relaxivity of per gadolinium atom because of an increase in rotational correlation time. On

the other hand, macromolecular MRI contrast agents may show prolonged intravascular

retention due to its bulky molecular volume, it can be used clinically as a blood pool contrast

agent. In addition, when an organ-targeting group, for example, PM is attached to this

macromolecular metal complex, it can be endowed with liver-targeting property [45-58].

Macromolecular liver-targeting Gd(III) chelates have been developed by the

incorporation of Gd-DTPA and pyridoxanine into polyasparamides, dendrimers and

polyester. Relaxivity studies showed that the chelates possessed obviously higher relaxation

effectiveness than that of Gd-DTPA. MR imaging of the liver in rats and experimental data of

biodistribution in mice indicated that they exhibited liver-targeting properties and enhanced

the contrast of MR images in the liver.

Polyester Liver-Targeting MRI Contrast Agents

Water-soluble polyester ligands were synthesized by the polycondensation of

diethylenetriaminepentaacetic dianhydride (DTPAA) with protected polyalcohol 3-O-benzylsn-glycerol (3-O-Bz-GLYC), monobenzaldehyde-pentaerythritol (S-Bz-PETO) and Nbenzyl-diethanolamine (N-Bz-DEA), respectively, and the protecting groups were then

removed by hydrogenation to give polyesters P(DTPA-GLYC), P(DTPA-PETO) and

P(DTPA-DEA). In the same manner, by adding ethylene glycol (EG) monomer into the

polymerization system, polyesters P(DTPA-GLYC-EG), P(DTPA-PETO-EG) and P(DTPADEA-EG) were also synthesized (Table 1). Pyridoxamine as a liver-targeting group was first

chlorocarbonylated and then incorporated into polyesters. The polyester ligands containing

pyridoxamine group thus prepared were further reacted with GdCl3 in water at room

temperature to give the corresponding hepatic-targeting polyester gadolinium complexes

(Figure 4) [11].

O O

CCH2NCH2CH2NCH2CH2NCH2C

COOCOO- COO-

( )

n

=CH2CH

CH2OOCNHCH2

HO

CH2OH

N

CH3

=CH2CH2NCH2CH2

CONHCH2

HO

CH2OH

N

CH3

= CH2 C CH2

CH2OOCNHCH2

CH2OH

HO

CH2OH

N

CH3

P(Gd-DTPA-GLYC-PM)

P(Gd-DTPA-DEA-PM)

P(Gd-DTPA-PETO-PM)

Gd3+

ORO

R

R

R

Figure 4. Structural formula of polyester gadolinium complexes.

158 Guo-Ping Yan, Xiao-Yan Wang, Li-Li Mei

Table 1. Molecular weight of polyesters

Polymer Mn

( ×10 3 )

Mw

( ×10 3 )

Polydispersity

P(DTPA-GLYC) 7.51 9.08 1.21

P(DTPA-PETO) 10.9 11.4 1.04

P(DTPA-DEA) 8.33 10.6 1.27

P(DTPA-GLYC-EG) 8.73 16.8 1.92

P(DTPA-PETO-EG) 9.47 15.9 1.68

P(DTPA-DEA-EG) 19.7 37.6 1.95

Table 2 Experimental data of relaxivity

Gadolinium complexes [Gd3+](mmol· l-1) T1obsd (s) R1(mmol· l-1·s) -1

Gd-DTPA 1.240 0.138±0.0071 5.6

P(Gd-DTPA-GLYC-PM) 1.3048 0.0631±0.0096 11.91

P(Gd-DTPA-PETO-PM) 1.6195 0.0577±0.0052 10.51

P(Gd-DTPA-DEA-PM) 1.5905 0.0531±0.0073 11.65

P(Gd-DTPA-GLYC-EG-PM) 1.8333 0.0441±0.0053 12.20

P(Gd-DTPA-PETO-EG-PM) 1.3643 0.0476±0.0063 15.17

P(Gd-DTPA-DEA-EG-PM) 1.3950 0.0575±0.0076 12.25

Temp: 25 oC; NMR Frequency: 80MHz; T1d =3.23±0.021s.

