JP2015515445A - Methods of treating cancer by administering low levels of heat shock protein 70 (Hsp70) - Google Patents

Abstract

The present invention relates to a method of treating cancer by administering heat shock protein 70 (Hsp70) to a subject suffering from cancer.

Description

[Cross-reference of related applications]
This application claims the benefit of priority of US Provisional Application No. 61 / 583,535, filed Jan. 5, 2012, the disclosure of which is incorporated herein by reference in its entirety.

  Heat shock proteins are a class of functionally related proteins involved in the folding and unfolding of other proteins. There are several different heat shock proteins, named according to their weight. Heat shock proteins are expressed when cells are exposed to high temperatures or other stresses such as infection, inflammation, exposure to toxins, starvation and water deprivation, hypoxia, radiation exposure, and the like.

  Various heat shock proteins exist and are named according to their approximate molecular weight. Hsp60, Hsp70, Hsp90 and Hsp100 are different proteins, and their numbers reflect their approximate molecular weight (in kilodaltons). Major heat shock proteins are expressed at high levels in stressed cells, but are present at low to moderate levels in unstressed cells. As an example, Hsp70 is hardly detected at room temperature, but is highly expressed in heat shocked cells. In contrast, Hsp60 and Hsp90 are more highly expressed at standard conditions, and their expression is further induced by heat.

  Heat shock protein 70 (Hsp70) is a chaperone protein with an approximate weight of 70 kilodaltons and acts as a quality control mechanism that protects amino acids when they are aligned and become proteins. It appears to be upregulated in transformed cells, consistent with the trend that transformed cells show an increased metabolic rate compared to normal cells. Because they are clearly associated with cancer, heat shock proteins are of great interest as cancer prevention and treatment targets.

  In particular, some researchers believe that heat shock proteins may be involved in binding protein fragments from dead malignant cells and presenting them to the immune system. Heat shock proteins have been used as immunological adjuvants in enhancing the response to vaccines.

  Since such heat shock proteins are overexpressed in cancer cells and are thought to be important for cancer cell survival, heat shock protein inhibitors are also considered promising anticancer therapeutics Yes. Didelot, et al. “Anti-cancer therapeutic approaches based on intracellular and extracellular heat shock proteins”. See Curr. Med. Chem. 14 (27): 2839-47 (2007). See also Solit, et al. “Hsp90: a novel target for cancer therapy”. Curr Top Med Chem 6 (11): 1205-14 (2006).

  Multhoff, US Pat. No. 7,700,737 discloses anti-Hsp70 antibodies for use in diagnosing and treating primary and metastatic neoplastic diseases. A potential limitation of such therapies is that treating cancer patients with anti-Hsp70 monoclonal antibodies can sufficiently destroy cancer cell targets while damaging healthy cells in which Hsp70 activity is essential.

The disclosure of Srivastava U.S. Pat. Administration in combination with therapy (eg, tyrosine kinase inhibitors (imatinib mesylate, Gleevec ™)) is disclosed. In particular, Hsp preparations are said to contain “HSP-peptide complexes exhibiting antigenicity of infectious disease pathogens” (column 8, lines 31-33). The dose of HSP is stated to be in the range of 0.1 μg to 1000 μg per dose, and the preferred dose of Hsp70 is in the range of 10 to 600 μg per dose for intradermal administration. It is stated. (Column 50, lines 36-49)
The disclosure of Srivastava US Pat. No. 7,666,581 is also interesting and is directed to methods of treating infectious diseases and cancer by complexing antigenic proteins or peptides with heat shock proteins.

  In addition, Srivastava, US Pat. No. 8,029,808, administers a combination of a heat shock protein and other anti-cancer therapeutics (including complexes of heat shock proteins and such anti-cancer therapeutics). It is disclosed. The dose of HSP is stated to be in the range of 0.1 μg to 1000 μg per dose, and the preferred dose of Hsp70 is in the range of 10 to 600 μg per dose for intradermal administration. It is stated.

  Thus, there is still interest in alternative therapies that can reduce Hsp70 production in cancer cells and possibly induce selective killing of the population.

U.S. Patent 7,700,737 U.S. Patent No. 6,984,389 U.S. Patent 7,666,581 U.S. Pat.No. 8,029,808

Didelot, et al. “Anti-cancer therapeutic approaches based on intracellular and extracellular heat shock proteins”. Curr. Med. Chem. 14 (27): 2839-47 (2007) Solit, et al. “Hsp90: a novel target for cancer therapy”. Curr Top Med Chem 6 (11): 1205-14 (2006)

  The present invention relates to the discovery that administration of low doses of HSP70 is useful for the treatment of various cancers. While not wishing to be bound by any particular theory of the present invention, introducing a low level of HSP70 induces a negative feedback loop in the cell such that endogenous HSP70 production in the transformed cell is reduced. It is thought that it can be done. Such a reduction will make the cancer cells more susceptible to attack by the subject's own immune system and other cancer therapies.

