Hemoglobin-Targeted Drug Delivery For The Treatment of Cancer

Abstract

The use of hemoglobin as a targeting carrier for drugs is disclosed. Such hemoglobin-drug complexes have utility in the treatment of cancer, in particular cancers of the liver and colon. Said drug is preferably a nucleoside analog, in particular floxuridine and is covalently attached to the hemoglobin in a molar drug ratio of between 1 and 20.

Claims

1: A method for treating liver and non-liver tumors comprising contacting the tumor with a hemoglobin-drug complex, or mimic of a hemoglobin-drug complex, to thereby affect the growth of the tumor and survival of the host.

2: The method of claim 1 comprising contacting the tumor with the hemoglobin-drug complex or mimic to cause incorporation of the complex or mimic into cells within tumors bearing receptors for hemoglobin or via other mechanisms of uptake and thereby affect growth and survival of the tumor.

3: The method of claim 1 comprising contacting the tumor with a hemoglobin-drug complex or mimic to effect incorporation into cells within tumors bearing receptors for hemoglobin.

4: A method for the treatment colon cancer tumors comprising contacting the tumor with a hemoglobin-drug complex or mimic a hemoglobin-drug complex to cause incorporation of the complex or mimic into the tumor cells and/or cells within tumors bearing receptors for hemoglobin or via other mechanisms of uptake.

5: The method of claim 4 comprising contacting the tumor with the hemoglobin-drug complex or mimic to affect incorporation of the complex or mimic into cells within tumors bearing receptors for hemoglobin.

6: A method according to claim 1, wherein the drug is a nucleoside analog.

7: A method according to claim 1, wherein the drug is a nucleoside analog anticancer drug.

8: A method according to claim 1, wherein the hemoglobin-drug complex is hemoglobin-floxuridine.

9: A method according to claim 8 wherein the hemoglobin-floxuridine has a molar drug ratio of between 1 and 20.

10: A method according to claim 1, wherein the mimic of a hemoglobin-drug complex comprises a hemoglobin mimic.

11: A method according to claim 10 wherein hemoglobin mimic is an antibody or antibody fragment.

12: A method according to claim 1 wherein the conjugate is a hemoglobin-drug conjugate.

13: A method according to claim 12, wherein the hemoglobin is >99% pure hemoglobin sub-type A0.

14: A method according to claim 1, wherein the hemoglobin, hemoglobin-drug complex or hemoglobin mimic binds to CD163.

15: A method according to claim 4, wherein the drug is a nucleoside analog.

16: A method according to claim 4, wherein the drug is a nucleoside analog anticancer drug.

17: A method according to claim 4, wherein the hemoglobin-drug complex is hemoglobin-floxuridine.

18: A method according to claim 17, wherein the hemoglobin-floxuridine has a molar drug ratio of between 1 and 20.

19: A method according to claim 4, wherein the mimic of a hemoglobin-drug complex comprises a hemoglobin mimic.

20: A method according to claim 19, wherein hemoglobin mimic is an antibody or antibody fragment.

Description

EXAMPLE 1. HEMOGLOBIN-FLOXURIDINE CONJUGATE

[0028] Hb-FUdR is a synthetic conjugate of purified human hemoglobin (Hb) and the fluoropyrimidine anticancer drug floxuridine (FUdR), in which FUdR is covalently attached to Hb. The purified Hb used in the production of Hb-FUdR consists of >99% HbA.sub.0, the predominant human Hb phenotype. Hb was extensively purified to >99% purity using a process described in U.S. Pat. No. 5,439,591 (1995).

Synthesis of FdUMP-Im

[0029] Hb-FUdR was synthesized according to Scheme 1 and as described below.

##STR00001##

[0030] 5-fluoro-2′-deoxyuridine-5′-monophosphate sodium salt (FdUMP, Sigma-Aldrich, 6.3 mg, 20 μmop was stirred in 1.0 mL of dimethyl sulfoxide DMSO in a pre-dried vial under a stream of dry N.sub.2. In a separate pre-dried vial under dry N.sub.2, 40 mg of carbonyl diimidazolide (CDI) was dissolved in 1.0 mL of DMSO. In a separate, third pre-dried vial under dry N.sub.2, 30 mg of imidazole (Im) was dissolved in 2.0 mL of DMSO. To the stirred FdUMP suspension was added 470 μL of the CDI solution (116 μmol, ie. a 5.8-fold excess) and 500 μL of the Im solution (110 μmol, i.e. a 5.5-fold excess) under dry N.sub.2. The suspension was rapidly stirred at RT. The final concentration of FdUMP was 3.2 mg/mL in DMSO. An additional 10 mg each of CDI and Im were dissolved in the reaction mixtures, which were then re-charged with N.sub.2 and stirred at RT overnight. The CDI and Im additions (10 mg each) were repeated at 20, 22, and 26 h to complete the reaction by 27 h, as indicated by HPLC.