0 2000 4000 6000 8000 10000 12000 14000 16000 18000

0

20

40

60

80

100

Viability Relative to Control (%)

Concentration (? g/mL)

P(DTPA-PETO-PM)

P(Gd-DTPA-PETO-PM)

Gd-DTPA

Figure 5. Cytotoxicity assay of anticancer drugs in L-02 cells.

Relaxivity studies showed that these polyester gadolinium complexes possess higher

relaxation effectiveness than that of the clinically used small molecular gadolinium complex

Gd-DTPA (Table 2). At the concentration (4280μg ml-1) of polyester ligand and its

Vitamin B6 as Liver-targeting Group in Drug Delivery 159

gadolinium complex in the growth medium (RPMI-1640 media (10% foetal bovine serum

(Gibco Co, USA), 100units ml-1 penicilium, 100μg ml-1 streptomycin)) , the viability of

human normal liver cells (L-02) incubated with P(DTPA-PETO-PM) and P(Gd-DTPAPETO-PM) retained 57.7% and 58.2%, respectively, relative to the control. It illustrated that

possess low cytotoxicity to L-02 cells (Figure 5).

In comparison to the signal intensity (SI) of the liver in the rat injected of Gd-DTPA(0.1

mmol/kg) and P(Gd-DTPA-PETO) (0.1 mmol/kg) without the liver-targeting group PM, the

signal intensity of the liver in the rat injected of P(Gd-DTPA-PETO-PM) (0.1mmol/kg) was

obviously enhanced, the irradiated portion of the liver was brighter and the demarcation

became clearer at the same time intervals during the detection time. This result illustrated that

P(Gd-DTPA-PETO-PM) can greatly enhance the contrast of MR images of the liver after

injection (Figure 6-8). Thirty minutes after injection of P(Gd-DTPA-PETO-PM), the signal

enhancement of the liver (black cycle) is 176% (Table 3). It is better than that of Gd-DTPA

(119%). On the other hand, P(Gd-DTPA-PETO-PM) has prolonged intravascular duration

time for approximately one hour (127%). These results indicated that polyester gadonilium

complexes containing pyridoxamine group can be targeted to the liver.

 A 1 Control B 1 15 min C 1 30min D 1 45 min

Figure 6. A1 is the T1-weighted image of the rat received no injection of MRI contrast agent; B1, C1 and

D1 are the T1-weighted images of the rat received injection of Gd-DTPA (0.1 mmol/kg, Magnevist)

after 15 min, 30min and 45min.

Table 3. Enhancement (%) in the signal of the liver in different time after injection

Time after injection

 ( min )

Gd-DTPA P(Gd-DTPA-PETO) P(Gd-DTPA-PETO-PM)

Control 100 100 100

5 107 107 111

15 114 112 160

30 119 114 176

45 115 115 154

60 114 116 127

160 Guo-Ping Yan, Xiao-Yan Wang, Li-Li Mei

 A 2 Control B 2 15 min C 2 30 min

 D 2 45 min E 2 60 min F 2 75 min

Figure 7. A2 is the T1-weighted image of the rat received no injection of MRI contrast agent; B2, C2 , D2,

E2 and F2 are the T1-weighted images of the rat received injection of P(Gd-DTPA-PETO) (0.1

mmol/kg) without the liver-targeting groups PM after 15min, 30min, 45min, 60min and 75min.

 A 3 Control B 3 15 min C 3 30 min

 D 3 45 min E 3 60 min F 3 75 min

Figure 8. A3 is the T1-weighted image of the rat received no injection of MRI contrast agent; B3, C3 , D3,

E3 and F3 are the T1-weighted images of the rat received injection of P(Gd-DTPA-PETO-PM) (0.1

mmol/kg) after 15min, 30min, 45min, 60min and 75min.

Vitamin B6 as Liver-targeting Group in Drug Delivery 161

Polyaspartamide Liver-Targeting MRI Contrast Agents

Polyaspartamide is a biologically water-soluble synthetic polymer with a protein-like

structure. It has been used as a plasma extender and a drug carrier and for some other

biomedical applications because it is nontoxic, nonantigenic and degradable in living

systems, and modified easily by reactions with the side chain. Antiviral drugs and antiinflammatory agents have been covalently linked to (poly-α,β-[N-(2-hydroxyethyl)-D,Laspartamide] (PHEA) forming drug-polymer conjugates capable of increasing drug stability

and bioavailility [59-67].

