMENTAL SECTION 2.1. Materials Celastrol, carbamazepine and glycyrrhetinic acid with purity of 98.0% were obtained from Chengdu Must Biotechnology Co. Ltd. (Chengdu, China). N, N-dimethyl ethanolamine (DMEA) and N, N-dimethyl-1,2-diaminoethamine(DMEDA) were supplied by Tokyo Chemical Industry (Tokyo, Japan). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, USA). All other chemicals were of analytical grade and obtained commercially. 2.2. Animals and cell cultures Male Sprague-Dawley (SD) rats weighed 180 to 220 g were purchased from the Institute of laboratory Animals of Sichuan Academy of Medical Sciences & Sichuan Provincial People`s Hospital (Chengdu, China) and were maintained under standard housing conditions. All animal experiments were performed according to the institutional guidelines and approved by the Institutional Animal Care and Ethic Committee of Sichuan University.


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AR42J cells (rat pancreatic acinar cell line), A549 cells (human pulmonary alveolar epithelial cell line), HK-2 cells (human proximal tubule epithelial cell line) and HepG2 cells (human liver cells) were obtained from ATCC and cultured in DMEM with high glucose (Hyclone, USA) supplemented with 10% fetal bovine serum (Hyclone, USA), 1% penicillin and streptomycin. Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2, and the culture medium was changed every other day. 2.3.

Synthesis, characterization and in vitro stability assay of ligand–celastrol conjugates The conjugates were synthesized as illustrated in Scheme S1. The detailed methods were

presented in the supporting information (SI). The structures of the drug-ligand conjugates were confirmed and characterized by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (1H-NMR). The pKa of CTA was detected based on the published spectrophotometric indicator method in which p-nitrophenol was selected as an acid-base indicator27. Also the partition coefficient (logP) values of CTA and CLT were measured in an octanol-water system at 25 ± 0.5 °C. The stability of CP and CTA in PBS (pH7.4 and pH7.0), serum, lung homogenates and pancreas homogenates from SD rats was investigated at 37 °C. At predetermined time intervals, 100 µL samples were collected and the content of CTA was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, the detailed method was presented in SI. 2.4.

Sample preparation and LC-MS/MS analysis. Cell lysate, tissue homogenates and plasma samples for LC-MS/MS analysis were

performed by liquid–liquid extraction method. Briefly, to enrich CLT from biological samples, each 100 µL aliquot of these samples spiked with 10 µL of glycyrrhetinic acid (internal standard, IS) working solution was extracted with 1 mL ethyl acetate. The extraction was performed for 10


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min and centrifuging at 12000 rpm for 5 min. 750 µL aliquot of the supernatant was harvested and the residue underwent another extraction. The upper organic layer of two-time extraction was subsequently collected and completely evaporated at 40 °C under a stream of nitrogen. The dried residue was reconstituted with 200 µL of the mobile phase and filtered for further LCMS/MS injection. While in the case of CTA and CP, 10 µL of carbamazepine working solution was used as IS and dichlormethane was chosen as extraction solution. The preparation procedures were similar to those in CLT. To accurately trace the amount of CLT, CTA or CP in biological samples, a rapid and sensitive LC-MS/MS method was developed. 1µL volume of each sample were injected into a Diamonsil ODS column (50 mm × 4.6 mm, 1.8 µm) protected by the corresponding guard column (ODS, 5 µm) using a SL auto-sampler equipped to an Agilent 1200 series RRLC system, which is coupled with an Agilent triple-quadrupole mass spectrometer. For the determination of CLT, the mobile phase consisted of acetonitrile and 0.5% formic acid (80:20, v/v). The flow rate was 0.4 ml/min at 30 °C. Analytes were detected using an ESI source interface in positive ion mode. Multiple reaction monitoring (MRM) was used to monitor precursor to product ion transitions at m/z 451.3-201.1 (CLT) and m/z 471.3-135.1 (IS). Voltages of fragmentor potential and collision energy were 113 eV and 36 eV for CLT, and 615 eV and 40 eV for IS. Dwell time was 550ms. For CTA determination, the mobile phase was adjusted to methanol and 0.1% formic acid (70:30, v/v). The total run time was 4 min at a flow rate of 0.4 mL/min at 30 °C. The transitions for the protonated analytes were CTA (m/z 521.3→201.1) and carbamazepine (m/z 237.2→194). The mass spectrometer was operated in positive ion electrospray mode. Voltages of fragmentor potential and collision energy were 159 eV and 48 eV for CTA, and 106 eV and 16 eV for IS. For CP quantification, the mobile phase comprised methanol and 0.1% formic acid (78:22, v/v) and the flow rate was 0.4 mL/min at 30