[0031] Monitoring by HPLC: 20 μL of reaction mixture was mixed with 180 μL of 200 mM NaHCO.sub.3/Na.sub.2CO.sub.3 pH 9.5 buffer to quench and dissolve prior to injection (50 μL injection volume) onto an analytical C18 RP Aqua-Luna Phenomenex column, 66 mM K.sub.2HPO.sub.4 elution buffer pH 7.35, isocratic, 1 mL/min flow rate, absorbance monitored at 210, 280, and 254 nm.

[0032] The reaction mixture was transferred to a test tube and the reaction was stopped by freezing in liquid N.sub.2. The frozen sample was lyophilized to remove DMSO. The lyophilized sample (a waxy, yellow solid) was sealed under N.sub.2 and frozen at −20° C. until work-up and reaction with Hb

Work-Up of FdUMP-Im

[0033] The waxy residue of the reaction mixture was dissolved in 500 μL of anhydrous ethanol (EtOH) to quench the excess CDI. To the anhydrous EtOH supernatant was added 1 mL of anhydrous ether (Et.sub.2O) to cause precipitation/crystallization. The tube was put at −20° C. for 2 h to complete precipitation. The product was a fluffy white precipitate. This was centrifuged down to a pellet (5 min) and the supernatant was removed. The product was washed with 1 mL of Et.sub.2O, centrifuged, and the Et.sub.2O removed. This process was repeated 3 times. The final off-white powder product was dried under a gentle stream of dry N.sub.2 in the tube and the final weight was determined (4.6 mg, 12.3 μmol, 63% yield).

[0034] Approximately ⅔.sup.rd of the product was dissolved immediately in 300 μL of 200 mM NaHCO.sub.3/Na.sub.2CO.sub.3 pH 9.5 buffer in preparation for reaction with COHb.

[0035] The other ⅓rd of the product was kept in a dried vial, charged with N.sub.2, at −20° C., for supplemental addition to the conjugation reaction mixture at the 48 h mark.

Conjugation of FdUMP-Im to COHb

[0036] In an eppendorf tube was placed 70 μL of 10 g/dL carbonmonoxyhemoglobin (COHb) in WFI (7 mg, 108 nmol). To this was added the 300 μL of FdUMP-Im solution followed by an additional 200 mL of 200 mM NaHCO.sub.3/Na.sub.2CO.sub.3 pH 9.5 buffer. The reaction mixture was vortexed and the pH was monitored to maintain above pH 9.3 with 200 mM Na.sub.2CO.sub.3 pH >11 solution. The pH meter electrode was rinsed with 400 μL of 200 mM NaHCO.sub.3/Na.sub.2CO.sub.3 pH 9.5 buffer (total reaction volume 970 μL). This was CO charged as best as possible, sealed with parafilm, and placed in a 37° C. water bath. After 3 h the pH was still 9.3. After 49 h of reaction, the remaining ⅓.sup.rd portion of the FdUMP-Im solution was added to the reaction solution.

[0037] Progress of the reaction was monitored periodically over the course of 1 week by Anion Exchange HPLC using a Poros HQ 10 cm column eluted with a pH gradient (achieved using Tris and bis-Tris buffers outlined in Chart 1), a 4 mL/min flow rate, and a 504 injection volume.

TABLE-US-00001 CHART 1 Anion Exchange Chromatography Elution Conditions % % Duration to Solvent Solvent achieve next Time (min) A B gradient (min) 0 100 0 1 1 0 100 7 8 0 100 2 10 100 0 2 14 stop data stop data — Solvent A: 25 mM Tris pH 8.3 buffer Solvent B: 25 mM bis-Tris pH 6.3 buffer

[0038] After one week (190 h) the reaction was considered complete with <10% unreacted Hb remaining; the reaction mixture was CO charged and frozen at −80° C. until further characterization.

[0039] Characterization by methods described in Brookes et al. 2006 confirmed the test article Hb-FUdR to have a molar drug ratio (MDR) of 4-8.