The effects of PHEA and PAEA on cell growth and metabolism of HeLa cells in vitro

were determined as a function of polymer concentration and compared to polylysine. The

preliminary results show that over the concentration range tested, the cells incubated with

PHEA and poly-α,β-[N-(2-amino ethy1)-L-aspartamide] (PAEA) retain 55.1% and 61.9%

viabilities, while in the presence of polylysine (PLys), HeLa cells show no viability under the

same concentration (100µg/mL). At a higher concentration (200µg/mL) of PHEA and PAEA,

the cells still retain 62.4% and 49.8% viabilities respectively, relative to control (Figure 9).

Thus polyaspartamides are the good polymeric carriers for MRI contrast agent and drug

controlled release system [11].

0 20 40 60 80 100 120 140 160 180 200

0

20

40

60

80

100

120

Viability Relative to Control (%)

Concentration ( ¦Ì g /mL )

 PLys

 PAEA

 PHEA

Figure 9. Cytotoxicity assay of PLys, PHEA and PAEA in HeLa cells.

Pyridoxamine (PM)-containing diethylenetriaminepentaacetic acid mono(Nhydroxysuccinimide) ester (SuO-DTPA-PM) was prepared by reacting PM with DTPA

bis(N-hydroxysuccinimide) ester. The PM-containing DTPA active ester thus obtained was

further incorporated into poly-α,β-[N-(2-hydroxyethy1)-L-aspartamide] (PHEA) and polyα,β-[N-(2-amino ethy1)-L-aspartamide] (PAEA) to give liver-targeting macromolecular

ligands PHEA-DTPA-PM and PAEA-DTPA-PM. Finally, by the metalation of the ligands

162 Guo-Ping Yan, Xiao-Yan Wang, Li-Li Mei

with gadolinium Gd(Ⅲ), two kinds of polyaspartamide gadolinium complexes were

synthesized [11].

Relaxivity studies showed that these polyaspartamide gadolinium complexes possess

higher relaxation effectiveness than that of Gd-DTPA. In vitro cytotoxicity assay showed that

polyaspartamide gadolinium complexes have low cytotoxicity. Magnetic resonance imaging

showed that the signal intensity (SI) of the liver in rat injected with PHEA-Gd-DTPA-PM

(the average percent value of linked of polymeric repeat unit in gadolinium complexes

(wt%): Gd 2.65) or PAEA-Gd-DTPA-PM (the average percent value of linked of polymeric

repeat unit in gadolinium complexes (w%): Gd 11.04) was obviously enhanced. Experimental

data of biodistribution in Kunming mice indicated that the rapid decrease of the

polyaspartamide MRI contrast agent in blood, heart and spleen correlated with its increasing

capture by the liver, indicating that these polyaspartamide MRI contrast agents containing

pyridoxamine were taken up specifically by hepatcocytes.

DTPA and pyridoxamine (PM) as a liver-targeting group were both incorporated into

polyaspartamides i. e. (poly-α,β-[N-(2-hydroxyethyl)-L-aspartamide] (PHEA), poly-α,β-[N-

(3-hydroxypropyl)-L-aspartamide] (PHPA), poly-α,β-[N-(2-aminoethy1)-L-aspartamide]

(PAEA) and poly-α,β-[N-(6-aminohexyl)-L- aspartamide] (PAHA) to obtain the

polyaspartamide ligands. The polyaspartamide containing both DTPA ligands and

pyridoxamine groups thus prepared were further reacted with gadolinium chloride to give the

corresponding polyaspartamide gadolinium complexes with different amount of gadolinium

ions PHEA-Gd-DTPA-PM, PHPA-Gd-DTPA-PM, PAEA-Gd-DTPA-PM and PAHA-GdDTPA-PM [12].

 The polyaspartamide gadonilium complexes containing pyridoxamine groups possess

obviously higher relaxation effectiveness than that of Gd-DTPA. PHEA-Gd-DTPA-PM (the

average percent value of linked of polymeric repeat unit in gadolinium complexes (mol%):

Gd-DTPA 5.30, PM 0.80) possesses the low intravenous acute toxicity and LD50/7days

(intravenous, mouse) to IRC mice is 5.2g/kg±0.5g/kg.

