PEPT1-m ed iated p rod rug strategy for oral d elivery of p eram ivir

Yonging Sun ,Wei Gn ,Mingdo Lei,Wei Jing ,Meng Cheng ,Junwei He,Qi Sun ,Wn Liu ,Lvjing Hu ,Yi Jin ,∗∗

a Division of Pharmaceutics,National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine,Jiangxi University of Traditional Chinese Medicine,56 Yangming Road,Nanchang 330006,China

b Department of Pharmacy,Jiangxi Maternal and Child Health Hospital,NO 318 Bayi Road,Nanchang 330001,China c Research Center of Natural Resources of Chinese Medicinal Materials and Ethnic Medicine,Jiangxi University of

Traditional Chinese Medicine,1688 Meiling Road,Nanchang 330004,China

d Aff iliated Hospital of Jiangxi University of Traditional Chinese Medicine,NO 445 Bayi Road,Nanchang 330006,China

Keywords:Peramivir Prodrug Peptide transporter 1 Pharm acokinetics Oral bioavailability

A B S T R A C T

1. Introd uction

Peramivir w as a novel and highly potent neuraminidase inhibitor developed by Bio Cryst Pharm aceuticals Inc.for the treatm ent of inf luenza A and B[1].Like oseltam ivir and zanam ivir,it can bind to the active site of the inf luenza virus neuraminidase to prevent the spread of viruses.Peramivir show ed a distinct in vitro resistance prof ile w hen com pared w ith oseltam ivir and zanam ivir,and som e viral variants w ith in vitro oseltam ivir and zanam ivir resistance could be susceptible to peram ivir[2,3].FDA issued an Em ergency Use Authorization(EUA)on 23 October 2009,authorizing peram ivir for treatm ent of certain hospitalized adults and children w ith p H1N1 in the United States till 23 June 2010,w hich w as the f irst tim e that an investigational drug w as authorized for clinical use under an EUA[4,5].Due to the high polarity(log P of-1.4)and the low m em brane perm eability across the intestine,peram ivir had a very low oral bioavailability(only 3%).Oral peram ivir w as associated w ith reduced viral titers,but no signif icant decrease w as observed to relief of sym ptom s,w hich could be attributed to a low oral bioavailability in hum ans[2].Therefore,peramivir can only be given as an intravenous injection to the patients w ith inf luenza in clinic,w hich lim ited the clinical utility of peram ivir to treat inf luenza infections.Hence,the development of an oral alternative to intravenous administration of peram ivir is indispensable for reducing costs dictated by hospital treatm ent and im proving patient com pliance.

Many attem pts had focused on the structural m odif ication of peram ivir w ith the aim to im prove the oral bioavailability.Wang et al.have synthesized a series of peram ivir phosphonate derivatives as orally available anti-inf luenza drugs[6].But none of them w ere in routine clinical use at present.

With the developm ent of m olecular biology,m any m embrane transporters have been identif ied,for exam ple,peptide transporters 1(PEPT1)[7],am ino acid transporter B0,+(ATB0,+,SLC6A14)[8,9],the glucose transporter GLUT1[10].Prodrugs targeted to m em brane transporters have been extensively developed in order to im prove oral bioavailability and overcom e undesired biopharm aceutical properties of drugs.Thanks to the high expression on the apical membrane of the sm all intestine,and the broad substrate specif icity,PEPT1 has been the m ost prom ising and attractive target in the prodrug design am ong all nutrient transporters.It has been dem onstrated a posteriori that a 3 to 5-fold higher oral bioavailability of valacyclovir and valganciclovir com pared w ith acyclovir and ganciclovir,respectively,w as attributed to the transport by PEPT1[11–14].This PEPT1-targeted prodrug strategy has also been extended to LY2140023,an N-linked m ethionil prodrug of LY404039.LY2140023 im proved the oral bioavailability of the parent drug from 6.0%to 68.0%in hum an[15,16].In our previous study,w e have synthesized s series of am ino acid prodrug of cytarabine,and the candidate drug valcytarabine,the L-valyl prodrug of cytarabine,displayed a PEPT1-m ediated transport across the sm all intestine and increased the oral bioavailability of cytarabine from 21.8%to 60.0%.Valcytarabine has been allow ed to the clinic trial by CFDA in 2016(Clinical NO 2016L10399 and 2016L10400)[17].

In order to im prove the oral availability and develop the oral alternative to peram ivir,the structural m odif ication w as performed on the carboxyl group,and seven amino acid ester prodrugs and seven am ino acid am ide prodrugs w ere synthesized.The transport of these prodrugs w as com pared w ith peram ivir in Caco-2 cell m onolayers to screen the lead compound with the highest permeability.The uptake of peramivr-(CH2)2-L-Val and peram ivir-L-Ile in PEPT1-overexpressing MDCK(MDCK-PEPT1)cells w as perform ed to delineate the role of PEPT1 in the transport of peram ivr-(CH2)2-L-Val and peram ivir-L-Ile across the intestinal epithelium.We also reported the chem ical and enzym atic stability of the prodrugs in phosphate buffers,rat plasm a,tissue hom ogenates and gastrointestinal f luids.Finally,the pharmacokinetics(PK)of peram ivir-(CH2)2-L-Val and peram ivir-L-Ile w as evaluated in Sprague-Daw ley rats after oral adm inistration.All above results indicated the PEPT1-m ediated prodrug strategy has successfully enhanced the oral absorption of peramivir.

2. Materials and m ethod s

2.1. Materials

Peram ivir w as purchased from Hubei Xinkang Chem icals Co.Ltd(Wuhan,China).(1S,2S,3R,4R)-Methyl 3-((R)-1-acetam ido-2-ethylbutyl)-4-(tert–butoxycarbonyl) am ino)-2-hydroxycyclopentanecarboxylate (com pound 1, CAS:229,614-05-5)w as purchased from Acesys Pharm atech Co.Ltd (Nanjing,China).1,3-Bis(tert–butoxycarbonyl)-2-methylthiopseudourea(Boc TU)w as purchased from Shanghai Balm xy Pharm aceutic Co.Ltd(Shanghai,China).HgCl2,LiOH and 4-dim ethylam inopyridine(DMAP)w ere purchased from Shandong Xiya Chem icals Co.Ltd(Jinan China).3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride(EDC)w as purchased from Zhejiang Kangpu Chem icals Co.Ltd(Quzhou China).1-Hydroxybenzotriazole(HOBT)w as purchased from Chengdu Best Reagents Co.Ltd(Chengdu China).All amino acids were purchsed from GL Chemical Ltd(Shanghai,China).PEPT1-overespressing MDCK cell(MDCKPEPT1)w as established by Cellbio Biology Co.Ltd(Shanghai China).All other chem icals used w ere of the highest purity available.

