Hyaluronan as a cytotoxic agent, drug pre-sensitizer and chemo-sensitizer in the treatment of disease

a cytotoxic agent and cytotoxic technology, applied in the direction of biocide, heavy metal active ingredients, drug compositions, etc., can solve the problems of chemotherapeutic agents that are inherently refractory, chemotherapeutic agents that suffer from acquired resistance, and not all chemotherapeutics are readily used, so as to reduce the primary tumour mass, improve the effect of well-being and reduce the number of tumours

Inactive Publication Date: 2003-09-25
ALCHEMIA ONCOLOGY PTY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

0030] FIGS. 2a-2f shows exponentially growing breast cancer cells that were exposed to 750,000 dalton HA for 30 min, 1 h, or 24 h at which stage the cells were varying concentrations of adriamycin. These figures also illustrate the effect of HA / drug co-incubation for the period of 1 or 3 days. These diagrams illustrate that HA can "pre-sensitise" and / or chemosensitise cells to therapeutic drugs.
0031] FIGS. 3a-3d shows exponentially growing breast cancer cells exposed to varying concentrations of 750,000 dalton hyaluronan for 1 h, 24 h or 3 days followed by treatment with 40 nM Adriamycin for varying time periods of 1 h, 24 h or 3 days. These figures show that a wide concentration range of hyaluronan can act as a chemosenitiser or exert a cytotoxic effect.
0032] FIG. 4 shows that there was no treatment toxicity noted throughout the 6-week study. In comparison to the 5-FU treatment group the mice receiving HA therapy, that is as a sole agent or as a chemosensitizer, demonstrated enhanced well being where the animal did not loose weight, but maintained its body mass.
0033] FIG. 5 shows that at the end of the 6 week study, tumour mass was determined where the HA chemosensitizing therapy had significantly smaller tumours than the saline group, HA and 5-FU groups (p=0.005). HA as a sole agent also demonstrated its effect by reducing the primary tumour mass in comparison to the saline control. No significant differences in tumour response were noted in the initial 2 weeks of treatment, but thereafter the HA followed by 5-FU tumour growth was retarded in comparison to the other treatment groups. During the 6 weeks of treatment interesting differences were noted in the number of tumour doubling cycles. Mice receiving the saline treatment underwent an average of 4 tumour doublings, while the incorporation of HA into the treatment regimen significantly increased the tumour doubling time where HA / 5-FU animals underwent an average of one tumour doubling cycle, once again highlighting the effect of HA on 5-FU cytotoxicity.

Problems solved by technology

Unfortunately, not all chemotherapeutics are readily useable.
For example, some chemotherapeutic agents are inherently refractory in that animal cells do not readily respond to these agents, while other chemotherapeutics suffer from acquired resistance.
Moreover, some chemotherapeutics, while not affected by inherent or acquired resistance per se, are not effective in the treatment of certain diseases as they have innate problems with bioavailability.
More importantly there are no practical means of increasing bioavailability of chemotherapeutics without concomitant increase in toxicity or side effects.
When HA is bound to diseased cells such as tumour cells and internalised there could be a hyperosmotic effect resulting in cell lysis.
HA could exert oxidative membrane damage resulting in apoptosis.
HA internalisation could elevate the mitochondrial membrane potential which could result in cell death or increased drug retention.

Method used

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  • Hyaluronan as a cytotoxic agent, drug pre-sensitizer and chemo-sensitizer in the treatment of disease
  • Hyaluronan as a cytotoxic agent, drug pre-sensitizer and chemo-sensitizer in the treatment of disease
  • Hyaluronan as a cytotoxic agent, drug pre-sensitizer and chemo-sensitizer in the treatment of disease

Examples

Experimental program
Comparison scheme
Effect test

example 2

Testing the Effect of Hyaluronan on Cancer Cell Morphology

[0069] Human breast adenocarcinoma cell lines MDA-MB-468, MDA-MB-435 and MDA-MB-231 were selected based on HA binding affinity (Culty et al, 1994), and the expression of the HA receptors of CD44 and RHAMM (Wang et al, 1996). The characteristics of these cell lines are shown in Table 2.