Table 4. Enhancement (%) in the signal from the liver at different times after injection

Time after

injection

( min )

Gd-DTPA

(0.1mmol/kg )

PHEA-Gd-DTPAPM (M2,

0.025 mmol/kg )

Area 1 Area 2

PHEA-Gd-DTPAPM (M2,

0.05 mmol/kg )

Area 1 Area 2

PHEA-Gd-DTPAPM (M2,

0.075 mmol/kg )

Area 1 Area 2

PHEA-Gd-DTPAPM (M2,

0.1 mmol/kg )

Area 1 Area 2

Control 100 100 100 100 100 100 100 100 100

2 104 107 126 171 109 113 109 152 150

8 107 126 128 192 117 117 115 137 162

15 114 129 139 173 132 125 130 135 158

30 119 141 141 173 136 133 133 130 150

45 115 135 133 194 159 141 139 128 141

60 114 133 135 198 152 145 144 128 136

75 133 160 192 148 163 137 119 135

90 146 150 195 145 181 130 110 134

105 156 135 213 144 165 122 102 133

120 154 131 184 133 156 117 100 124

Vitamin B6 as Liver-targeting Group in Drug Delivery 163

MR imaging showed that the signal intensities (SI) of the liver in rat injected with low

dosage of PHEA-Gd-DTPA-PM (0.1mmol/kg, 0.075mmol/kg, 0.05mmol/kg and 0.025

mmol/kg) were obviously enhanced in comparison to that of the liver in the rat injected with

Gd-DTPA (0.1mmol/kg) (Figure 10). It greatly enhanced the contrast of MR image of the

liver and provided prolonged intravascular duration in the liver (Table 4). These results

indicated that the polyaspartamide gadonilium complex containing pyridoxamine groups

could be used as the candidate of specific MRI contrast agent for the liver.

 A4 Control B4 15 min

 C4 60 min D4 120 min

Figure 10. A4 is the T1-weighted image of the rats which received no MRI contrast agent; B4, C4 and D4

are the T1-weighted images of the rats which received injection with PHEA-Gd-DTPA-PM (the average

percent value of linked of polymeric repeat unit in gadolinium complexes (mol%): Gd-DTPA 5.30, PM

0.80, 0.05mmol/kg) after 15min, 60min and 120min. Indicated areas 1 and 2 were used to calculate

contrast enhancements listed in Table 4.

Dendritic Liver-Targeting MRI Contrast Agents

The conjugation of paramagnetic metal chelates to dendrimers is currently being

explored as a new potential macromolecular MRI contrast agents because dendrimers have

some advantages over other polymer carriers including the highly branched structure, low

polydispersity molecule, uniform surface chemistry and high numbers of reactive functional

groups per unit mass and volume for modification [68-80].

A series of liver-targeting dendritic gadolinium complexes were synthesized by

conjugation of diethylenetriaminepentaacetic acid (DTPA) and pyridoxamine to the terminal

amines of the dendrimers with 1,4,7,10-tetraazacyclododecane as the core (Generation: G1.0-

5.0) and chelation with gadoliniumn chloride (Figure 11). These dendrimer-metal chelate

conjugates have high ion relaxivities (of 13.0 - 23.5 (mmol/L)-1·s-1 at 300MHz, 17℃, and pH

of 7.4). MR imaging showed that the signal intensities (SI) of the liver in rats injected with

low doses of G4.0-Gd-DTPA-PM (the average mole ratio of attached Gd-DTPA and PM to

164 Guo-Ping Yan, Xiao-Yan Wang, Li-Li Mei

amine groups on the surface of the dendrimers (mol%): Gd-DTPA 10.84, PM 1.64) were

significantly enhanced (Figure 12). G4.0-Gd-DTPA-PM greatly enhances the contrast of MR

images of the liver, provides prolonged intravascular duration and produces highly contrasted

visualization of blood vessels in the liver. These novel dendritic gadolinium complexes

containing pyridoxamine groups demonstrate liver-targeting properties and show strong

potential as new liver-targeting MRI contrast agents [13].

( G2.0-DTPA-PM )

NH

HN

HN

NH

NH

R1

R2

R1

R1

N

H2N

H2N

N N

N N

NH2

NH

N N N

CH2

COO-

-

OOC COOCH2 N

CH3

CH2OH

HO

C

Gd3+

O

COOR1

R2

R1 =

= C

O

Figure 11. Structural formula of G2-Gd-DTPA-PM.

