PHOTODYNAMIC THERAPY COMPLEX

Abstract

The invention concerns a complex for delivering light-activated antimicrobial agents (photosensitising (PS) agents) into cells; use of said complex as a medicament in photodynamic therapy; use of said complex for treatment of a microbial infection; a method for the manufacture of a composition comprising said complex; a pharmaceutical or veterinary composition comprising said complex; a combination therapeutic comprising said complex and at least one other agent; a method of treatment employing the use of said complex, composition or combination therapeutic. The complex comprises a poly--amino ester and a photosensitizing agent.

Claims

1. A complex comprising a poly--amino ester (PBAE), or a derivative thereof, and at least one photosensitizing agent.

2. The complex according to claim 1 wherein said PBAE, or derivative thereof, is obtained by reaction of a diacrylate monomer with an amine monomer.

3. The complex according to claim 2 wherein said diacrylate monomer is a compound of formula (I) ##STR00044## where R.sup.1 is an optionally substituted divalent hydrocarbyl group, which may also be interposed with heteroatoms, with a primary or secondary amine monomer.

4. The complex according to claim 2 wherein said primary or secondary amine monomer is a compound of formula (II) or formula (III) ##STR00045## where R.sup.2 is an optionally substituted hydrocarbyl group; R.sup.3 and R.sup.4 are independently selected from optionally substituted hydrocarbyl groups; R.sup.5 is a divalent hydrocarbyl group which is optionally substituted and may also be interposed with heteroatoms; or R.sup.3 and/or R.sup.4 are linked together or to R.sup.5 to form one or more ring structures.

5. The complex according to claim 4 wherein optional substituents for the hydrocarbyl groups R.sup.3, R.sup.4 and R.sup.5 comprise one or more groups selected from hydroxyl, C.sub.1-6alkoxy, C.sub.1-6alkylsilane or heterocyclic groups or halo.

6. The complex according to claim 4 wherein optional substituents for R.sup.2 are one or more groups selected from hydroxyl, C.sub.1-6alkoxy, C.sub.1-6alkylsilane or heteroaryl groups or halo.

7. The complex according to claim 4 wherein R.sup.2 is an optionally substituted alkyl group, in particular an optionally substituted C.sub.1-6alkyl group.

8. The complex according to claim 4 wherein R.sup.3 or R.sup.4 are substituted alkyl groups.

9. The complex of claim 8 wherein R.sup.3 or R.sup.4 are optionally substituted alkyl groups, in particular unsubstituted C.sub.1-6alkyl.

10. The complex according to claim 4 wherein R.sup.3 and R.sup.4 are linked together to form a ring structure.

11. The complex according to claim 10 wherein said ring structure is a piperazine or piperidine ring.

12. The complex according to claim 4 wherein said amine monomer is selected from 5-amino-1-pentanol, 2-methoxyethylamine, 3-(dimethylamino)-propylamine, (3-aminopropyl) triethoxysilane, 2-(2-pyridyl)ethylamine, 3-methoxy-propylamine, 3-amino-1,2-propanediol, 1-amino-2 propanol, piperazine, 4,4-trimethylenedipiperidine and N,N-dimethylethyldiamine.

13. The complex according to claim 12 wherein the amine monomer is a compound of formula (III) or is a compound of formula (II) wherein R.sup.2 carries at least one substituent which includes a nitrogen atom and/or is a hydroxyl group.

14. The complex according to claim 13 wherein the amine monomer is selected from the group comprising: 4,4-trimethylenedipiperidine, 3-(dimethylamino)-propylamine 3-(dimethylamino)-propylamine, 2-(2-pyridyl)ethylamine and 1-amino-2 propanol.

15. The complex according to claim 1 wherein said PBAE is represented by formula (IV) and (V) ##STR00046## where R.sup.1 is an optionally substituted divalent hydrocarbyl group, which may also be interposed with heteroatoms, with a primary or secondary amine monomer, R.sup.2 is an optionally substituted hydrocarbyl group; and R.sup.3 and R.sup.4 are independently selected from optionally substituted hydrocarbyl groups; R.sup.5 is a divalent hydrocarbyl group which is optionally substituted and may also be interposed with heteroatoms; or R.sup.3 and/or R.sup.4 are linked together or to R.sup.5 to form one or more ring structures; and n is a integer greater than 2.