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°C. MRM of m/z 522.3→201.1 was used to monitor CP. Voltages of fragmentor potential and collision energy were 174 eV and 54 eV for CP, and 106 eV and 16 eV for IS. Instrumental parameters were as follows: gas temperature of 350 °C, gas flow of 8 mL/min, nebulizer of 30 psi, capillary of 4000 v. 2.5. Cellular uptake studies 2.5.1. Dose-dependent cellular uptake The indicated cell lines were seeded in 6-well culture plates at a density of 5 × 105 cells per well in 2 mL culture medium and cultured for 48h at 37 °C to reach 75% confluence. Then cells were incubated with 1mL CLT or CTA solution at increasing concentrations (2.22, 6.66, 11.10, 17.77, 22.2 nM respectively) at 37 °C for 4h. Thereafter, cells were washed with ice-cold PBS (pH7.4) three times to remove extracellular drugs. Cells were then trypsinized, collected and centrifuged at 2000 rpm for 3 min. The cell pellets were suspended in ultrapure water and lysed by five freeze-thawing cycles to release the intracellular drugs. The intracellular concentrations of CTA or CLT were determined by LC-MS/MS and the total protein of cell lysates was measured by BCA assay kit (Pierce, USA). Cellular uptake was expressed as the amount (nM) of CTA or CLT per 1 mg of total cellular protein. 2.5.2. Cellular uptake on different cell lines The AR42J, A549, HK-2, HepG2 cells were seeded in 6-well plates and incubated with CTA or CLT (11.10 nM) for 4 h at 37 °C, respectively. Then, cells were washed and collected for the determination of intracellular concentration. Here HK-2 and HepG2 cells were served as negative control cell lines 2.5.3. Effect of temperature and energy inhibitors on cellular uptake The indicated cells were seeded in 6-well culture plates and cultured for 48h. Then cells


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were exposed to CTA or CLT (11.10 nM) for 4 h at 37 °C, 4 °C, or in the presence of 0.1% NaN3. After cells were rinsed and collected carefully, the intracellular concentration was quantified. 2.6.

Mechanistic studies on intracellular delivery of CTA In the study of intracellular delivery mechanism of CTA, cells were pre-incubated with

various inhibitors for 1 h and co-cultured with CTA (11.10 nM) for another 4 h. Here, DMEDA was used as a competitive inhibitor. L-Arginine and L-Lysine were served as inhibitors of basic amino acid transporters. Spermine specifically blocked the alkaline polyamine transporters. Pyrilamine was a substrate of the pyrilamine-sensitive transporter. Tetraethylammonium (TEA) was used as a classic inhibitor of organic cationic transporters (OCTs). Choline was chosen to indicate the choline transport system. Imipramine served as a cationic inhibitor. The concentration of the inhibitors used were 10-times higher than that of CTA. Then cells were carefully washed and harvested to determine the intracellular concentration of CTA. 2.7.

Tissue distribution of CTA in rats To explore the tissue distribution profile of CTA, 50 male SD rats were randomized into

two groups. For the animal administration, CLT or CTA were first dissolved by anhydrous ethanol and then diluted using the polyethylene glycol 400 (PEG400) into the concentration of 1 mg/mL. A single dose of CLT (1 mg/kg body weight) and CTA (1.16 mg/kg body weight, which is equivalent to CLT) were injected intravenously. Five rats from each group were sacrificed at 0.25, 0.5, 1, 2, 4 h post administration. The blood plasma was obtained and major tissues (hearts, livers, spleens, lungs, kidneys, and pancreases) were quickly harvested, washed with physiological saline and dried with filter paper carefully and then stored at −40 °C. Before assay, the organs were thrown to room temperature, accurately weighed and homogenized with two volumes of cold physiological saline (based on the organ weight) on the Precellys 24 lysis


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instrument (Bertin, France). The concentration of CLT and CTA were measured by LC-MS/MS. The biodistribution of the two drugs in each organ was normalized by the weight of the selected tissues. The pharmacokinetic parameters, including area under the curve (AUC0-t) and maximal concentration (Cmax) were calculated using Data and Statistics Software (DAS 3.0; Shanghai, China). Relative uptake efficiency (Reorgan) and concentration efficiency (Ceorgan) were adopted to evaluate the dual pancreas- and lung-targeting property of CTA after intravenously injection. Reorgan and Ceorgan were defined as follows: e = , ,

= , ,

2.8.