 A5 Control B5 15 min

 C5 30 min D5 60 min E5 120 min

Figure 12. A5 is the T1-weighted image of the rat which received MRI contrast agent; B5, C5, D2 and E5

are the T1-weighted images of the rat which received injections of dendritic G4-Gd-DTPA-PM (0.1

mmol/kg) after 15min, 30min, 60min and 120min. Indicated areas I and 2 were used to calculate

contrast enhancements listed in Table 5.

Vitamin B6 as Liver-targeting Group in Drug Delivery 165

Table 5. Enhancement (%) in the signals from the liver at different times after injection

Time after

injection ( min )

Gd-DTPA

( 0.1 mmol/kg )

G4.0-Gd-DTPA-PM

( 0.05 mmol/kg )

Area 1 Area 2

G4.0-Gd-DTPA-PM

( 0.1 mmol/kg )

Area 1 Area 2

Control 100 100 100 100 100

8 107 137 107 131 135

15 114 147 118 145 152

30 119 154 126 148 179

45 115 160 131 153 176

60 114 165 148 169 172

75 159 147 166 169

93 155 144 162 166

105 149 128 145 165

120 148 127 137 164

( G2.0 )

N

H2N

N

N N

NH2

H2N

N

N

N

N

N

N

H2N

H2N

H2N

NH2

NH2

NH2

NH2 H2N NH2

0

1

2

Gen.

( G2.0-Gd-DTPA-PM )

N

HN

N

N N

NH

HN

N

N

N

N

N

N

H2N

H2N

HN

NH2

NH

NH

NH

H2N NH2

0

1

2

Gen.

R1

R2

R2

R1

R1

R1 R1

N N N NH

CH2

COO-

-

OOC COOCH2 N

CH3

CH2OH

HO

C

Gd3+

O

COO- R2 R1 = = C

O

Figure 13. Structural formula of PAMAM-Gd-DTPA-PM.

In addition, diethylenetriaminepentaacetic acid (DTPA) and pyridoxamine (PM) were

also both incorporated to the amine groups on the surface of the ammonia core

poly(amidoamine) dendrimers (PAMAM, Generation 2.0-5.0) to obtain the dendritic ligands

(Figure 13). These dendritic ligands were further reacted with gadolinium chloride to yield

the corresponding dendritic gadolinium complexes. They also have high relaxivity and higher

intravascular retention time, and greatly enhance the contrast of MR images of the liver.

Animal tests showed that small doses of this dendrimer contrast agent could promise a highly

resolved and contrasted visualization of blood vessels. So the results suggest that this new

and powerful class of contrast agents have the potential for diverse and extensive application

in MR imaging for the liver [14].

166 Guo-Ping Yan, Xiao-Yan Wang, Li-Li Mei

LIVER-TARGETING ANTICANCER CONJUGATES

Polymer-based drug delivery systems are used to optimize the therapeutic properties of

drugs and render them safer, more effective and reliable. Moreover, polymeric drugs with

macromolecules used as drug carriers can be easily synthesized at low cost, freely watersoluble, non-toxic, non-immunogenic and well characterized from the physico-chemical point

of view. Now the development of biomedical polymers for drug controlled release was laid

emphasis on the temporal control, distribution control and responsive drug delivery systems

[81,82].

One important approach in drug delivery design is that the attached drugs can be targeted

to specific organs, tissues or cells by the incorporation of a drug into a polymer containing

organ or tissue-targeting group or moiety. By this method, the toxic side effects of the drugs

can be suppressed and the distribution of drugs can be improved and reduce the drugs dose

[83].

5-Fluorouracil (5-Fu) was chosen as a drug model because its structure and mode of

action are well described and it is widely utilized in cancer chemotherapy. Anticancer

conjugates of 5-fluorouracil and polyaspartamides containing pyridoxamine moiety were

prepared by conjugating anticancer drug 5-fluorouracil and hepatocyte-targeting group

pyridoxamine to the polyaspartamides with different side chains (poly-α,β-[N-(2-

hydroxyethyl)-L-aspartamide] (PHEA), poly-α,β-[N-(2-aminoethy1)-L-aspartamide] (PAEA),

poly-α,β-[N-(3- hydroxypropyl)-L-aspartamide] (PHPA) and poly-α,β-[N-(6- aminohexyl)-Laspartamide] (PAHA). When the mole ratio of 5-Fu to the polymeric units in the feed of the

reaction increased, the conversions of 5-Fu in polymeric drugs increased from 5.9wt% to

25.6wt%. Their properties in vitro and in vivo were also evaluated [64].