16. The complex according to claim 15 wherein n is in the range of from 10-100.

17. The complex according to claim 16 wherein n is in the range of 30-70 or 45-55.

18. The complex according to claim 1 wherein said PBAE is a derivative having a functional group at an end of the polymer chains.

19. The complex according to claim 18 wherein said functional groups are introduced by reacting the PBAE with a primary or secondary diamine compound.

20. The complex according to claim 19 wherein said primary or secondary diamine compound is of formula (VI) ##STR00047## where R.sup.6 is an alkylene chain, and R.sup.7 and R.sup.8 are the same and are selected from hydrogen or a C.sub.1-6alkyl group.

21. The complex according to claim 20 wherein said primary or secondary compound is selected from the group consisting of: ethylene, diethyleneamine, and diethylenetriamine.

22. The complex according to claim 18 wherein said PBAE derivatives of the invention may be represented by formula (VII) or (VIII) ##STR00048## wherein R.sup.1 is an optionally substituted divalent hydrocarbyl group, which may also be interposed with heteroatoms, with a primary or secondary amine monomer; R.sup.2 is an optionally substituted hydrocarbyl group; R3 and R4 are independently selected from optionally substituted hydrocarbyl groups; R.sup.5, R5 is a divalent hydrocarbyl group which is optionally substituted and may also be interposed with heteroatoms; or R3 and/or R4 are linked together or to R5 to form one or more ring structures. R.sup.6, R6 is an alkylene chain; and R7 and R8 are the same and are selected from hydrogen or a C1-6alkyl group.

23. The complex according to claim 1 wherein said photosensitizing agent is selected from the group consisting of: thiazine compound, porphyrins, chlorins, and xanthenes or derivatives thereof.

24. The complex according to claim 23 wherein said photosensitizing agent is selected from the group consisting of: (7-amino-8-methyl-phenothiazin-3-ylidene)-dimethyl-ammonium (TBO), Sn(IV)-chlorine-e6 (SnCe6), and -Aminolevulinic acid (ALA), Rose Bengal (RB), Eosin Y (EOS) and Erythrosine B (ERI).

25-28. (canceled)

29. A pharmaceutical or veterinary composition comprising a complex according to claim 1 together with a pharmaceutically or veterinary acceptable excipient or carrier.

30. The pharmaceutical or veterinary composition according to claim 29 wherein said composition is formulated for topical application.

31. A combination therapeutic comprising the complex according to claim 1 in combination with at least one other therapeutic.

32. (canceled)

33. A method of treating a microbial infection by photodynamic therapy comprising administering the complex according to claim 1, a pharmaceutical or veterinary composition comprising the complex and a pharmaceutically or veterinary acceptable excipient or carrier, or a combination therapeutic comprising the complex and at least one other therapeutic, to a subject having or suspected of having a microbial infection.

34. The method according to claim 33 wherein said subject is a mammal selected from the group comprising: human, equine, canine, feline, porcine, ovine, ungulate or any other domestic or agricultural species.

35. The method according to claim 34 wherein said subject is human.

36. The method according to claim 33 wherein said microbial infection is a viral, bacterial, prion, fungus, viroid, or parasitic infection.

37. The method according to claim 36 wherein said microbial infection is a bacterial infection.

Description

[0078] The invention will now be described by way of example only with reference to the Examples below and to the following Figures wherein:

[0079] FIG. 1. Antimicrobial activity of TBO (.circle-solid.) and A3-e1-TBO () against (a), E. coli (b), P. aeruginosa, (c) A. baumannii, (d) S. epidermidis, (e) MRSA and (f) S. pyogenes;

[0080] FIG. 2. Log.sub.10 reduction of viable cells after 30 sec laser exposure of (a) E. coli and (b) S. epidermidis for different PBAE-TBO and pure TBO;

[0081] FIG. 3. Uptake of TBO for (a) E. coli and (b) S. epidermidis for different PBAE-TBO, having butandiole diacrylate (A), 1,6-hexanediol diacrylate (B) or tetra(ethylene glycol) diacrylate (C) shown in Table 2 below, compared to pure TBO;

[0082] FIG. 4. ROS produced by different PBAE-TBO, having butandiole diacrylate (A), 1,6-hexanediol diacrylate (B) or tetra(ethylene glycol) diacrylate (C) shown in Table 2 below, after 1 minute irradiation compared to pure TBO measured in a solution of PBAE-TBO or pure TBO;