(1) (2)

Experimental acute pancreatitis (AP) A well-validated experimental severe acute pancreatitis (SAP) model in rats was adapted

as described before28, 29. Briefly, male SD outbred rats weighed 180 to 220g were anesthetized with 10% chloral hydrate (0.3 g/kg body weight, i.p. injection) and then sterile laparotomy was followed. After the bile-pancreatic duct was cannulated through the duodenum with a 25-G needle, 30 mg/kg body weight sodium taurocholate (5% in methyl blue-containing normal saline, pH 7.4) was retrograde intraductally infused in a pressure-controlled manner within 10 min. The abdominal wounds were immediately sutured under sterility subsequently. Sham-operated control rats are treated in exactly the same way as the experimental mice, but the infusion solution consists of saline only. Rats were recovered in a warm area and housed singly for the duration of the experiment. CLT and CTA were administered to rats via tail vein immediately after induction of AP. The optimal dose of CTA for treating sodium taurocholate induced AP was screened in preliminary experiments. Five rats from each group were sacrificed 6 h after the induction of AP. The time point was selected according to the kinetic of the disease. At the


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scheduled time point, 5 mL blood was obtained via femoral artery. The serum was separated from whole blood by centrifugation (3000 rpm × 5 min) and stored at −20 °C until analysis. Thereafter each rats underwent genital laparotomy and ascites were carefully pipetted and precisely weighted to evaluate the magnitude of edema associated with pulmonary and pancreas30. The pancreases and lungs were removed, rinsed with saline and blotted dry. To determine the pancreatic water content, one third pancreas was gained and the wet tissue weight was measured, tissues were dried overnight in 60 °C oven and the dry weight was obtained. Pancreatic water content was calculated as the difference between the wet and dry tissue weights, and was expressed as a percentage of the total (wet) weight. One third pancreatic tissue and the right lower lobe of the lung were fixed in 4% paraformaldehyde overnight for the histological examination. The other pancreatic and pulmonary tissues were snap frozen in liquid nitrogen and stored at −80 °C for biochemical quantification of myeloperoxidase (MPO) activity. 2.9.

Safety investigation After therapeutic CLT (3 mg/kg) and CTA (3.48 mg/kg) was administrated intravenously

into randomly grouped normal rats, the whole blood and major tissues were collected from 5 rats at predetermined time point. Blood was centrifuged at 5000 rpm for 10 min and the resulted serum was assayed on a Hitachi 7020 automatic biochemical analyzer (Hitachi, Japan) for the following analytes: uric acid (UA), creatinine (CREA), alanine transaminase (ALT), aspartate transaminase (AST), creatine kinase (CK), and lactate dehydrogenase (LDH). The harvest tissues were carefully washed, fixed with 4% paraformaldehyde, stained with hematoxylin and eosin (H&E) for histopathology evaluation and the lesions were photographed with a light microscope (Olympus BX53, Tokyo, Japan). 2.10.

Biochemical analysis and histological examination


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Blood amylase (AMY) levels was measured based on iodine-starch colorimetric method using Amylase Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer's instructions. One unit is defined as 10 mg starch digested by 100 mL serum in 30 min at 37 °C. The concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were determined using ELISA kit (R&D systems, Minneapolis, USA) following the manufacturer's procedure. To quantify the neutrophil infiltration in the pancreases and lungs, tissue MPO activity was determined by a spectrophotometric method using a commercial MPO assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. For histology evaluation, the fixed tissues were paraffin embedded, sectioned (5 mm thick) and stained with hematoxylins & eosin for morphological analyses. H&E-stained pancreas sections were scored in a blinded fashion by a pathologist, using previously published parameters31. To assess inflammatory cell infiltration in pancreas, a standard protocol for immunohistochemistry study was conducted and mouse anti-rat CD68 (ED-1) monoclonal antibody (BD Pharmingen, San Diego, CA) and rabbit anti-MPO polyclonal antibody (Merck Millipore Corporation) were used. The enumeration of MPO+ and ED1+ cells was done using ImageJ and it was expressed as positive cells per million pixels. 2.11.

Statistical analysis Data are presented as mean ± standard deviation (SD). An unpaired two-tailed Student’s

t-test was performed for two-group comparisons. One-way analysis of variance (ANOVA) with Tukey post hoc test was performed for multiple group comparisons. A p-value of < 0.05 was considered statistically different.