2.2. The synthesis procedure for prodrugs

2.2.1. Synthesis of N-Boc-protected peramivir(4)(Fig.1)

To the suspension of com pound 1(4.0 g,10 m m ol)in 20 m l ether at 0°C,4 M HCl-1,4-dioxane(10.0 m L,40 mm ol)and ether(40 m L)w as added.The reaction m ixture w as stirred overnight at room tem perature,and then ref luxed at 50°C for 2 h.The m ixture w as cooled to room tem perature and f iltered,and the f iltrate was concentrated in vacuo to give the w hite solid compound 2(3.0 g,100%).

To the solution of com pound 2(3.0 g,10 m m ol)in 30.0 m L N,N-Dim ethylform am ide(DMF),triethylam ine(TEA,4.2 m l,30 m m ol),Boc-TU(3.2 g,11 m m ol)and HgCl2(3.2 g,11 m m ol)w ere added under ice-cooled w ater.The reaction m ixture w as stirred at room tem perature for 2 h to give a w hite com pound 3(4.3 g,79%).

To the solution of com pound 3(4.3 g,7.92 m m ol)in ethanol(EtOH,30 m l)and tetrahydrofuran(THF,30 m l)under 0°C,5%NaOH solution(24 m l,30 m m ol)w as added.The reaction m ixture w as stirred at room temperature for 3 h,and EtOH and THFw ere evaporated under vacuum.Water(30 m l)and glacial acetic acid(2 m l)w as added to the residue and stirred to give com pound 4(3.6 g,85.9%)as a w hite solid.

2.2.2. Synthesis of amino acid ester prodrug of peramivir(8a–g)(Fig.2)

Boc-protected am ino acids(5,29.0 m m ol),ethylene glycol(9.0 g,140 m m ol)and DMAP(0.5 g,5.8 m m ol)and CH2Cl2(150 m l)w ere stirred under argon at 0°Cfor 10 m in.Then EDC(7.2 g,37.3 mmol)was added to the solution above and stirred for 20 h at room tem perature.The organic solvent w as evaporated in vacuo and the residue w as purif ied by colum n chrom atography(CH2Cl2/CH3OH)to give com pound 6.

Compound 4(1.06 g,2 mmol),compound 6(5 m mol)and DMAP(75 m g,0.6 m m ol)w ere dissolved in CH2Cl2(30 m l)and stirred under argon at room tem perature for 20 m in.EDC(0.58 g,3.0 m m ol)w as added to the solution above and stirred overnight at room tem perature.The organic solvent w as evaporated in vacuo and the residue w as purif ied by colum n chrom atography(ethyl acetate(EtOAc)/petroleum ether(PE))to give a w hite foam-like com pound 7.

Trif luoroacetic acid(TFA,2.0 m l)w as added to the solution of com pound 7(1.04 m m ol)in CH2Cl2(20 m L)and stirred at room tem perature for 16 h.The organic solvent w as evaporated under reduced pressure to give a w hite solid compound 8a–g.

2.2.3. Synthesis of amino acid amide prodrug of peramivir(11a–g)(Fig.3)

To the solution of com pound 4(1.06 g,2.0 m m ol)in N,Ndim ethylform am ide(DMF,15 m l),triethylam ine(TEA,0.56 m l,4.0 m mol),EDC(0.57 g,3.0 mmol),HOBt(0.41 g,3.0 mmol)were added sequentially,and stirred under argon at room tem perature for 10 m in.Then am ino acid m ethyl ester(2.4 m m ol)w as added to the above m ixture and stirred at room tem perature for 24 h.TEA and DMF w as evaporated under reduced pressure.The residue w as diluted w ith EtOAc(100 m l)and successively w ashed w ith w ater and brine.The organic layer was condensed and the residue was purif ied by column chrom atography(EtOAc/PE)to give the w hite com pound 9.

The solution of LiOH(0.126 g,3 m m ol)in w ater(3 m l)w as added to the solution of com pound 9(1.4 m m ol)in EtOH(10 ml)and THF(10 ml)and stirred overnight at room temperature.The reaction w as m onitoring by thin-layer chrom atography(TLC).EtOH and THFw ere evaporated under vacuum.Water(3 m l)and glacial acetic acid(3 m l)w as added to the residue and stirred under ice-cooled water to give a w hite compound10.

To the solution of 10(1.27 m m ol)in CH2Cl2(20 m L),TFA(2 m L)w as added and stirred at room tem perature for 16 h.The reaction mixture w as condensed under reduced pressure to give the w hite product 11a–g.

Peram ivir-(CH2)2-l-Val(8a):yield 28.2%,1H NMR(600 MHz,DMSO–d6,δppm)8.43(s,3H),7.96–7.81(m,2H),6.88(d,J=10.5 Hz,2H),4.52–4.46(m,1H),4.40(td,J=10.6,2.5 Hz,1H),4.34–4.24(m,3H),4.21–4.14(m,1H),3.85(dd,J=15.3,7.9 Hz,1H),2.75–2.71(m,1H),2.67–2.57(m,1H),2.20–2.06(m,2H),2.04(s,1H),1.82–1.74(m,2H),1.61(dd,J=13.3,6.6 Hz,0.5H),1.49–1.24(m,5H),1.11(dd,J=8.6,4.7 Hz,0.5H),0.99–0.82(m,12H);13C NMR(600 MHz,DMSO–d6,δppm)174.05,169.38,159.19,156.41,62.60,57.66,54.73,50.88,49.62,48.69,47.26,43.36,43.04,29.83,23.11,21.32,18.60,17.82,13.03,12.32,11.80.

Peram ivir-(CH2)2-d-Ala(8b):yield 23.3%,1H NMR(600 MHz,DMSO–d6,δppm)7.26(dd,J=10.0,3.5 Hz,4H),7.19–7.18(m,4H),5.61(d,J=4.8 Hz,1H),4.56(d,J=5.6 Hz,1H),4.32–4.21(m,4H),4.10(d,J=5.3 Hz,1H),3.44–3.40(m,1H),3.05(d,J=7.9 Hz,1H),2.88(d,J=7.5 Hz,1H),2.71(dd,J=8.6,5.1 Hz,1H),2.60–2.56(m,2H),2.07–2.01(m,2H),1.90(s,3H),1.66(ddd,J=13.7,8.1,3.4 Hz,2H),1.24(s,3H),0.93–0.83(m,6H);13CNMR(600 MHz,DMSO–d6,δppm)174.05,169.38,159.19,156.41,62.60,57.66,54.73,50.88,49.62,48.69,47.26,43.36,29.83,23.11,21.32,18.60,17.82,13.03,12.32,11.80.