Human Colon Cancer Model:

[0040] A study testing Hb-FUdR in mice orthotopically implanted with tumor fragments derived from colon cancer cells (transfected with green fluorescence protein for in-life tumor visualization was conducted as follows:

Study design: Fifty (50) tumor-bearing mice were randomly divided into five groups with ten (10) mice per group. Animals (10 mice per group) were treated with Hb-FUdR at 6, 32, and 161 mg Hb/kg (0.15, 0.74, and 3.7 mg FUdR/kg) twice weekly via intravenous injection (tail vein) at 5 mL/kg. Control untreated animals were treated with PBS at 5 mL/kg. Control FUdR treated animals were treated with 3.7 mg FUdR/kg.
Study endpoint: The study was terminated 47 days post tumor inoculation. All remaining mice in each group were sacrificed at this time point. All animals in the PBS treated group and approximately 50% of animals in other treated groups died before the study endpoint. Therefore, survival time between groups was considered one of the criteria to assess the efficacy of the test agents. Primary tumors were excised and weighted at necropsy for subsequent analysis.

Results:

[0041] The anti-tumor efficacy of Hb-FUdR against the implanted human colon cancer was evaluated by comparing the primary tumor sizes measured twenty-one days after treatment initiation, and the differences in survival time between the Hb-FUdR treated and PBS control groups.

[0042] Efficacy of treatment on tumor size: Average tumor volume measured in each Hb-FUdR treated group and the corresponding p value comparison to the PBS treated group (Group 1) are shown in Table 2 below. There were no statistically-significant differences in the tumor volumes in any of the Hb-FUdR treated groups by comparison to the PBS treated group. But a trend of reduction in tumor size could still be seen in all Hb-FUdR treated groups especially in the high dose treated groups. Curves of the mean tumor volume in each group can be seen in FIG. 1.

TABLE-US-00002 TABLE 1 Efficacy on tumor volume Average tumor Group Agent volume (mm.sup.3) 1 PBS 563.63 ± 413.91 2 Hb 161 mg/kg 274.99 ± 221.77 FUdR 3.7 mg/kg 3 Hb 32 mg/kg 351.53 ± 323.25 FUdR 0.74 mg/kg 4 Hb 6 mg/kg 301.27 ± 195.4  FUdR 0.15 mg/kg 5 Hb 0 mg/kg 254.58 ± 196.74 FUdR 3.7 mg/kg

[0043] Efficacy of treatment on tumor metastasis: Tumor metastases were found in the liver and mesentery lymph nodes. The incidence of tumor metastasis to the liver and local lymph nodes in each Hb-FUdR treated group was compared to the PBS treated group. Table 3 below shows the total number of lymph node and liver metastases in each group in comparison to the PBS treated group. There were no statistically significant differences in lymph node or liver metastases between any of the Hb-FUdR treated groups and the PBS treated group.

TABLE-US-00003 TABLE 2 Comparison of tumor metastases in each group No. of p-value No. of No. of mice (Fisher's mice mice with L.N. exact with liver Group Agents evaluated met. test) met. 1 PBS 9 6 — 4 2 Hb 161 mg/kg 10 7 1.0 4 FUdR 3.7 mg/kg 3 Hb 32 mg/kg 10 5 0.65 2 FUdR 0.74 mg/kg 4 Hb 6 mg/kg 10 4 0.37 1 FUdR 0.15 mg/kg 5 Hb 0 mg/kg 9 6 1.0 4 FUdR 3.7 mg/kg

[0044] Efficacy of treatment on survival time: The difference in the survival between each Hb-FUdR treated group and the PBS treated group was compared. Table 3 below shows the mean survival time in each Hb-FUdR treated group in comparison to the PBS control (group 1). Statistically-significant differences can be seen in all of the Hb-FUdR treated groups, except the Hb 6 mg/kg-FUdR 0.15 mg/kg treated group (group 4), by comparison to the PBS control (The survival curves of each group can be seen in FIG. 3).

TABLE-US-00004 TABLE 3 Comparison of survival time in each group Mean survival p-value Group Agents time (days) (log-rank test) 1 PBS 35.7 — 2 Hb 161 mg/kg 42.4 0.018 FUdR 3.7 mg/kg 3 Hb 32 mg/kg 44.1 0.014 FUdR 0.74 mg/kg 4 Hb 6 mg/kg 39.4 0.118 FUdR 0.15 mg/kg 5 Hb 0 mg/kg 43.9 0.003 FUdR 3.7 mg/kg

[0045] Estimation of compound toxicity: The mean body weight in all groups of mice was maintained within the normal range during the entire experimental period (Mean body weight in each group during the experimental period can be seen in FIG. 2). No weight losses were observed in any treated group during the experimental period. Other symptoms of related toxicity were absent by gross observation.

[0046] The data indicates that Hb-FUdR and FUdR significantly prolonged survival time of the treated mice without obvious toxicity.

EXAMPLE 2

[0047] TBI 304, an antibody to CD163, was labelled using the Alexa Fluor 488 dye with 3.4 fluors per protein and binding to a cell line engineered to express recombinant human CD163 was demonstrated by flow cytometry.