3. RESULTS 3.1.

Synthesis, characterization and in vitro stability of ligand–CLT conjugates


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The target compounds were synthesized by coupling tertiary amine group to CLT via an ester or amide bond (Scheme S1) and the detailed synthetic method was described in the SI. The structures of the conjugates were confirmed by ESI-MS and 1H-NMR (Figure S1, S2, S3, S4). Due to the poor solubility of CTA in water, the initial water system was substituted by an ethanol-water system. The pKa values of CTA in different ethanol-water systems was shown in Table 1. The pKa value of CTA was 8.04 in water and was obtained from the linear regression equation, pKa = − 0.053C + 8.0378, in which C represents the proportion of ethanol and the linearity of standard curve (R2) was greater than 0.99. According to the equation for weakly basic compound, pH = pKa + log[B]/[BH+], the ionic type of CTA under physiological conditions (pH 7.4) accounted for 81%. The logP of CTA was slightly increased when compared with CLT, which may improve the membrane permeability of the conjugate (Table 2).

Table 1. The pKa of CTA in ethanol-water system. Data represent mean ± SD (n = 5).

Ethanol (%, v/v) pKa

66.67 8.00 ± 0.27

50 8.01 ± 0.29

33.33 8.02 ± 0.20

25 8.03 ± 0.19

Table 2. The logP values of CTA and CLT at 25 ± 0.5°C, data represented mean ± SD (n=3).

ρ (mg/mL) CTA CLT

0.6 1.99 ± 0.04 1.65 ± 0.07

0.8 1.97 ± 0.04 1.68 ± 0.04

1.0 1.97 ± 0.05 1.65 ± 0.06

As shown in Figure 2A, about 95% CP degraded in plasma and PBS (pH7.0) within 15 min, which was unlikely to achieve sufficient delivery of the conjugates to the target site in vivo. In comparison, after 8 h incubation at 37 °C with rat plasma, ~80% of CTA remained unchanged in plasma, indicating that the amide bond remains stable in plasma. Over 90% of the total CTA


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remained unchanged in PBS after 8 h incubation, and ∼75% of the CTA remained in the pancreas or lung homogenates after incubation suggesting CTA is relatively stable and may undergo slow degradation at the target site in vivo (Figure 2B).

Figure 2. Stability of CP (A) and CTA (B) in plasma, pancreas homogenates, lung homogenates and 0.1 M PBS (pH7.4 or pH7.4) at 37 °C for 8 h. Data represent mean ± SD (n = 3).

3.2.

In vitro cell uptake To determine the cell uptake efficiency of CTA in pancreases and lungs, A549 and AR42J

cells were incubated with serial concentrations of CTA or CLT for 4 h at 37 °C, respectively. Revealed by a standard MTT assay, the cell viability was over 90% at the maximum concentration of 22.2 nM (Figure S5). For both cell lines, CTA treated groups displayed significantly higher levels of cellular uptake than CLT treated groups at all given concentrations (Figure 3A, 3B). Specifically, the intracellular concentration of CTA was 11.4 and 10.3 times higher than that of CLT at the dose of 17.77 nM in AR42J and A549, respectively. The uptake efficiency of CTA in HK-2 and HepG2 was decreased profoundly when compared with those in A549 (Figure 3E); in contrast, the cellular uptake of CLT showed no substantial difference between AR42J, A549 and HK-2 (Figure S6), demonstrating cell-specific delivery of CTA. Next,


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the effect of energy and temperature on the cellular uptake efficiency was explored. As shown in Figure 3F, the uptake

Figure 3. Enhanced intracellular delivery of CTA in AR42J (A) and A549 cells (B) after 4 h incubation, *p < 0.001 versus CLT. Competitive inhibition of cellular uptake of CTA into AR42J (C) and A549 (D) by different inhibitors. *p < 0.05 versus control. (E) Comparison of the cellular uptake of CTA in different cell lines. (F) Effects of energy and temperature on cellular uptake efficiency of CTA. Data represent mean ± S.D (n = 3).


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amount of CTA at 4 °C or in the presence of NaN3 was significantly lower than that at 37 °C, while the cellular uptake of CLT was not influenced by temperature or energy depletion (Figure S7). The temperature- and energy-dependent cellular uptake of CTA indicated that an active transport process may be involved in the cell uptake process. The cellular uptake mechanisms were examined after pre-incubating AR42J and A549 cells with various inhibitors. TEA, a classic inhibitor of OCTs, dramatically decreased the amount of intracellular accumulation of CTA in both cell lines (Figure 3C, 3D). As expected, DMEDA, a competitive inhibitor, also significantly reduced the cellular uptake of CTA in both cell lines. Pyrilamine-sensitive transporter might also mediate the intracellular uptake of CTA by AR42J cells, which is consistent with literature that the transporter likely contributed to the cellular delivery of tertiary amine based compounds32. Choline (a substrate of the choline transport system), alkaline amino acids (such as L-lysine, L-arginine, Spermine) and imipramine (cationic inhibitors) displayed no obvious influence on cellular uptake. These results suggest that OCTs and pyrilamine-sensitive transporter likely contributed to the cell-specific internalization of CTA in AR42J or A549 cells. 3.3.

