Table 1 Axonal transport experiments

A Synthesis, function and stability

   I Chemical synthesis

1

Dextran tripartites

Chemical

NMR, PLC, Mass Spec

30% loading

2

a. CM dex standard

b. CM dex-amino upgraded

c. CM dex-lysine extended

d. WGA-CM dex

e. WGA-CM dex-valacyclovir

f. CM dex-gabapentin

g. WGA-CM dex-gabapentin

1

Validate chemical basis of uptake amplification

2

Ferrite particle tripartites

chemical

MRI

useful effect on T2 at obtainable conc.

a. dex coated particles precip

b. periodate conjugation

c. affinity purification

Validate assembly of nanoparticulate carrier

d. DNA adhesion

e. relaxivity assessment

test tube cast gels

solenoid coil

7

Validate imageability in MRI

   II Intracellular release and drug activation

3

valacyclovir

BHK-Herpes

plaque count

control

Establish cellular capability of uptake and release

dex-val

BHK-Herpes

plaque count

steric decrease in uptake

WGA-dex-val

BHK-Herpes

plaque count, assay

improved delivery

B Interaction with Axon Terminals and Intra-axonal environment

   III Effects of polymer, linker and drug

4

Dextran 10K

symp gang

fluor mic

rapid uptake

Evaluate effects of tripartite size on uptake

Dextran 70K

symp gang

fluor mic

slower uptake

5

Dextran-FITC neut

symp gang

fluor mic

extensive uptake

3

Evaluate effects of tripartite charge on uptake

Dextran-FITC pos

symp gang

fluor mic

extensive uptake

Dextran-FITC neg

symp gang

fluor mic

limited uptake

6

Acylation

symp gang

fluor mic

no uptake

Evaluate effects of hydrophobicity on uptake

   IV Effects of Axonal Transport Facilitator

7

WGA-FITC

symp gang, Campenot

fluor mic

WGA and NGF well transported

Comparability of physiologic and non-phys ATF in vitro

NGF-TR

symp gang, Campenot

fluor mic

WGA and NGF well transported

4,17,18

8

Phage display

Campenot

pfu's

identification of synthetic ATF

Phage display for generation of synthetic ATFs

9

Transport inhibitor

symp gang, Campenot

fluor mic

blocked by inhibitors of transport

Confirmation of role of microtubule based transport

   V Effects of Intra-axonal Processing

10

Anti-gabapentin Ab

gastroc/ant tib - rat

excise, Ab stain

delivery of gabapentin antigenicity to DRG

Confirm delivery of gabapentin antigenicity

11

WGA-HRP

axial - M. fascicularis

HRP-TMB backlit

small IM inj to enzyme in cells in primates

5

Confirm that enzymatic function survives transport

12

WGA-dex-mag

gastroc - rabbit

EM

transport in axoplasm

6

Verify axonal transport compartment

13

59-Fe WGA-dex-mag

gastroc/ant tib-rabbit

autoradiography

gross sections

Confirm intraneural transport of particle tripartite

14

WGA-dex-mag

gastroc/ant tib-rabbit

MRI vs gel chamber

non-lysis of particle

8,19

Confirmation of transport of intact magnetite

15

WGA-dex-mag

forearm-rabbit

4.7T high res MRI

demonstration of nerve image

9

Confirmation of fast transport of intact magnetite

C Targeting and pharmacological efficacy

   VI Clinical target access

16

WGA-FITC

hindlimb - rat

excise, section, fl mic

ventral horn & intermediolateral

10

Assess access to relevant motor & autonomic targets

WGA-dex-FITC

hindlimb - rat

excise, section, fl mic

ventral horn & intermediolateral

Dex-FITC

hindlimb - rat

excise, section, fl mic

limited transport

17

WGA-FITC

hindlimb - rat

excise, section, fl mic

DREZ lamina I and II

11

Confirm access to dorsal root entry zone

WGA-FITC

hindlimb - rat

excise, section, fl mic

DRG axon dendrite

18

WGA-FITC

hindlimb - rat

fluor mic, cell count

good filling in DRG

12,13

Assess properties of access to dorsal root ganglia

WGA-TRITC

foot pad-rat

fluor mic, cell count

good filling in DRG

19

WGA-FITC

hindlimb - rat

fluor mic, peripherin

nociceptors reached

14

Verify access to nociceptors

dextran-FITC

hindlimb - rat

fluor mic, peripherin

no transport

   VII Distinctive pattern of distribution relative to trans-vascular

20

125-I WGA

forelimb/hindlimb - rat

dissect, count

saturable, time consistent, selective

15

Characterize expected whole body distrib

125-I NGF

hindlimb - rat

dissect, count

differences in distribution due to ATF

Evaluate effects of ATF on distribution

59-Fe WGA-dex-mag

forelimb - rat

dissect, count

large particles comparable

Evaluate effects of large agent size on distribution

21

131-I WGA

gastroc/ant. tib-rabbit

gamma camera image

voxel size causes averaging and inj site bright

Examine local injection area effects

22

14C-gbp-dex

hindlimb - rat

dissect, count

minimal

Validate delivery of therapeutic dose

14C-gbp-dex-WGA

hindlimb - rat

dissect, count

transported in high conc.

   VIII Unique pharmacologic effects not obtainable by trans-vascular

23

gbp-dex-WGA

foot pad/hindlimb - rat

withdrawal latency

prolonged decrease in hyperalgesia

16

Validate pharmacological efficacy

gbp-dex

foot pad/hindlimb - rat

withdrawal latency

minimal

16

gbp

foot pad/hindlimb - rat

withdrawal latency

minimal

16

gbp-dex-WGA

cervical fat pad - rat

withdrawal latency

minimal

16