[0083] FIG. 5. Log.sub.10 reduction of viable cells (a) E. coli after 3 min laser exposure and (b) MRSA NCTC12494 after 5 sec for different PBAE-Rose Bengal and pure Rose Bengal (RB);

[0084] FIG. 6. Uptake of Rose Bengal (RB) for (a) E. coli and (b) MRSA NCTC12494 for different PBAE-RB and pure RB;

[0085] FIG. 7. Log.sub.10 reduction of viable cells (a) E. coli after 3 min laser exposure and (b) MRSA NCTC12494 after 5 sec for different PBAE-Erythrosin B (ERY) and pure ERY;

[0086] FIG. 8. Uptake of Erythrosin B (ERY) for (a) E. coli and (b) MRSA NCTC12494 for different PBAE-ERY and pure ERY;

[0087] FIG. 9. Logic) reduction of viable cells (a) E. coli and (b) P. aeruginosa after 3 min laser exposure; (c) A. baumannii after 2 min laser exposure; (d) MRSA NCTC12494 and (e) MRSA 59275 after 5 sec for different PBAE-Rose Bengal and pure Rose Bengal;

[0088] FIG. 10. Uptake of RB for cells (a) E. coli, (b) P. aeruginosa, (c) A. baumannii, (d) MRSA NCTC12494 and (e) MRSA 59275 for different PBAE-Rose Bengal (RB) and pure RB;

[0089] Table-1. List of amine monomers used to generate acrylate terminated poly(-amino ester)s;

[0090] Table-2. Structure of 1,4-butandiole diacrylate (A), 1,6-hexanediol diacrylate (B) or tetra(ethylene glycol) diacrylate (C) used to generate poly(-amino ester)s when mixed with the amine monomers of table 1;

[0091] Table-3. End-capping reagents used to generate PBAE derivatives;

[0092] Table-4. Structure of Toluidine Blue 0 (TBO), Rose Bengal (RB) and Erythrosin B (ERY).

[0093] Methods and Materials

[0094] Synthesis of PBAE

##STR00006##

[0095] Acrylate-terminated poly(-amino ester)s were synthesized by mixing 1,4-butandiole diacrylate (A), 1,6-hexanediol diacrylate (B) or tetra(ethylene glycol) diacrylate (C) shown in Table 2 below, with a range of amine monomers as shown in Table 1 below a 1.1:1 ratio diacrylate:amine in Dichloro-methane (DCM) at a concentration of 5 ml of DCM each 3.7 mmol of acrylate. In the above scheme, the letter R is indicative of the particular organic group present in the monomers.

[0096] The polymerisation was then performed under stirring at 50 C. for 48 hours. PBAEs were precipitated through pouring the reaction mixture in about 10 times the volume of diethyl-ether under vigorous mixing; the solvent was removed under vacuum.

[0097] Preparation of End-Capped PBAE Derivatives

[0098] Acrylate-terminated polymers of were dissolved in DCM at a concentration of 31.13% w/w. A range of end capping agents shown in Table 3 below were then added.

##STR00007##

[0099] Specifically, amine capping reagents such as (either ethylene diamine (e1), diethylenetriamine (e2) were dissolved in DCM to a concentration of 0.25 mol/l). The capping reactions were performed by mixing the polymer/DCM solution with amine solution at a ratio of 800 l per 321 mg of polymer solution; the mixture was kept for 24 hours at room temperature.

[0100] End-capped PBAEs were recovered through precipitation in diethyl-ether under vigorous mixing, the unreacted amine were removed centrifuging the suspension of PBAE in diethyl-ether/DCM for 2 min at 1155 g. The supernatant was removed and the PBAEs washed twice with diethyl-ether. The end-capped PBAEs were then dried under vacuum.

[0101] The PBAEs and derivatives obtained were coded through the constituents of the back bone using a capital letter to indicate the acrylate (A being 1,4-butanediol acrylate) and a number to indicate the amine as shown in Table 1;

[0102] the further end capping is described by a number preceded by the letter e. For example, A2-e1 is the PBAE obtained from 1,4-Butanediol diacrylate and 4,4-Trimethylenedipiperidine then capped with ethylene-diamine.