In vitro drug release properties studies showed that these anticancer conjugates can

sustain in vitro release rate 5-Fu in PBS. A steady release rate of the drug was maintained for

more than 80h (Figure 14). 5-Fu-PHEA-PM and 5-Fu-PHPA-PM were released faster than 5-

Fu-PAEA-PM and 5-Fu-PAHA-PM because the hydrolysis rate of –NCOO- from the side

chains of 5-Fu-PHEA-PM and 5-Fu-PHPA-PM was faster than that of –NCONH- from the

side chains of 5-Fu-PAEA-PM and 5-Fu-PAHA-PM.

In vitro cytotoxicity assay exhibited that polymeric drugs possess low cytotoxicity to the

human liver cells (L-02) than Fluorofur and 5-fluorouracil (Figure 15). FT-207 is a derivative

of 5-fluorouracil and possesses lower toxicity than 5-fluorouracil. At 225µg/ml of anticancer

drugs in the growth medium, the L-02 cells incubated with FT-207 and 5-Fu-PAEA-PM,

respectively, retained above 47.1% and 63.2% viabilities relative to control.

The distribution of PAEA-DTPA in mice tissues at various time points was shown that

the agent was distributed to all the different tissues (such as the blood, liver, lung, heart and

small intestine) showed no tissue-targeting property. It was rapidly cleared by the kidney,

liver and small intestine, with the predominant excretion route of PAEA-DTPA is through

kidney. The distribution of 5-Fu-PAEA-(DTPA)-PM in mice tissues at various time points

was indicated that the majority of the polymeric drug was transferred from the blood into the

liver within 20 min after injection. Very little amount of polymeric drug was found in the

heart, spleen, lung, small intestine, muscle and bone at any time. This result shows the agent

entered the liver from the blood and was excreted by kidneys later. A high content of 5-Fu-

Vitamin B6 as Liver-targeting Group in Drug Delivery 167

PAEA-(DTPA)-PM stayed in the liver for 90 min after injection. The incorporation of

pyridoxamine in the polymers increased the pinocytic uptake by the liver.

0 20 40 60 80 100 120

0

20

40

60

80

100

Cumulative percent release (%)

Time (h)

 5-Fu-PHEA-PM(1)

 5-Fu-PHEA-PM(2)

 5-Fu-PHPA-PM(1)

 5-Fu-PHPA-PM(2)

0 20 40 60 80 100 120

0

20

40

60

80

100

Fraction Release ( % )

Time ( h )

 5-Fu-PAEA-PM(1)

 5-Fu-PAEA-PM(2)

 5-Fu-PAHA-PM(2)

 5-Fu-PAHA-PM(1)

Figure 14. Release profiles 5-Fu of the polymeric drugs.

 

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In: Vitamin B: New Research ISBN: 978-1-60021-782-1

Editor: C. M. Elliot, pp. 153-174 © 2008 Nova Science Publishers, Inc.

Chapter IX

VITAMIN B6 AS LIVER-TARGETING

GROUP IN DRUG DELIVERY

Guo-Ping Yan∗ , Xiao-Yan Wang and Li-Li Mei

School of Material Science and Engineering, Wuhan Instituite of Technology,

Wuhan 430073, P. R. China.

ABSTRACT

Vitamin B6 includes a series of compounds containing the pyridoxal structure, such

as pyridoxol, pyridoxamine, pyridoxaldehyde and their derivatives. The pyridoxal

structure，the catalytically active form of vitamin B6, possesses specific hepatocyte

uptake by the pyridoxine transporter at the sinusoidal pole because the pyridoxine

transporters that exist in hepatocytes can selectively recognize and bind to the pyridoxal

structure, and transport it into the cells via a member transport system. Thus pyridoxine

can be adopted as a liver-targeting group and be incorporated into the low molecular

weight compounds and macromolecules for the use as magnetic resonance imaging

(MRI) contrast agents and anticancer conjugates. The research progress of liver-targeting

drug delivery system is discussed briefly. Previous researches have demonstrated that the

incorporation of pyridoxine into these molecules can increase their uptake by the liver,

and that these molecules containing pyridoxine groups exhibit liver-targeting properties.