Tissue distribution and bioavailability of CTA in rats To explore the in vivo profiles and tissue distribution pattern of CTA, the biodistribution

of CTA was further examined in rats following systemic administration. Compared with CLT, CTA displayed lower plasma concentrations and a higher clearance rate from plasma, indicating that CTA could rapidly distribute to tissues, organs, and the target sites (Figure 4A and Table S2). CTA showed significantly higher accumulation than CLT in lung and pancreas, while


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significantly lower accumulation than CLT in heart and spleen (Figure 4B and Figure S8). Specifically, 15 min after intravenous administration, the concentration of CTA showed a 2.59and 3.31-fold increase over CLT in the pancreases and lungs, respectively (Figure 4B). The mean concentration of CTA in pancreases and lungs were significantly higher than CLT at all given time points (Figure 4C, 4D). Together, introducing tertiary amine group into CLT led to enhanced BPB penetration and tissue-specific deposition, which suggests improved therapeutic efficacy and reduced off-target toxicity in vivo. Major pharmacokinetic parameters of CLT and CTA, including AUC0–t, Cmax, Re and Ce in pancreases and lungs were provided in Table 3. The Cmax and AUC0–t of CTA in pancreases and lungs were remarkably higher than that of CLT. CTA increased the Re 3.13-fold in pancreases and 5.47-fold in lungs, respectively. The Cmax of CTA was 2.63 times and 3.30 times higher than that of CLT in pancreases and lungs respectively. All these results suggest that tailoring the structure of CLT with a tertiary amine group improved the bioavailability of the therapeutics in the pancreases and lungs.

Table 3. Pharmacokinetic parameters of CTA in pancreases and lungs after i.v. injection in rats

CLT

CTA

Parameters Lungs

Pancreases

Lungs

Pancreases

AUC0-t (nM/L*h)

1.55 ± 0.16

0.89 ± 0.095

8.48 ± 0.46

2.79 ± 0.26

Cmax (nM/L)

1.44 ± 0.15

0.40 ± 0.035

4.75 ± 0.36

1.05 ± 0.073

Re

-

-

5.47

3.13

Ce

-

-

3.30

2.63


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Figure 4. (A) The mean plasma concentration-time profiles of CTA and CLT in rats after i.v. injection. (B) Tissue distribution of CTA and CLT in rats 15 min after intravenously injected. The concentration of CTA and CLT in (C) pancreases and (D) lungs at different time points (values were normalized to total tissue weight).

3.4.

Therapeutic Effect of CTA on local and systemic complications of AP in Rats. Next, the efficacy of CTA in protecting against organ injury was evaluated in a well-

validated rat SAP model that generates secondary organ dysfunction within 6 h of the initial insult33. After anesthetized and laparotomized, rats were given a pressure-controlled retrograde biliopancreatic infusion of sodium taurocholate. PBS, CTA and CLT were injected intravenously into

rats

immediately

after

the

abdominal

wound

was

sutured.

The

optimal


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Figure 5. The therapeutic effect of CTA on local and systemic inflammation in SAP rats. The serum amylase (A), IL-6 (J) and TNF-α (K) level, the pancreas (B) and lung (I) MPO activity, the water content (C) in pancreas was greatly downregulated following CTA treatment. (D) Representative tissue photomicrographs from H&E stained lung and pancreas. The intensive acinar necrosis (black arrow) and inflammatory cells infiltration (red arrow) in pancreas was spotted. Scare bar, 200 µm. (E) Representative immunohistochemistry images showing neutrophil infiltration (MPO+ cells and macrophage infiltration (ED1+ cells) into pancreas tissue from AP mice. Scare bar, 100 µm. (F) Composite histological pancreas injury score. (G) MPO+ cell counts per 106 pixels in pancreas. (H) Enumeration of ED1+ cells per 106 pixels in pancreas tissue. (L) The exact ascites weight evaluating the protective effect against systemic inflammation.