[0103] The Preparation of Photosensitiser-Polymer Complex

[0104] 10 mg of TBO, for example, photosensitiser (PS) was dissolved in DCM (25 ml) along with 100 mg of end-capped polymer; the solutions were immediately covered with aluminium foil and stirred for 24 hours at room temperature. The PBAEs-PS, e.g. PBAE-TBO, derivatives were recovered through precipitation in diethyl-ether under vigorous mixing, the unreacted molecules were removed centrifuging the suspension of PBAE in diethyl-ether/DCM for 2 min at 1155 g. The supernatant was removed and the PBAE-PS, e.g. PBAE-TBO, washed twice with a diethyl-ether/DCM mixture 4:1. The end-capped PBAEs were then dried under vacuum.

[0105] Antimicrobial Protocol

[0106] The organisms used in this study were Escherichia coli (NCTC10418) and Staphylococcus epidermidis (ATCC12228), Pseudomonas aeruginosa (NCIMB10548), Methicillin Resistant Staphylococcus aureus (NCTC12493), Streptococcus pyogenes (ATCC19615) and Acinetobacter baumannii (NCIMB9214); they were stored at 80 C. These organisms were subcultured, when needed, on Brain Heart Infusion (BHI) Agar overnight aerobically at 37 C. The plates were then stored at 4 C. for no more than two weeks. Bacterial suspensions used for the experiments were grown in Brain Heart Infusion (BHI) broth after inoculation with a loopful of cells from a single colony on a BHI plate and incubated aerobically for 24 h at 37 C. statically. These overnight cultures were then diluted 1 in 100 in PBS. The resulting bacterial suspension contained approximately 10.sup.6 CFU/mL. Each cell suspension was used to disperse pure PS, e.g. PBAE-TBO, to concentrations of 0.2 mg/mL or PBAEs-PS derivatives to an equivalent PS concentration.

[0107] 200 L aliquots of the resulting suspension were immediately poured in a GREINER 96 U-BOTTOM well plate. The well plate was then irradiated with a laser light (633 nm) using a 500 mW red laser (RRL-635 nm-500 mW-1080060, Changchun New Industries Optoelectronics, China) or a 500 mW green laser (MGL-FN-532 nm-500 mW-15097031, Changchun New Industries Optoelectronics, China) for different time periods (between 5 sec to 3 min). The red laser was employed for TBO while the green laser was used for RB and ERY.

[0108] After exposure the bacterial cells (L+S+) were counted through serial dilutions and then plating on BHI Agar. Along this test, different experiments were performed including the testing of dark toxicity (labelled with LS+), samples exposed uniquely to laser light (L+S) or samples not expose to either laser light and PBAE (LS).

[0109] Through the experiment prepared samples, well plates and inoculated plates were immediately covered with aluminium foil to reduce biased measuring results due to exposure of any light besides the laser. All plates were incubated after the experiment for 24 hours at 37 C. All experiments were performed on three independent cultures.

[0110] Reactive Singlet Oxygen Species

[0111] The generation of reactive oxygen species (ROS) was assessed using a singlet oxygen Sensor Green reagent (SOSG) (Molecular Probes, Unites States). The SOSG was stored at 20 C. and protected from light prior to use. The working solution was prepared with methanol to a final concentration of 5 mM. The prepared reagent was further diluted in methanol (1:100) before each experiment. Each sample was dispersed in PBS to a concentration equivalent to 0.2 mg/mL of pure PS, e.g. PBAE-TBO. 100 L of diluted sample, 100 L PBS and 20 L reagent were immediately poured in two adjoining wells of a GREINER 96 U-BOTTOM well plate. The irradiation of one well (L+) was in each case done for a period of one minute with a 500 mW red laser (RRL-635nm-500nW-1080060, Changchun New Industries Optoelectronics, China); whilst the other well was covered with aluminium foil (L). After exposure the ROS were determined using the FLUOstar OPTIMA (BMG Lab technologies, Germany).

[0112] Cell Uptake

[0113] 10 mL of fresh sterile BHI broth were inoculated with a loopful of cells from a single colony on a BHI plate and incubated aerobically for 24 h at 37 C. statically. The bacterial suspension was then centrifuged with an Avanti J-20XP Centrifuge (Beckmann and Coulter, United States) for duration of 3 min at 2938 g, afterwards the supernatant was disposed. After one wash and centrifuge with PBS, 1 mL of sample containing either PBAE-PS or pure PS at an equivalent concentration of pure TBO or dye of 0.2 mg/mL was added to the precipitated cells. The resulting solution was vortexed and centrifuged after 3 min exposure. The exposure of samples to cells was limited to 3 min to ensure comparability to other experiments. After two more wash- and centrifugation-runs with PBS, the cells were dissolved in 1 mL of 0.1 M NaOH and 1% Sodium dodecyl sulfate (SDS) and then incubated at 37 C. for 24 h to lyase the cells.