Keywords: vitamin B6, liver-targeting, drug delivery, magnetic resonance imaging (MRI)

∗

 Correspondence concerning this article should be addressed to: Guo-Ping Yan, School of Material Science and

Engineering, Wuhan Instituite of Technology, Wuhan 430073, P. R. China. E-mail address:

guopyan@hotmail.com.

154 Guo-Ping Yan, Xiao-Yan Wang, Li-Li Mei

INTRODUCTION

Vitamin B6, also known as pyridoxine, is water-soluble and is required for both mental

and physical health. Vitamin B6 includes a series of compounds containing the pyridoxal

structure, such as pyridoxol, pyridoxamine, pyridoxaldehyde and their derivatives.

The liver has both a unique blood supply (arterial, venous and portal-venous) and

specific cells that are capable of transporting/accumulating bulk amounts of both endo- and

exobiotic substances [1-3]. The pyridoxine transporters that exist in hepatocytes at the

sinusoidal pole can selectively recognize and bind to the pyridoxal structure, and transport it

into the cells via a member transport system. The pyridoxal structure, the catalytically active

form of vitamin B6, possesses specific hepatocyte uptake by the pyridoxine transporter. Thus

pyridoxine can be adopted as a liver-targeting group and be incorporated into the low

molecular weight compounds and macromolecules for the use as magnetic resonance imaging

(MRI) contrast agents and anticancer conjugates [4-7]. The research progress of livertargeting drug delivery system is discussed briefly. Previous researches have demonstrated

that the incorporation of pyridoxine into these molecules can increase their uptake by the

liver, and that these molecules containing pyridoxine groups exhibited liver-targeting

properties [8-19].

LIVER-TARGETING MRI CONTRAST AGENTS

Over the last three decades, nuclear magnetic resonance (NMR) has been perhaps the

most powerful method for the non-invasive investigation of human anatomy, physiology and

pathophysiology. Developed in 1973 by Paul Lauterbur [20], magnetic resonance imaging

(MRI) has become widely used as the diagnosis and treatment of human diseases in hospitals

around the world, since it received FDA approval for clinical use in 1985. It is a non-invasive

clinical imaging modality, which relies on the detection of NMR signals emitted by hydrogen

protons in the body placed in a magnetic field. In 2003, Paul C. Lauterbur and Sir Peter

Mansfield won the Nobel Prize in physiology and medicine for their discoveries concerning

MRI because it can be widely used for the diagnosis and treatment of human diseases, such

as necrotic tissue, infarcted artery and malignant disease [21,22].

One important way to improve the contrast in MRI is to introduce contrast agents. MRI

contrast agents are a unique class of pharmaceuticals that enhance the image contrast between

normal and diseased tissue and indicate the status of organ function or blood flow after

administration by increasing the relaxation rates of water protons in tissue in which the agent

accumulates [8,9]. Paramagnetic substances, superparamagnetic and ferromagnetic materials

have been used as MRI contrast agents because paramagnetic substances have a net positive

magnetic susceptibility, having the ability to become magnetized in an external magnetic

field. Some MRI exams include the use of contrast agents. The categorizations of currently

available contrast agents have been described according to their effect on the image,

magnetic behavior and biodistribution in the body, respectively [23].

Subsequently proper ligands have been designed and complexed with paramagnetic

metal ions to form strong water-soluble chelates as the first generation MRI contrast agents,

Vitamin B6 as Liver-targeting Group in Drug Delivery 155

for example, gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA, Magnevist®,

Schering AG, Germany) (Figure 1) [24]. Some clinically used MRI contrast agents are small

ionic molecules such as Gd-DTPA and gadolinium 1,4,7,10-tetraazacyclododecane-N, N’,

N’’, N’’’-tetraacetic acids (Gd-DTOA, Dotarem®, Guerbet SA, France) (Figure 2) [25,26] that

can diffuse freely through the extracellular space and excreted rapidly by the kidney. Then

their biodistribution are nonspecific although Gd-DTPA works well in organs such as the

brain and spinal cord, where the normal brain parenchyma has a barrier to permeability of the

contrast agent and pathologic conditions such as cancer do not. The injection of large

quantities of the ionic complex will raise ion concentration in vivo and cause localized

disturbances in osmolality, which, in turn leads to cellular and circulatory damage. Most

commonly, Gd-DTPA and Gd-DOTA have been modified to form neutral molecules, which

thus exhibited much lower osmolality and higher LD50s in animals [27-31].