effective dosage of CTA was evaluated based on the decrease of serum AMS level (a key indicator of the severity of SAP) and the pancreas MPO (the maker to quantify the neutrophil infiltration) activity. High dose of CTA (3.48 mg/kg) significantly decreased the serum AMS and the MPO activity, while an equivalent dose of CLT (3 mg/kg) did not ameliorate the hyperamylasemia and the intensive neutrophil infiltration (Figure S9). Accordingly, 3.48 mg/kg was selected as the optimal dosage in the subsequent experiments. The serum amylase level in SAP group was 3.1-fold higher than that in sham group, which confirmed the establishment of the SAP model. The magnitude of the serum amylase upregulation was substantially suppressed by CTA treatment (Figure 5A). However, no significant differences in the serum α-amylase level were observed between CLT treated group and SAP group. As expected, pancreases from shamoperated rats showed normal architecture and no signs of inflammation, whereas, the sodium taurocholate challenge induced pronounced morphological lesion in pancreases, e.g. acinar cell necrosis, interlobular edema and inflammatory infiltration. These damages were relieved to a less degree when treated with CTA (Figure 5D, 5F). The MPO activity in pancreases was elevated


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when rats were insulted by sodium taurocholate, which was presented to a lower extent in the pancreases of SAP rats treated with CTA (Figure 5B). Surprisingly, CLT treatment also reduced the MPO activity in pancreases, which may be attributed to the inhibition of neutrophils recruitment into pancreases. The intensive inflammatory cell infiltration in pancreases consisted of murine macrophages positive for ED1 antigen and MPO-positive neutrophils was observed in experimental SAP, which was less obvious in CTA treated group (Figure 5E, 5G, 5H). To accurately predict the magnitude of edema, the pancreas water content was captured. As shown in Figure 5C, CTA treatment markedly alleviated the edema in pancreas when compared with CLT. Lung is the most commonly affected extra-pancreatic organ system in humans with SAP. The histological changes in lung tissues from experimental SAP rats were consistent with moderate acute respiratory distress syndrome (ARDS). The thickening of the alveolar walls, interstitial edema and leukocyte infiltrates could be clearly noted in SAP rats, this pathology was obviously remitted when CTA was administered (Figure 5D). The elevation of MPO activity in lungs indicating the overwhelming neutrophil infiltration was essentially prevented by CTA, while the aggravation was not sufficiently addressed by CLT (Figure 5I). To evaluate the antiinflammation effect of CTA, systemic concentration of TNF-α and serum IL-6 (another reliable maker of the severity of SAP in humans) were quantified using a standard ELISA assay. Compared to SAP group, the treatment with CLT failed to efficiently reduce the levels of TNF-α and IL-6, while CTA substantially decreased the serum level of TNF-α and IL-6, (Figure 5J, 5K). Ascitic fluids was previously suggested to be involved in the pathogenesis of lung injury in SAP partly through the activation of NF-κB30 and convinced to be a lethal factor associated with multiple organ failure (MOF)34. The CTA treatment greatly decreased the weight of ascites in


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comparison with SAP models, suggesting the alleviation of histological edema (Figure 5L). 3.5.

The in vivo toxicity of CTA. To assess the possibility of further application of this drug-ligand conjugate in protecting

against SAP, the in vivo toxicity was evaluated. No obvious inflammation and tissue damages were observed in major organs except for heart (Figure 6). CLT treatment caused severe atrophy of myocardial cells and myofibrillar loss in the heart, while the cardiac toxicity of CTA was greatly compromised as evidenced by the remarkably decreased serum CK and LDH level compared with CLT group (Table 4), which are common indices for the diagnosis of cardiac diseases35, 36. Due to the high accumulation in the liver, CTA may show hepatotoxicity. Serum ALT and AST level (important indicator of liver function in clinic37) from CTA treated rats was marginally but not statistically significantly higher than normal rats. Serum CREA and UA level was small in magnitude and equivalent in normal rats and CTA treated rats, indicating the preservation of renal function38. All these results demonstrate that CTA be safe for in vivo administration.

Figure 6. Representative tissue photomicrographs (H&E stain) from normal rats and rats administrated with therapeutic dosage of CLT and CTA. Magnification, 100X. (n = 5).


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Table 4. Biochemical examination of control and CTA treated rats. Data represent mean ± SD (n = 5).