Peram ivir-(CH2)2-l-Ala(8c):yield 25.4%,1H NMR(600 MHz,DMSO–d6,δppm)7.26(dd,J=10.0,3.5 Hz,4H),7.19–7.18(m,4H),5.61(d,J=4.8 Hz,1H),4.56(d,J=5.6 Hz,1H),4.32–4.21(m,4H),4.10(d,J=5.3 Hz,1H),3.44–3.40(m,1H),3.05(d,J=7.9 Hz,1H),2.88(d,J=7.5 Hz,1H),2.71(dd,J=8.6,5.1 Hz,1H),2.60–2.56(m,2H),2.07–2.01(m,2H),1.90(s,3H),1.66(ddd,J=13.7,8.1,3.4 Hz,2H),1.24(s,3H),0.93–0.83(m,6H);13CNMR(600 MHz,DMSO–d6,δppm)174.05,169.38,159.19,156.41,62.60.57.66,54.73,50.88,49.62,48.69,47.26,43.36,29.83,23.11,21.32,18.60,17.82,13.03,12.32,11.80.

Peram ivir-(CH2)2-d-Leu(8d):yield 25.4%,1H NMR(600 MHz,DMSO–d6,δppm)7.29–7.23(m,1H),7.20–7.13(m,1H),7.04(s,1H),4.42–4.39(m,1H),4.33–4.27(m,1H),4.20–4.14(m,1H),4.02–3.94(m,1H),3.62(q,J=5.2 Hz,1H),3.45(q,J=7.0 Hz,2H),1.73–1.59(m,4H),1.52–1.43(m,1H),1.40–1.25(m,2H),1.22–1.13(m,1H),1.07–1.04(m,5H),1.03–0.98(m,1H),0.89(ddd,J=15.2,8.3,5.3 Hz,9H);13C NMR(600 MHz,DMSO–d6,δppm)174.05,169.38,159.19,156.41,62.60,57.66,54.73,50.88,49.62,48.69,47.26,43.36,30.35,29.83,23.11,22.53,21.32,18.60,17.82,13.03,12.32,11.80Peramivir-(CH2)2-l-Leu(8e):yield 20.6%,1H NMR(600 MHz,DMSO–d6,δppm)7.29–7.23(m,1H),7.20–7.13(m,1H),7.04(s,1H),4.42–4.39(m,1H),4.33–4.27(m,1H),4.20–4.14(m,1H),4.02–3.94(m,1H),3.62(q,J=5.2 Hz,1H),3.45(q,J=7.0 Hz,2H),1.73–1.59(m,4H),1.52–1.43(m,1H),1.40–1.25(m,2H),1.22–1.13(m,1H),1.07–1.04(m,5H),1.03–0.98(m,1H),0.89(ddd,J=15.2,8.3,5.3 Hz,9H);13C NMR(600 MHz,DMSO–d6,δppm)174.05,169.38,159.19,156.41,62.60.57.66,54.73,50.88,49.62,48.69,47.26,43.36,30.35,29.83,23.11,22.53,21.32,18.60,17.82,13.03,12.32,11.80.

Peram ivir-(CH2)2-l-Phe(8f):yield 17.8%,1H NMR(600 MHz,DMSO–d6,δppm)7.32–7.13(m,5H),δ7.26(dd,J=10.0,3.5 Hz,4H),7.19–7.18(m,4H),5.61(d,J=4.8 Hz,1H),4.56(d,J=5.6 Hz,1H),4.32–4.21(m,4H),4.10(d,J=5.3 Hz,1H),3.44–3.40(m,1H),3.29(d,J=7.9 Hz,2H),3.05(d,J=7.9 Hz,1H),2.88(d,J=7.5 Hz,1H),2.71(dd,J=8.6,5.1 Hz,1H),2.60–2.56(m,2H),2.07–2.01(m,2H),1.90(s,3H),1.66(ddd,J=13.7,8.1,3.4 Hz,2H),0.93–0.83(m,6H);13C NMR(600 MHz,DMSO–d6,δppm)174.05,169.38,159.19,156.41,141.69,137.34,136.31,129.82,128.75,126.28,62.60,57.66,54.73,50.88,49.62,48.69,47.26,43.36,30.35,29.83,23.11,22.53,21.32,18.60,17.82,13.03,12.32,11.80.

Peram ivir-(CH2)2-l-Pro(8g):yield 23.7%,1H NMR(600 MHz,DMSO–d6,δppm)7.26(dd,J=10.0,3.5 Hz,4H),7.19–7.18(m,4H),5.61(d,J=4.8 Hz,1H),4.56(d,J=5.6 Hz,1H),4.32–4.21(m,4H),4.10(d,J=5.3 Hz,1H),3.44–3.40(m,1H),3.05(d,J=7.9 Hz,1H),2.88(d,J=7.5 Hz,1H),2.80(m,2H),2.71(dd,J=8.6,5.1 Hz,1H),2.60–2.56(m,2H),2.07–2.01(m,2H),1.96(m,2H),1.90(s,3H),1.66(ddd,J=13.7,8.1,3.4 Hz,2H),1.64(m,2H),0.93–0.83(m,6H);13CNMR(600 MHz,DMSO–d6,δppm)174.05,169.38,159.19,156.41,62.60,57.66,54.73,50.88,49.62,48.69,47.26,46.87,43.36,30.30,29.83,25.31,23.11,21.32,18.60,17.82,13.03,12.32,11.80.

Peram ivr-l-Val(11a):yield 45.2%;1H NMR(600 MHz,DMSO–d6,δppm)12.51(s,1H),8.13(dd,J=36.3,8.5 Hz,1H),7.82(s,1H),7.48(dd,J=79.6,8.1 Hz,1H),7.26(s,1H),6.79–6.68(m,1H),5.15(d,J=4.9 Hz,0.5H),4.37(dd,J=10.5,8.4 Hz,0.5H),4.16–4.11(m,1H),3.84(dd,J=8.4,6.1 Hz,0.5H),3.39–3.33(m,0.5H),2.93–2.78(m,1H),2.75–2.70(m,0.5H),2.58–2.53(m,0.5H),2.16(td,J=10.4,4.5 Hz,0.5H),2.14–1.99(m,3H),1.76(s,1H),1.56–1.02(m,6H),0.99(dt,J=14.8,7.2 Hz,1H),0.95–0.77(m,12H);13C NMR(600 MHz,DMSO–d6,δppm)173.50,169.97,159.06,156.17,74.48,59.48,48.85,48.08,47.63,43.39,30.25,23.16,21.33,19.60,12.40.