HOOCCH2

-

OOCCH2

CH2COOH

CH2COO- CH2COOGd3+

N N N

Figure 1. Structural formula of Gd-DTPA.

-

OOCCH2

-

OOCCH2 CH2COOH

CH2COOGd3+

N

N N

N

Figure 2. Structural formula of Gd-DOTA.

Nowadays ideal MRI contrast agent is focused on the neutral tissue- or organ-targeting

materials with high relaxivity and specificity, low toxicity and side effect, suitable long

intravascular duration and excretion times, high contrast enhancement with low doses in vivo,

and minimal cost of procedure [8,9,27,28]. In general, tissue or organ-specific contrast agents

consist of two components: a magnetic label capable of altering the signal intensity on MR

images and a target-group molecule having a characteristic affinity for a specific type of cell

or receptor. Some suitable residues have been incorporated into either the acetic side-arms or

the diethylenetriamine backbone of Gd-DTPA and Gd-DOTA to obtain the tissue or organspecific contrast agents. For example, liver-targeting agents such as gadobenate dimeglumine

(Gd-BOPTA, Gadobenate, Multihance®, Bracco Imaging, Italy) and gadolinium

ethoxybenzyltriamine pentaacetic acid (Gd-EOB-DTPA, Gadoxetate; Eovist®, Schering AG,

156 Guo-Ping Yan, Xiao-Yan Wang, Li-Li Mei

Germany) have been developed, which can accumulate in the liver site, increasing contrast

concentration, and producing greater signal in the MR images [32-42].

Low Molecular Weight Liver-Targeting MRI Contrast Agents

Manganese dipyridoxyl-diphosphate (mangafodipir, Mn-DPDP, Teslascan®, Nycomed

Amersham Imaging, Princeton, NJ) is a contrast agent developed for imaging of the

hepatobiliary system (Figure 3). Unlike Gd-DTPA, Mn-DPDP is an intracellular agent that is

taken up specifically by hepatocytes and pancreas, and excreted in the bile since the ligand

consists of two linked pyridoxal-5’-phosphate groups, the catalytically active form of vitamin

B6. Thus, it was thought that Mn-DPDP was a potential candidate for specific hepatocyte

uptake by the pyridoxine transporter at the sinusoidal pole. However, it was reported that the

complex dissociated both in the blood and in the liver and the uptake mechanism did not

depend on the pyridoxine transporter [4-6].

N

H3 C

-

OPOCH2

N

H+

CH2OPO -

N

H+

CH3

Mn

O

O O

O

O

O

C

C

N

OH OH

O O

Figure 3. Structural formula of Mn-DPDP.

Other liver-targeting DTPA derivates containing vitamin B6 groups have also been

prepared according to the liver-targeting property of Mn-DPDP. A series of DTPA

derivatives ligands containing pyridoxol groups have been synthesized by the reaction of

DTPA dianhydride with the pyridoxol derivatives with the different space groups. Compared

with Gd-DTPA, their non-ionic bulky Gd3+ complexes have higher relaxivities, lower

stability constants and the liver-targeting property. Moreover, Gd-DTPA and Gd-DOTA are

modified to form neutral molecules, which thus exhibit much lower osmolality, while these

neutral agents have been shown to have higher LD50s in animals [7,43,44].

Macromolecular Liver-Targeting MRI Contrast Agents

Macromolecular MRI contrast agent can be prepared by the incorporation of a low

molecular weight paramagnetic metal cheated complex such as Gd-DTDA or Gd-DOTA, to

the backbone or the pendant chains of macromolecule. It usually exhibits more effective

relaxation than that of the low molecular weight metal complex alone and improves the

Vitamin B6 as Liver-targeting Group in Drug Delivery 157