Parameters

Control

CLT

CTA

ALT (U/L)

39 ± 5

54 ± 7

42 ± 8

AST (U/L)

114 ± 15

153 ± 21

137 ± 16

CREA (µM/L)

28 ± 3

41 ± 6

34 ± 7

UA (µM/L)

117 ± 10

110 ± 12

101 ± 7

CK (U/L)

415 ± 27

702 ± 47

536 ± 25

LDH (U/L)

1336 ± 323

1747 ± 359

149638

4. DISCUSSION Our group previously reported that a phenolic propanediamine moiety with a linear or cyclic tertiary amine residue markedly enhanced the pancreatic and pulmonary drug localization in rats16, 17. In this study, the tertiary amine residue was selected as a novel ligand and coupled to CLT through an amide bond to afford CTA with excellent pancreas and lung targeting efficiency. Regarding the structure of small molecule ligand drug conjugates, a cleavable bridge is generally preferred in the design of ligand-drug conjugate to release the therapeutic cargoes at the target site39. Such self-cleaving linkers should remain intact during transit, but lyse rapidly to achieve site-specific release of the therapeutic cargo. Thus, CP was designed and synthesized by conjugation via an ester bond, which was further proven to undergo rapid hydrolysis in plasma (Figure 2B). The poor plasma stability of CP could not afford the conjugate with sufficient circulation time to reach the target organs. To address this dilemma, a relative stable amide bond


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was employed in the later work of CTA. The remarkable chemical and enzymatic stability of CTA in plasma allowed successful delivery of the conjugate to target organs via systemic administration without encountering quick hydrolysis or degradation in the plasma. Currently, rat pancreatic acinar cell line AR42J is the only available cell line that maintained features of normal pancreatic acinar cells and has been widely used as an in vitro model to study the exocrine pancreas40, 41. Bearing most morphologic and biochemical features of the human pulmonary alveolar type 2 cell, A549 cells (human alveolar epithelial cells) are widely used in vitro for cellular mechanistic studies of lungs42. The intracellular delivery efficiency of CTA in AR42J and A549 was prominently superior than that in HepG2 and HK-2 (Figure 4E), illustrating the potential of cell-specific delivery. The energy- and temperature-dependent cellular uptake reveled by the mechanism study suggest an active transport process may be involved in CTA uptake (Figure 4F). The following competitive inhibition experiments certified our assumption that OCTs and pyrilamine-sensitive transporters mediated the cellular accumulation process (Figure 4C, D), which is in line with previous report16, 17. The excellent cell delivery efficiency and the cell selectivity suggested a possibility of outstanding dual pancreas- and lung- targeting ability of CTA in vivo. The tissue distribution profile in rats certified our assumption. The conjugated tertiary amine residue remarkably increased the tissue accumulation of CTA throughout the entire time course (Figure 4C,4D) and meanwhile improved the Cmax and AUC0-t (Table 3). It is well accepted that the bio-distribution profile of compound was governed to a large extent by its physicochemical property43, e.g. pKa and logP. The slightly increase of lipophilicity by coupling to tertiary amine allowed CTA to rapidly traverse through lipid membranes by passive diffusion. Modified by the basic residue, CTA displayed a moderately strong basic property with a pKa value of 8.04. As determined by a


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spectrophotometric indicator method, the percentage of un-ionized CTA at pH 7.4 and pH 7.0 was 19.0%, and 8.2%, respectively. Partially driven by the well-known pH gradient between the plasma (pH7.4) and the tissues, the molecular form of CTA preferentially migrated to acidic pancreas (pH7.0). Being internalized into pancreatic cells by passive diffusion and active transport, CTA became ionized and then sequestered in the acidic compartment. While CLT remained unionized and could freely diffuse outside the cells, thus the content of CTA in pancreas was higher than that of CLT at each time point. The amine-contained moderately strong basic compound was reported to preferably accumulated into lungs because of the interaction with the abundantly distributed negatively charged acidic phosphatidylserine (PS) in cell membrane44. The positively charged CTA under physiological conditions specifically interacted with negatively charged PS, leading to the excellent lung-specific accumulation. While the negatively charged CLT failed to selectively bind to the anchors in cell membrane, leading to poor absorption and a relative higher plasma level (Figure 7). To sum up, when tailored by a tertiary amine group, CTA displayed a profound increase of tissue selectivity over CLT. Thus, we concluded that the specific targeting accumulation was mainly mediated by the tertiary amine. Inflammation plays an important role in the pathology of AP. The manifestations of the disease are mediated by various inflammatory cytokines released during the course of pancreatitis. Masamune45 and Cuzzocrea46 reported that therapies against TNF-α, IL-6 showed a protective effect in experimental animal models of SAP. CLT had been reported for the beneficial and curative effects in various models of inflammation. By inhibiting the translocation of NF-κB to nucleus, CLT significantly downregulated the production of TNF-α, IL-6 and IL-8 in Crohn’s disease patients47. CLT also showed great beneficial effect in a mouse allergic asthma model through decreasing the phosphorylation of NF-κB21. Thereby, the reduction of pro-inflammatory


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cytokines such as TNF-α, IL-6 was likely due to the similar signaling pathway, which remained to be explored in our future study.