Peram ivir-l-Phe(11b):yield 39.4%;1H NMR(600 MHz,DMSO–d6,δppm)12.51(s,1H),7.32–7.13(m,5H),δ7.26(dd,J=10.0,3.5 Hz,4H),7.19–7.18(m,4H),5.61(d,J=4.8 Hz,1H),4.56(d,J=5.6 Hz,1H),4.10(d,J=5.3 Hz,1H),3.44–3.40(m,1H),3.29(d,J=7.9 Hz,2H),3.05(d,J=7.9 Hz,1H),2.88(d,J=7.5 Hz,1H),2.71(dd,J=8.6,5.1 Hz,1H),2.60–2.56(m,2H),2.07–2.01(m,2H),1.90(s,3H),1.66(ddd,J=13.7,8.1,3.4 Hz,2H),0.93–0.83(m,6H);13C NMR(600 MHz,DMSO–d6,δppm)174.05,169.38,159.19,156.41,141.69,137.34,136.31,129.82,128.75,126.28,57.66,54.73,49.62,48.69,47.26,43.36,30.35,29.83,23.11,22.53,21.32,18.60,17.82,13.03,12.32,11.80.

Peram ivir-d-Phe(11c):yield 36.1%,1H NMR(600 MHz,DMSO–d6,δppm)12.51(s,1H),7.32–7.13(m,5H),δ7.26(dd,J=10.0,3.5 Hz,4H),7.19–7.18(m,4H),5.61(d,J=4.8 Hz,1H),4.56(d,J=5.6 Hz,1H),4.10(d,J=5.3 Hz,1H),3.44–3.40(m,1H),3.29(d,J=7.9 Hz,2H),3.05(d,J=7.9 Hz,1H),2.88(d,J=7.5 Hz,1H),2.71(dd,J=8.6,5.1 Hz,1H),2.60–2.56(m,2H),2.07–2.01(m,2H),1.90(s,3H),1.66(ddd,J=13.7,8.1,3.4 Hz,2H),0.93–0.83(m,6H).13C NMR(600 MHz,DMSO–d6,δppm)174.05,169.38,159.19,156.41,141.69,137.34,136.31,129.82,128.75,126.28,57.66,54.73,49.62,48.69,47.26,43.36,30.35,29.83,23.11,22.53,21.32,18.60,17.82,13.03,12.32,11.80.

Peram ivir-l-Ile(11d):yield 29.7%,1H NMR(600 MHz,DMSO–d6,δppm)12.51(s,1H),8.26(dd,J=7.8,6.5 Hz,1H),7.82(s,1H),7.57(d,J=7.3 Hz,1H),7.33(dd,J=8.9,7.0 Hz,1H),6.82–6.70(m,1H),5.12–5.03(m,1H),4.37(t,J=10.6 Hz,1H),4.27–4.24(m,1H),3.86(dd,J=8.6,5.6 Hz,1H),2.74–2.67(m,1H),2.05(d,J=5.5 Hz,3H),1.76(s,2H),1.67–1.56(m,1H),1.55–1.44(m,2H),1.35(ddd,J=14.4,9.0,3.5 Hz,2H),1.31–1.24(m,1H),1.18(dt,J=11.5,7.1 Hz,1H),1.13–1.05(m,1H),0.98(dd,J=14.0,8.1 Hz,1H),0.91–0.82(m,12H).13C NMR(600 MHz,DMSO–d6,δppm)174.74,172.02,169.74,159.20,65.91,55.18,48.75,47.91,46.86,42.69,24.77,23.35,21.75,20.86,12.38.

Peramivir-l-Pro(11e):yield 23.1%,1H NMR(600 MHz,DMSO–d6,δppm)174.05,7.26(dd,J=10.0,3.5 Hz,4H),7.19–7.18(m,4H),5.61(d,J=4.8 Hz,1H),4.56(d,J=5.6 Hz,1H),4.10(d,J=5.3 Hz,1H),3.44–3.40(m,1H),3.05(d,J=7.9 Hz,1H),2.88(d,J=7.5 Hz,1H),2.80(m,2H),2.71(dd,J=8.6,5.1 Hz,1H),2.60–2.56(m,2H),2.07–2.01(m,2H),1.96(m,2H),1.90(s,3H),1.66(ddd,J=13.7,8.1,3.4 Hz,2H),1.64(m,2H),0.93–0.83(m,6H);13C NMR(600 MHz,DMSO–d6,δppm)174.05,169.38,159.19,156.41,57.66,54.73,49.62,48.69,47.26,46.87,43.36,30.30,29.83,25.31,23.11,21.32,18.60,17.82,13.03,12.32,11.80.

Peram ivir-d-Leu(11f):yield 30.6%,1H NMR(600 MHz,DMSO–d6,δppm)12.51(s,1H),8.26(dd,J=7.8,6.5 Hz,1H),7.82(s,1H),7.57(d,J=7.3 Hz,1H),7.33(dd,J=8.9,7.0 Hz,1H),6.82–6.70(m,1H),5.12–5.03(m,1H),4.37(t,J=10.6 Hz,1H),4.27–4.24(m,1H),3.86(dd,J=8.6,5.6 Hz,1H),2.74–2.67(m,1H),2.05(d,J=5.5 Hz,3H),1.76(s,2H),1.67–1.56(m,1H),1.55–1.44(m,2H),1.35(ddd,J=14.4,9.0,3.5 Hz,2H),1.31–1.24(m,1H),1.18(dt,J=11.5,7.1 Hz,1H),1.13–1.05(m,1H),0.98(dd,J=14.0,8.1 Hz,1H),0.91–0.82(m,12H);13C NMR(600 MHz,DMSO–d6,δppm)174.74,172.02,169.74,159.20,65.91,55.18,48.75,47.91,46.86,42.69,24.77,23.35,21.75,20.86,12.38.