2TABLE 2 Hyaluronan Binding And Receptor Expression Of Human Mammary Carcinoma Cell Lines HA Receptor Type of breast Degree of HA Expression.sup.b Cell Line cancer Binding.sup.a CD44 RHAMM MDA-MB-231 adenocarcinoma ++ +++ +++ MDA-MB-468 adenocarcinoma ++++ ++++ ++ MDA-MB-435 ductal + +++ ND carcinoma .sup.aCulty et al, 1994 .sup.bWang et al, 1996

[0070] Cell lines MDA-MB-468, MDA-MB-435 and MDA-MB-231 were routinely grown and subcultured as a monolayer in 175 cm.sup.2 culture flasks in Leibovitz L-15 Medium supplemented with 10% Foetal calf serum (FCS) and antibiotic / antimycotic reagents at 37.degree. C. in humidity controlled incubator with 100%...

example 3

Efficacy of Hyaluronan In Vivo

[0085] Based on the results from the in vitro drug sensitivity experiments in Example 2, evaluation of the treatment efficacy of hyaluronan as a sole agent, and as a chemosensitizer in the treatment human breast carcinomas in vivo was undertaken.

[0086] From the results in Example 2 the carcinoma cell line MDA-MB-468 was selected as the cancer cell inoculant for the generation of any nude mouse human tumour xenografts. Cells were routinely grown and subcultured as a previously described in Example 2. For injection into mice, cells were grown to 100% confluency, trypsinised in 0.025% trypsin / 0.01% EDTA solution, washed twice by centrifugation in a Beckman TJ-6 bench centrifuge at 400 g.sub.av for 10 min, counted using a Model-ZM Coulter counter and resuspended in serum-free Leibovitz L-15 medium at 1.times.10.sup.8 cells / ml.

[0087] Six to eight weeks old athymic CBA / WEHI nude female mice, purchased from the Walter and Eliza Hall Research Institute, Melbour...

example 4

Effect of Hyaluronan Concentration on the In Vitro Efficacy of 5-FU

[0121] MDA-MB 468, MDA-MB 435 and MDA-MB 231 cells were cultured as described in Example 2. When the cultures had reached 70-80% confluency they were washed in 1.times. HBSS at 37.degree. C. and trypsinised in 10 ml of 0.25% trypsin / 0.05% EDTA until cells have fully detached. After add 1 ml of FCS to neutralise trypsin the cells were counted, centrifuged at 1,200 rpm for 5 min and resuspended as follows:

[0122] MDA-MB 231: 12,000 cells / ml of media;

[0123] MDA-MB 468: 25,000 cells / ml of media; and

[0124] MDA-MB 435: 12,000 cells / ml of media.

[0125] Cells were then plated into 48-well plates and incubated in accordance with suppliers' instructions. After 24 h media was removed and replaced with the following test media:

[0126] MDA-MB 468: 40 nM adriamycin;

[0127] MDA-MB 231: 50 nM adriamycin; and

[0128] MDA-MB 435: 10 nM adriamycin

[0129] 40 nM Adriamycin media:450 ml (Stock adriamycin is 1.7 mM, therefore 1,700,000 / 40=42,500;...

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Abstract

The present invention relates to the enhancement of bioavailability of chemotherapeutic agents for the treatment of disease. In particular the present invention relates to a method of enhancing the bioavailability of a chemotherapeutic agent comprising the step of administering to a subject in need thereof a therapeutically effective amount of hyaluronan.

Description

[0001] The present invention relates to the enhancement of bioavailability of chemotherapeutic agents for the treatment of disease. In particular the present invention relates to the use of hyaluronan either alone or in combination with a chemotherapeutic agent to enhancement the bioavailability of the chemotherapeutic agent for treatment of disease. The present invention also relates to the treatment of a drug resistant disease whereby the drug resistance is overcome or alleviated with the use of hyaluronan either alone or in combination with a chemotherapeutic agent.BACKGROUND TO THE INVENTION[0002] Many diseases that afflict animals, including humans, are treated with chemotherapeutic agents. For example, chemotherapeutic agents have proven valuable in the treatment of neoplastic disorders including connective or autoimmune diseases, metabolic disorders, and dermatological diseases, and many of these agents are highly effective and do not suffer from any bioavailability problems....

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K9/00A61K31/728A61K45/00A61K45/06A61K47/36A61P35/00A61K47/46A61P35/04
CPCA61K9/0014A61K9/0019A61K31/728A61K45/06A61K47/36A61K2300/00A61P35/00A61P35/04A61K31/337A61K31/4745A61K31/525A61K31/704A61K31/7048
Inventor BROWN, TRACEYFOX, RICHARD
Owner ALCHEMIA ONCOLOGY PTY LTD
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