[0114] The optical density of samples was measured at a 650 nm for TBO or 550 nm for RB and ERY using a plate reader (Labtech LT5000MS) against a calibration curve prepared using the corresponding bacterial lysate.

[0115] The protein content of the entire cell extract was determined by a modified Lowry method using bovine serum albumin (BSA) dissolved in 0.1 M NaOH/1% SDS to construct calibration curves. Results are expressed as nmol of PS/mg of cell protein.

[0116] Results

[0117] 1.0 Antimicrobial Activity

[0118] We investigated the effect of tested samples in each bacterium upon bacterial growth. Furthermore, the comparison to diluted TBO is illustrated to evaluate effectiveness of each PS-polymer.

[0119] The CFU decreased gradually with the increase of irradiation time, which is emphasized by comparison to control samples. PBAE delivered photosensitiser was more potent in all bacterial samples tested compared to photosensitiser alone. Notably, no reduction in viable cells count was observed in samples exposed to PBAE-TBO without irradiation (L-S+).

[0120] 1.1 A3-e1 Resulted in Significant Cell Death in Gram Positive and Gram Negative Bacteria

[0121] FIG. 1 displays the comparison of all tested microorganisms using pure TBO and A3-e1-TBO after exposure to laser light. In all cases, samples exposed to the polymer-TBO complex or to pure TBO without irradiation (L) did not return any cell reduction over the time. In comparison, irradiated (L+) samples (L) show tendencies of progressive reduction. The comparison with pure TBO showed an obvious improvement in the antimicrobial effect of laser exposure in all cases when the same amount of photosensitiser was employed along with A3-e1. Furthermore, when cells were exposed to laser light without the presence of photosensitiser (L+S) did not reveal any antimicrobial activity. Generally, Gram+ were more readily inactivated than Gram-.

[0122] 1.2 Alternative PBAEs Also Lead to Increased Photosensitisation

[0123] The comparison of the efficacy of other PBAE as delivery system of TBO for antimicrobial PDT against an example of Gram (E. coli) and an example of Gram+(S. epidermidis) was tested in FIG. 2 using similar amine and end-capping agents for the preparation of PBAE. Generally, PBAE-TBO complex improved photosensitivity and cell death of the two bacteria tested with the acrylate B more effective than acrylate A; acrylate C largely returned the least improvement of the TBO activity.

[0124] 2.0 PBAE Agents Improve Cell Uptake and Delivery of Photosensitisers

[0125] All PBAE tested increased the uptake of TBO in both E. coli and S. epidermidis (FIG. 3) cells suggesting that PBAE improves cell delivery. Acrylate B performed better than acrylate A in most of the cases and acrylate C the least effective. The overall efficiency of PBAE-TBO as delivery vehicle for TBO into bacterial cells depends on all three components of the polymer (acrylate, amine, and end-capping). The adjuvant effect of PBAE is remarkably greater in S. epidermidis than in E. coli, confirming the observed increased antimicrobial effect observed in Gram positive bacteria (FIG. 1).

[0126] 3.0 Efficacy of PBAEs in Combination with Rose Bengal and Erythrosin

[0127] The red laser light (around 630 nm) is suitable to PS such as TBO, however other compounds require photons at different wavelength in order to act as PS.

[0128] For example, RB and ERY are excited by green laser light at around 530 nm. These PS were also found to exhibit antimicrobial activity against E. coli and MRSA, an example of Gram- and Gram+, respectively (FIGS. 5 and 7).

[0129] All PBAE tested in combination with RB were capable of improving the inactivation kinetic against both bacteria (FIG. 5), in many cases for MRSA the number of viable cells after irradiation fell below detection limit. No dark toxicity was detected for any of the PBAE.

[0130] ERY was not as effective as RB as light activated antimicrobial agent, however some of the PBAE tested did result in enhanced antimicrobial activity (FIG. 7).

[0131] Cell uptake of either RB or ERY (FIGS. 6 and 8) was increased when in combination with all the PBAE prepared, moreover no correlation was evident between cell uptake of the PS and inactivation rate.