Peram ivir-l-Leu (11 g):yield 43.1%,1H NMR(600 MHz,DMSO–d6,δppm)12.51(s,1H),δ8.32–8.01(m,1H),7.82(s,1H),7.46(d,J=8.2 Hz,1H),7.24(s,1H),6.76–6.66(m,1H),4.48–4.35(m,1H),4.23–4.19(m,1H),4.12–4.06(m,1H),3.84(dt,J=14.5,8.7 Hz,1H),2.82(m,1H),2.74–2.65(m,1H),2.07–2.03(m,1H),1.86–1.71(m,2H),1.65–1.45(m,5H),1.40–1.35(m,1H),1.33–1.28(m,1H),1.26(dd,J=9.9,8.3 Hz,1H),1.21–1.14(m,1H),1.13–1.03(m,2H),1.03–0.94(m,1H),0.94–0.80(m,12H);13CNMR(600 MHz,DMSO–d6,δppm)174.59,172.64,169.73,159.92,74.51,56.48,50.69,48.09,47.32,43.00,27.66,24.84,21.30,19.45,12.40.

2.3. Caco-2 culture

Caco-2 cells w ere obtained from the Am erican Tissue Culture Collection(Rockville,MD)and w ere grow n routinely on 75 cm2culture f lasks in Dulbecco’s m odif ied Eagle’s m edium(DMEM,4500 m g/L glucose)as previously described[17].

2.4. Caco-2 permeability

For transport experim ents,Caco-2 cells w ere seeded onto polycarbonate m em brane(0.6 cm2grow th area,0.4μm pore size,Millipore,MA)at a density about 1.0×105cells/cm2and allow ed to grow for 21–25 d[17].Perm eability studies were conducted w ith the m onolayers that developed the transepithelial electrical resistance(TEER)values above 250Ωcm2.Sam ples(100μl)at 15,30,45,60,90,120 m in w ere taken from the basolateral(BL)solution,and the volum e w as replaced w ith prew arm ed Hanks’balanced salt solution(HBSS).Because the am ino acid prodrugs w ere partially m etabolized to peram ivir and amino acids during the transport across the Caco-2 m onolayers,the prodrugs transported w as calculated as the total sum of the unchanged prodrugs and peramivir.

2.5. Gly-Sar uptake inhibition

For uptake experim ents,Caco-2 cells w ere seeded onto 24-well plastic cluster trays at about 1.0×105cells/cm2for 15 days after seeding.After w ashing tw ice w ith HBSS buffer,Caco-2 w ere incubated w ith 20μM Gly-Sar along w ith various concentrations of peram ivir-(CH2)2-L-Val or peram ivir-LIle(0.05-10 m M)at 37°C for 10 m in[18,19].After 10 m in,the m edium w as rem oved,and the cells w ere rapidly rinsed tw ice w ith 1 m l of ice-cold uptake buffer(p H 6.0).The cells w ere collected and hom ogenized in 0.25 ml water.The homogenates w ere centrifuged at 1500 g for 6 m in,and gly-sar in the supernatant w as analyzed by HPLC/MS/MS[18].The protein concentration of each sam ple w as determ ined by Coom assie Brilliant Blue assay using a bovine serum albumin as standard.IC50was determ ined using nonlinear data f itting.

2.6. Uptake by MDCK-h PEPT1 cells

MDCK-PEPT1 and MDCK m ock cells w ere grow n in DMEM as described previously[19].The uptake of peram ivir-(CH2)2-LVal or peram ivir-L-Ile by MDCK-PEPT1 cells and MDCK cells in the presence or in the absence of inhibitor was evaluated to study the role of PEPT1.The sam ples w ere analyzed by HPLC.

The concentration-dependent uptake of peram ivir-(CH2)2-L-Val or peram ivir-L-Ile was also studied at p H 6 over a concentration range of 0.01–50 m M.The m ock cells and the MDCK-PEPT1 cells w ere incubated w ith each individual substrate for 10 m in.The m ock cell line kinetics values w ere subtracted from those observed in MDCK-PEPT1 cells[19].

2.7. Hydrolysis stability study

2.7.1. Chemical study

The chem ical study of peram ivir-(CH2)2-L-Val or peram ivir-LIle w as determ ined in p H 1.2 hydrochloric acid solution and phosphate buffers(p H 4.5,6.8,7.4)at 37°C for 4 h.At every tim e point,100μL of the sam ples w as taken and analyzed by HPLC.

2.7.2. Hydrolysis in rat gastric juices and intestinal f luids

The gastric juices and intestinal f luids for stability studies w ere collected from m ale Sprague-Daw ley rats.The experim ents w ere carried out by adding 200μl of a stock solution of compound peramivir-(CH2)2-L-Val or peramivir-L-Ile to 1.8 m l of gastric juices or intestinal f luids preheated to 37°C,and the concentration of peram ivir-(CH2)2-L-Val or peram ivir-L-Ile in the biological m edia w as about 80μg/m l and 100μg/m l,respectively.Hydrolysis of the prodrugs w as studied at 37°C for a period of 4 h.Sam ples(100μl)w ere taken at various tim e points and quenched w ith 300μl of ice-cold m ethanol,then centrifuged at 2500 g and 4°C for 10 min.The supernatants w ere analyzed by HPLC.

2.7.3. Stability in intestinal and liver homogenates

The jejunum segm ent and liver w ere rem oved from the euthanized Sprague-Dawley rat and w ashed w ith ice-bath buffer C(25 m M KCl,5 m M MgCl2and 10 m M HEPES,p H 7.4)several tim es to rem ove blood,then hom ogenized w ith a tissue hom ogenizer and centrifuged at 2000 g and 4°C for 10 m in.The resulting supernatant w as collected,and the total protein am ount w as determ ined as above.The hydrolysis experim ent w as carried out by addition of drug solutions to the hom ogenates at 37°C.Peram ivir-(CH2)2-L-Val,peram ivir-L-Ile and protein concentrations in the m ixture w ere 100μg/m l,100μg/m l,and 200μg/m l,respectively.The sam ple w as pretreated as above,and the supernatant w as analyzed using HPLC.

2.7.4. Stability in rat plasma

Plasm a w as obtained from rat after centrifugation at 2500 g for 10 min.One volume of drug stock solution was mixed with nine volum es of plasm a preheated to 37°C.Aliquot sam ples w ere collected at various tim e points(0,30,60,90,120 m in).Extraction and analysis m ethods w ere sim ilar to those for the gastrointestinal f luids experim ent.