Figure 7. Schematic representation shows the proposed mechanism underlying the tissue selectivity.

5. CONCLUSION In sum, a novel tertiary amine derivative of CLT was synthesized by covalently conjugating N, N-dimethyl-1,2-ethanediamine via an amide bond. The newly obtained conjugate CTA displayed sufficient in vitro stability and marginal cytotoxicity. The excellent cellular uptake efficiency of CTA in AR42J or A549 cells was likely mediated by OCTs in an active manner. CTA preferentially accumulated in the pancreas and lung after systemic administration, showing greatly improved tissue selectivity and penetration profile in vivo. Furthermore, CTA effectively ameliorated local and systemic complication in SAP induced rats with reduced adverse effect. Taken together, conjugation of lipophilic therapeutics with tertiary amine may represent a promising strategy for dual pancreas- and lung-specific drug delivery. ASSOCIATED CONTENT


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Supporting Information. Additional methods, tables and figures including the synthesis of CP and CTA, the measurement of pKa and LogP, cytotoxicity assay, 1H-NMR spectra and ESI-MS of compounds, the in vitro cytotoxicity, the biodistribution profiles and main pharmacokinetic parameters in rats and the protect efficacy of CTA and CLT against AP. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Zhirong Zhang Email: [email protected] Tel: +86-28-85501566; Fax: +86-28-85501615 *Yao Fu Email: [email protected] Tel: +86-28-85503798 Author Contributions Zhirong Zhang conceived and designed the study. Shi Luo carried out the experiments, generated and analyzed data, and wrote the original manuscript. Peiwen Li and Zhengwu Du helped with animal and cell studies, Sha Li and Xun Hu helped with sythesis studies. Yao Fu and Zhirong Zhang helped with manuscript editing. Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENT We are very grateful for the financial support from the National Science Foundation of China (No.81473169). ABBREVIATIONS AP, acute pancreatitis; MODS, multiple organ dysfunction syndrome; BPB, blood pancreas barrier; CLT, celastrol; CP, N, N-dimethyl ethanolamine-celastrol conjugate; CTA, N, Ndimethyl-1,2-diaminoethamine-celastrol

conjugate;

MTT,

3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide; pKa, ionization constant; logP, partition coefficient; ESI-MS, electrospray ionization mass spectrometry; NMR, nuclear magnetic resonance; IS, internal standard; MRM, multiple reaction monitoring; TEA, tetraethylammonium; OCTs, organic cationic transporters; AUC0-t, area under the curve; Cmax, maximal concentration; Reorgan, Relative uptake efficiency; Ceorgan, concentration efficiency; SAP, severe acute pancreatitis; MPO, myeloperoxidase; UA, uric acid; CREA, creatinine; ALT, alanine transaminase; AST, aspartate transaminase; CK, creatine kinase; LDH, lactate dehydrogenase; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; AMS, amylase; ARDS, acute respiratory distress syndrome; MOF, multiple organ failure.

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(45) Masamune, A.; Shimosegawa, T. Anti-cytokine therapy for severe acute pancreatitis. Nihon Rinsho 2004, 62, (11), 2116-2121. (46) Cuzzocrea, S.; Mazzon, E.; Dugo, L.; Centorrino, T.; Ciccolo, A.; McDonald, M. C.; de Sarro, A.; Caputi, A. P.; Thiemermann, C. Absence of endogenous interleukin-6 enhances the inflammatory response during acute pancreatitis induced by cerulein in mice. Cytokine 2002, 18, (5), 274-285. (47) Pinna, G. F.; Fiorucci, M.; Reimund, J.-M.; Taquet, N.; Arondel, Y.; Muller, C. D. Celastrol inhibits pro-inflammatory cytokine secretion in Crohn’s disease biopsies. Biochem. Biophys. Res. Commun. 2004, 322, (3), 778-786.


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For Table of Contents Use Only

Title: N, N-Dimethyl tertiary amino group mediated dual pancreas- and lung-targeting therapy against acute pancreatitis Author: Shi Luo, Peiwen Li, Sha Li, Zhengwu Du, Xun Hu, Yao Fu*, Zhirong Zhang* Institution: Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu 610041, China


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abstract graphic 88x32mm (300 x 300 DPI)


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N,N-Dimethyl Tertiary Amino Group Mediated Dual Pancreas- and

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