[0132] 4.0 Role of End-Capping Agent in Efficacy of PBAE-RB

[0133] The improvement in bacterial inactivation resulting from the combination of PS and PBAE depends not only on the nature of two monomers (acrylate and amine) but also on the end capping agent used. We investigated the effect of various chemicals suitable for PBAE end-capping on the efficacy of A16 (chosen based on the results in FIG. 5) combined with RB against a variety of Gram and Gram+ bacteria also including a clinical isolate of MRSA; longer exposures were required for Gram compared to Gram+ as the former are known to be more resistant to antimicrobial treatments.

[0134] FIG. 9 shows the log 10 reduction after exposure to green laser light for pure RB and for complexes A16-RB. The role of the end-capping agent on the modulation of the overall antimicrobial outcome is clearly noticeable. Some end-capping agents can enhance the antimicrobial activity of RBI; when the sulphur containing compound was used the product was not soluble hence no antimicrobial activity was displayed.

[0135] Cell uptake of RB (FIG. 10) was increased when in combination with some of the PBAE prepared, moreover no correlation was evident between cell uptake of the PS and inactivation rate.

[0136] 5.0 Reactive Oxygen Species

[0137] The amount of ROS generated by pure TBO or by PBAE-TBO complexes is shown in FIG. 4 and reveals a strong dependence on the presence and structure of the PBAE. In most of the cases, acrylate B performed better than acrylate A.

[0138] No ROS were detected when only PBAE were exposed to laser light, hence PBAE are not photosensitisers. This demonstrates that PBAE delivery does not only act in enhancing TBO uptake by cells but also have a direct role in the mechanism of free radical formation by PDT.

DISCUSSION

[0139] Light-activated antimicrobial agents (photosensitisers) are promising alternatives to antibiotics particularly, though not exclusively, for the treatment of skin infections and wounds through Photo Dynamic Therapy (PDT). Despite numerous benefits PDT applicability is limited by low efficacy requiring long light exposure time. We have developed a combination of photosensitisers with Poly-beta-amino esters (PBAEs) in order to enhance the photosensitiser uptake and ROS generating efficiency to reduce the exposure time required to achieve the targeted antimicrobial reduction. The overall performance of photosensitisers/PBAE complex is the result of two phenomena both of which PBAE is responsible for, one is cellular uptake and the other is the enhanced generation of ROS by the irradiated photosensitiser.

TABLE-US-00001 TABLE 1 1 [00008] piperazine 2 [00009] 4,4-Trimethylenedipiperidine 3 [00010] 5-amino-1-pentanol 4 [00011] 2-methoxyethylamine 5 [00012] 3-(dimethylamino)-1- propilamine 6 [00013] (3-aminopropyl) triethoxysilane 7 [00014] 2-(2-pyridy)ethylamine 8 [00015] 3-methoxy-propylamine 9 [00016] N,N-dimethylethyldiamine 10 [00017] 4-(2-aminoethyl)morpholine 12 [00018] 3-Amino-1,2-propanediol 13 [00019] DL 1-amino-2-propanol 14 [00020] trans-4-amino-hexanol 15 [00021] Cyclopentylamine 16 [00022] 3-amino-1-propanol 17 [00023] 1-(3-Aminopropyl)imidazole 18 [00024] 1-(2-aminoethyl)piperidine 19 [00025] 1-(2-aminoethyl)pyrrolidine 20 [00026] N,N-bis[(3-methylamino) propyl]methylamine

TABLE-US-00002 TABLE 2 A [00027] 1,4-Butanediol diacrylate B [00028] 1,6 Hexanediol diacrylate C [00029] Tetra(ethylene glycol) diacrylate

TABLE-US-00003 TABLE 3 [00030] Ethylen diamine e1 [00031] Diethylentriamine e2 [00032] Tris-(2-aminoethyl) amine e3 [00033] 2-aminoethanol c2 [00034] 3-amino-1-propanol c3 [00035] 4-amino-1-butanol c4 [00036] 5-amino-1-pentanol c5 [00037] 2-mercaptoethanol s2 [00038] 3-methoxy- ethyllamine e4 [00039] Cyclopentylamine e15 [00040] 1-(3-Aminopropyl) imidazole e17

TABLE-US-00004 TABLE 4 [00041] Toluidine Blue O (TBO) [00042] Rose Bengal (RB) [00043] Erythrosin B (ERY)