2.8. Pharmacokinetics study

Male Sprague-Daw ley rats ranging from 180 to 220 g w ere used for the pharm acokinetic study.The anim al experim ent w as approved by the University Com m ittee on Use and Care of Animals,Jiangxi University of Traditional Chinese Medicines.Anim als w ere housed under standard conditions of tem perature(25±2°C),hum idity(65±10%)and light.The rats w ere allow ed free m ovem ent and had access to food and w ater for 7 d before the experiments.Rats were fasted overnight w ith free access to w ater before the day of the experim ent.The rats(n=5 to 6 per treatm ent)w ere adm inistered by gavage of peram ivir,peram ivir-(CH2)2-L-Val or peram ivir-L-Ile(160 mg/kg calculated as peramivir)in aqueous solution,respectively.Serial blood sam ples(0.2 m l)w ere obtained from orbital plexus at 5,15,30,45 m in and 1,1.5,2,3,4,6,8,10,24,48 h after oral adm inistration.The solution of peram ivir w as also intravenously adm inistered to 6 rats at 8 mg/kg.During sam pling,rats w ere anesthetized w ith ether.The blood samples w ere centrifugated at 1200 g and 4°C for 10 m in,and the plasm a w as collected and frozen at-40°Cuntil analytes w ere determ ined by HPLC/MS/MS.

2.9. Analytical method

(a).HPLCAnalysis.Peram ivir and am ino acid prodrugs w ere analyzed on SHIMADZU LC-20AT HPLC system consisting of a SPD-M20A DAD,a SIL-20A autosam pler and a LabSolution workstation.The analytes w ere separated on ODSC18(4.6 m m×250 m m,5μm)w ith a w avelength of 210 nm and the m obile phase w as the m ixture of acetonitrile:w ater(containing 0.1%glacial acetic acid).The colum n tem perature w as set at 25°C.(b).HPLC-MS/MS analysis.The analytes w ere determ ined

by SHIMADZU LCMS-8050 liquid chrom atograph m ass spectrometer system.HPLC-MS/MS method w as fully validated in selectivity,linearity,precision and accuracy,extraction recovery,m atrix effect and stability according to Guidance for Industry,Bioanalytical,Method Validation US F ood and Drug Adm inistration.Quantif ication w as achieved by constructing a calibration curve w ith the least-square linear regression.

For determ ination of peram ivir,peram ivir and internal standard berberine w ere extracted from rat plasm a w ith the protein precipitation.A reversed-phase C18 colum n(Phenom enex,2.1 mm×50 mm,1.7μm)w as used to retain and separate the analytes from the endogenous com ponents.The colum n w as eluted by the gradient of acetonitrile and w ater containing 0.1%form ic acid.ESIw as set in positive ionization mode.Quantif ication was performed using multiple reaction m onitoring(MRM)and the optim ized MRM transitions w ere 329.1→270.1 for peram ivir and 336.2→292.0 for berberine.

The determ ination of peram ivir-L-Ile w as sim ilar to peramivir,but peram ivir-L-Ile was pretreated from the plasma w ith the liquid-liquid extraction.The optim ized MRM transition of 442.2→383.3 w as used for peram ivir-L-Ile.

2.10. Data analysis

(a).Thepermeability coeff icient(Papp)w as calculated using the follow ing equation:

w here d Q/dt is the steady-state appearance rate of the test com pound on the receiver side,C0is the initial concentration of the test com pound on the donor side,and A is the m onolayer grow th surface area.

(b).Plasma pharmacokinetic parameters w ere calculated by noncom partm ental analysis.The plasm a concentration at different times was expressed as mean±standard deviation(S.D.),and the m ean concentration-tim e prof iles w ere plotted.The Cmaxand Tmaxw ere observed directly from the concentration–tim e curves.The area under the plasma concentration–time prof iles(AUC)was calculated using the linear-trapezoidal rule.

(c).Statistical Analysis.The statistical differences w ere tested using a one-tailed Student t test at the P＜0.05 level.

3. Results and discussion

3.1. Synthesis of amino acid prodrugs of peramivir

To avoid the potential side reactions,Boc-peram ivir w as synthesized and then used as the starting m aterials for prodrug synthesis(Figs.1–3).The structures of all prodrugs w ere conf irmed by NMRand MS.

3.2. Caco-2 permeability

The apical to basolateral perm eability for peram ivir and 14 prodrugs w as in Fig.4A.All prodrugs w ere of higher perm eability than the parent drug,peram ivir.Peramivir-(CH2)2-LVal exhibited the highest perm eability in all ester prodrugs and its perm eability w as approxim ately 10.9-fold higher than peramivir.The permeability of Peramivir-L-Ile w as the highest in all am ide prodrugs,w hich w as about 9.1-fold higher than peram ivir.Therefore,in the subsequent experim ents,peram ivir-(CH2)2-L-Val and peram ivir-L-Ile w ere selected as the representative compounds for further study.

The extent of prodrug hydrolysis across Caco-2 m onolayer w as listed in Fig.4B.The am ide prodrugs w ere all m ore stable than the ester prodrugs.The am ino acid prom oiety and the stereochem istry had a really im portant effect on the hydrolysis rate of the ester prodrugs.

Fig.1–Synthesis of N-Boc-p rotected p eram ivir.(i)HCl-1,4-dioxane,ether,room tem perature,overnight;(ii)triethylam ine,Boc-TU,Hg Cl2,DMF,room tem perature,2 h;(iii)5%NaOH,EtOH and THF,room tem p erature,3 h.

3.3. Inhibition of gly-sar uptake by Caco-2 cells

An inhibition effect of peram ivir-(CH2)2-L-Val and peram ivir-L-Ile for PEPT1 w as determ ined by studying their ability to inhibit the uptake of gly-sar by Caco-2 cells.The initial rate tim e point(10 m in)for the uptake of gly-sar w as selected because the accum ulation of gly-sar w as linear up to 20 m in after incubation in Caco-2 cells(data not show n).As show n in Fig.5,gly-sar uptake w as inhibited by Peram ivir-(CH2)2-L-Val and Peram ivir-L-Ile in a concentration-dependent m anner,and the IC50w as 1.34±0.31 m M and 1.78±0.48 m M,respectively.

3.4. Uptake of peramivir-(CH2)2-L-val and peramivir-L-ile by MDCK-h PEPT1 cells

To further conf irm that Peram ivir-(CH2)2-L-Val and Peram ivir-L-Ile w ere the substrates of PEPT1,the uptake of the tw o compounds by MDCK-h PEPT1 cell and MDCK m ock cell w as investigated.Peram ivir-(CH2)2-L-Val and Peram ivir-L-Ile show ed 5.5-fold and 4.6-fold increase in uptake by MDCK-h PEPT1 cells when com pared w ith the mock cells,respectively.This kind of uptake can be inhibited by excess of gly-sar,a w ell-know n substrate of PEPT1.In contrast,no PEPT1-mediated transport of peram ivir w as observed(Fig.6A).

Fig.2–Synthesis of am ino acid ester prod rugs of peram ivir(8a–8g).(i)Ethylene glycol,DMAP,EDCand CH2Cl2,room tem perature,20 h;(ii)DMAP,EDC,overnight,room tem perature;(iii)TFA,CH2Cl2,room tem p erature,16 h.

Fig.3–Synthesis of am ino acid amid e p rodrugs of peramivir(11a–g).(i)TEA,EDC,HOBt,am ino acid m ethyl ester,DMF,room tem perature,24 h;(ii)LiOH,EtOH,THF and w ater,overnight,room tem p erature;(iii)TFA,CH2Cl2,room tem p erature,16 h.

Concentration-dependent prof iles of Peram ivir-(CH2)2-LVal and Peram ivir-L-Ile determ ined in MDCK-h PEPT1 cells after correcting for m ock cells w ere show n in Fig.6B.Vmaxvalues for Peram ivir-(CH2)2-L-Val and Peram ivir-L-Ile w ere 14.89±0.65 and 14.07±0.54 nm ol/m g protein/10 m in,respectively.Kmvalues for Peram ivir-(CH2)2-L-Val and Peramivir-L-Ile w ere 4.23±0.11 and 5.92±0.14 m M,respectively.

Table 1–The Stability of Peram ivir-(CH2)2-L-Val and Peram ivir-L-Ile in Different Phosphate buffer,in Rat Tissue Hom ogenate,Plasm a,Gastric and Intestinal f luids(Mean±SD,n=3).

Fig.4–(A)The apical-to-basolateral perm eability(Papp)for the am ino acid prodrugs(0.5 m M)in Caco-2 cells(m ean±SD,n=3).∗,P＜0.05,com pared w ith p eram ivir.(B)Percent prod rug rem aining intact in receiver side at 120 m in across Caco-2 cell m onolayer(m ean±SD,n=3).

Fig.5–Inhibition of Peram ivir-(CH2)2-L-Val and Peram ivir-L-Ile on gly-sar up take by Caco-2 cells.Caco-2 cells w ere incubated w ith 20μM gly-sar at 37°C for 10 m in in the p resence of various concentrations of peram ivir-(CH2)2-L-Val or peram ivir-L-Ile(0.05-10 m M).After the incubation,the concentrations of gly-sar w ere determined by HPLC-MS/MS.Data are presented as m ean±SD,n=3.

3.5. Stability studies

The stability of Peramivir-(CH2)2-L-Val and Peramivir-L-Ile was studied in p H 1.2 hydrochloric acid solution,different p H phosphate buffers,intestinal and liver hom ogenates,gastric and intestinal f luids,rat plasm a.The estim ated half-lives(t1/2)were calculated from linear regression of pseudo-f irst-order plots of the concentration vs.tim es.From Table 1,Peram ivir-LIle w as m ore stable than Peram ivir-(CH2)2-L-Val under all conditions,w hich m ay be that the am ide bond w as m ore stable than the ester bond.The chem ical hydrolysis of tw o prodrugs w as in a p H-dependent m anner,and they w ere m ore stable in the acidic environm ent than at the neutral and alkaline p H.In the biological f luids,Peramivir-(CH2)2-L-Val and Peramivir-L-Ile hydrolyzed at a higher speed(shorter t1/2)than in the phosphate buffer w ith the sam e p H,w hich indicated that a catalytic effect of enzym e m ay arise from these biological f luids.

Fig.6–(A)Up take of Peram ivir-(CH2)2-L-Val and Peram ivir-L-Ile by MDCK-h PEPT1 cells.The MDCK-h PEPT1 cells w ere incubated w ith Peram ivir-(CH2)2-L-Val or Peram ivir-L-Ile(0.5 m M)for 10 m in,p H 6.0,in the presence or absence of gly-sar(10 m M).After incubation,the concentration of Peram ivir-(CH2)2-L-Val and Peram ivir-L-Ile w as d eterm ined by HPLC.∗,P＜0.05,com pared w ith the presence of gly-sar.(B)Concentration-d epend ent uptake of Peram ivir-(CH2)2-L-Val and Peram ivir-L-Ile in MDCK-h PEPT1 cells.Each value w as calculated after subtracting the endogenous transp orters contributions observed in the m ock MDCK cells.Data are p resented as m ean±SD,n=3.

3.6. Pharmacokinetics study

The pharm acokinetics perform ance of Peram ivir-(CH2)2-L-Val and Peram ivir-L-Ile w as studied in rats to determ ine w hether the am ino acid prodrug strategy could im prove the oral bioavailability of peram ivir in vivo.The aqueous solution of peram ivir,Peram ivir-(CH2)2-L-Val and Peram ivir-L-Ile(all 160 m g/kg calculated as peram ivir)w ere orally adm inistered to rats,respectively.Peram ivir was also intravenously injected to rats at a dose of 8 m g/kg.PK param eters w ere presented in Table 2 and plasm a-concentration curves w ere show ed in Fig.7.

After Peram ivir-(CH2)2-L-Val w as orally adm inistered to the rats,Peram ivir-(CH2)2-L-Val could not be found in plasm a because of the rapid hydrolysis.As show n in Table 2,the AUC for peram ivir after dosing of Peram ivir-(CH2)2-L-Val and peramivir was 47,107.7 and 2955.9 ng h/ml respectively.The absolute oral bioavailability of peram ivir follow ing oral adm inistration of peram ivir-(CH2)2-L-Val and peram ivir w as 65.3%and 4.1%,respectively.

After Peramivir-L-Ile w as administered to the rats,only portion of Peram ivir-L-Ile w as hydrolyzed to peram ivir.The AUC for peram ivir-L-Ile and peram ivir w as 20,030.3 and 26,914.5 ng h/m l,respectively.Therefore,the oral bioavailability of peramivir after dosing of peramivir-L-Ile w as 37.3%,w hich w as 9.24-fold increase w hen com pared w ith oral adm inistration of peram ivir.

Fig.7–Mean(±SD)plasm a concentration-tim e of p eram ivir and Peram ivir-L-Ile(n=5 to 6).( , ,):the concentration of peram ivir after oral ad m inistration of Peram ivir-(CH2)2-L-Val,Peramivir and Peram ivir-L-Ile,respectively,(160 m g/kg calculated as peram ivir);():the concentration of Peram ivir-L-Ile after oral adm inistration of Peram ivir-L-Ile(160 m g/kg calculated as p eram ivir).

3.7. Discussion

Peram ivir has been approved for the treatm ent of inf luenza A and B by many countries.But the intravenous administration lim ited the patients’com pliance because of the high polarity and low oral bioavailability resulting from the carboxyl and the guanidine group.The intestinal peptide transporter,PEPT1,w as abundant in the epithelium of the gastrointestinal tract,and it w as a prom ising target for oral drug delivery strategy for the broad substrate specif icity[20–23].Therefore,a lot of am ino acid prodrugs of peram ivir targeted to PEPT1 have been synthesized w ith the aim to im prove the oral bioavailability and develop the oral alternative to peram ivir in the present study.

Amino acid amide prodrugs targeted to PEPT1 have been applied to m any successful cases,for exam ple LY2140023(m ethionine prodrug of LY404039)[15,16],lisdexam fetam ine dimesylate(the lysine am ide of D-amphetamine)[24,25],LY544344[26,27]and m idodrine[28].The am ide linkage could provide suff icient stability in the gastrointestinal tract w hen com pared w ith the ester bond,and it m ay exhibit potentially better oral absorption.Therefore,seven am ino acid am ide prodrugs w ere also synthesized in the present study,except for seven am ino acid ester prodrugs.The transport experim ent across Caco-2 cells w as perform ed to evaluate their transport across the intestinal m em branes.Peram ivir-(CH2)2-L-Val and Peram ivir-L-Ile w ere of the highest perm eability in all ester prodrugs and in all am ide prodrugs,respectively.The L-valine prodrug was the most permeable drug in most cases,which could be attributed to that the L-valine m ay have the optim al com bination of chain length and branch at theβ-Cposition of am ino acid for the intestinal transport[13].But as the am ide prodrugs were concerned in the present study,Peramivir-LIle had the highest perm eability,and it w as slightly higher than Peram ivir-L-val.Therefore,peram ivir-(CH2)2-L-Val and peram ivir-L-Ile w ere selected as the candidate com pounds for the next studies.

Table 2–Mean pharm acokinetic p aram eters of peram ivir after oral ad m inistration of Peram ivir-(CH2)2-L-VAL,Peram ivir-L-Ile and Peram ivr(160 m g/kg calculated as p eram ivir)and intravenous injection(8 m g/kg)to the Male Sp rague-Daw ley rats,respectively(n=5 to 6).

From gly-sar uptake inhibition by Caco-2 cells,it can be concluded that Peram ivir-(CH2)2-L-Val and Peram ivir-L-Ile com peted w ith gly-sar to interact w ith PEPT1.In the MDCKh PEPT1 cells,the direct uptake of Peramivir-(CH2)2-L-Val and Peram ivir-L-Ile w ere 5.5-fold and 4.6-fold higher than in m ock MDCK cells,respectively,and this uptake can be inhibited by excess of gly-sar.The uptake of peram ivir w as not(statistically)signif icant different between MDCK-h PEPT1 and MDCK m ock cells[19].These results show ed h PEPT1 contributed to the uptake of these tw o prodrugs by the sm all intestine.The uptake of Peram ivir-(CH2)2-L-Val and Peram ivir-L-Ile in MDCK-h PEPT1 cells conform ed to Michaelis–Menten Equation,w hich further conf irm ed the transport and uptake of tw o prodrugs w as a PEPT1-m ediated process.Kmvalues for peramivir-(CH2)2-L-Val were smaller than peramivir-L-Ile(4.23 m M vs 5.92 m M),w hich show ed peram ivir-(CH2)2-LVal had a higher aff inity to PEPT1 than peram ivir-L-Ile,and these results w as consistent w ith the gly-sar uptake inhibition by Caco-2 cells and the direct uptake by MDCK-h PEPT1 cells.

In the pharm acokinetics studies,no Peram ivir-(CH2)2-L-Val could be found in the plasm a,w hich m ight result from rapid degradation during the f irst-pass process[29].These were consistent w ith the stability experim ent result.The t1/2in the intestinal hom ogenate,liver hom ogenate and rat plasm a w as less than 80 m in,w hich suggested the f irst-pass m etabolism might be striking.After oral adm inistration of Peramivir-(CH2)2-L-Val,the oral bioavailability of peram ivir w as 65.3%,w hich w as 15.9-fold higher than the oral doing of peram ivir.The oral availability of peram ivir after oral adm inistration of Peramivir-L-Ile was 37.3%,w hich w as 9.24-fold increase w hen com pared w ith oral adm inistration of peram ivir.But the AUC for Peram ivir-L-Ile w as 20,030.3 ng h/m l,w hich w as comparable to the AUC of peram ivir(26,914.5 ng h/m l).Though the aff inity of Peramivir-L-Ile to PEPT1 was sim ilar(slightly sm aller)to Peram ivir-(CH2)2-L-Val,Peram ivir-L-Ile could not im prove the oral bioavailability of peram ivir to the sam e extent as Peram ivir-(CH2)2-L-Val because of the lim ited in vivo bioactivation,w hich can be forecasted from the stability results of Peram ivir-L-Ile.Therefore,to im prove the oral bioavailability of peram ivir w ith am ide prodrug,m ore am ino acids prodrugs,such as some unnatural am ino acids,must be attem pted in the future to com bine the im prove of perm eability,the stability in the gastrointestinal tract w ith the rapid bioactivation.

4. Con clusions

In summ ary,the prodrug strategy targeted to PEPT1 described in the present study has been very successfully in im proving the oral bioavailability of peram ivir and developing its oral alternative.Peram ivir-(CH2)2-L-Val w ith good oral prof iles and rapid conversion to the parent drug might be a promising prodrug for the further clinic developm ent.The present studies also corroborated the idea that the PEPT1-m ediated prodrug approach has enorm ous prom ise for im proving the oral absorption of poorly absorbed drug[21,22].

Conf lict of interest

The authors report no conf licts of interest.The authors alone are responsible for the content and w riting of this article.

Acknow led gm ents

This work w as supported by National Natural Science Foundation of China(81360485 and 81560577),and National Natural Science Foundation of Jiangxi(20132BAB215023).