  Specifically, the present invention provides a method of treating cancer in a subject in need of treatment, comprising administering an effective amount of heat shock protein 70 (Hsp70) to the subject. The subject can be any mammal, including a human, and the cancer is a brain tumor (meningioma, glioblastoma multiforme, anaplastic astrocytoma, cerebellar astrocytoma, other high grade Or low-grade astrocytoma, brainstem glioma, oligodendroglioma, mixed glioma, other glioma, neuroblastoma, craniopharyngioma, diencephalon glioma, germinoma, marrow Blastoma, ependymoma, choroid plexus tumor, pineal parenchymal tumor, ganglioglioma, neuroepithelial tumor, neuronal or mixed neuroglial tumor), lung tumor (small cell carcinoma, epidermoid carcinoma) , Adenocarcinoma, large cell carcinoma, carcinoid tumor, bronchial adenocarcinoma, mesothelioma, sarcoma or mixed tumor), prostate cancer (adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, prostate chamber carcinoma or carcinosarcoma) Breast cancer (including adenocarcinoma or carcinoid tumor), or gastric cancer, intestinal cancer or tuberculosis Cancer (including adenocarcinoma, invasive ductal carcinoma, invasive or invasive lobular carcinoma, medullary carcinoma, in situ ductal carcinoma, in situ lobular carcinoma, colloidal carcinoma or Paget disease of the nipple), skin cancer (Including melanoma, squamous cell carcinoma, tumor progression of human skin keratinocytes, basal cell carcinoma, perivascular cell carcinoma and Kaposi's sarcoma), lymphoma (including Hodgkin's disease and non-Hodgkin's lymphoma), sarcoma (osteosarcoma, chondrosarcoma) And including fibrosarcoma).

  Preferably, the Hsp70 is administered in the absence of a vaccine, and more preferably is not complexed with any other molecule.

  The method is particularly useful because Hsp70 is administered at a relatively low dose. According to one aspect of the present invention, the Hsp70 can be administered at a daily dose of 0.0003 microgram to 3 microgram / day, with a daily dose of 0.003 microgram to 0.3 microgram / day being particularly preferred. . Preferably, the dose is administered by administration once or more per day, and it is particularly preferred that the total of four doses is about 0.003 microgram / day.

  The Hsp70 can be administered by a variety of modes including a mode selected from the group consisting of sublingual, buccal, oral liquid, subcutaneous, intramuscular, intradermal or intravenous administration, although sublingual administration is particularly preferable.

FIG. 1 shows the transcription level of Hsp70 mRNA in cancer cell lines treated with Hsp70 protein.

  The present invention relates to the discovery that administration of low doses of HSP70 is useful for the treatment of various cancers. In some embodiments, the cancer is a brain tumor (meningiomas, glioblastoma multiforme, anaplastic astrocytoma, cerebellar astrocytoma, other high-grade or low-grade astrocytes , Brainstem glioma, oligodendroglioma, mixed glioma, other glioma, brain neuroblastoma, craniopharyngioma, diencephalon glioma, germinoma, medulloblastoma, ependymoma, choroid plexus Tumors, pineal parenchymal tumors, gliomas, neuroepithelial tumors, neuronal or mixed neuroglial tumors), lung tumors (small cell carcinoma, epidermoid carcinoma, adenocarcinoma, large cell carcinoma, Carcinoid tumor, bronchial gland tumor, mesothelioma, sarcoma or mixed tumor), prostate cancer (including adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, prostate chamber carcinoma or carcinosarcoma), breast cancer (adenocarcinoma) Or carcinoid tumors), or gastric cancer, intestinal cancer or colon cancer (adenocarcinoma, invasive gland) Carcinomas, including invasive or invasive lobular carcinomas, medullary carcinomas, in situ ductal carcinomas, in situ lobular carcinomas, colloidal carcinomas, or Paget disease of the nipple, skin cancers (melanoma, squamous cell carcinoma) , Including human skin keratinocyte tumor progression, including basal cell carcinoma, perivascular angioma and Kaposi's sarcoma), lymphoma (including Hodgkin's disease and non-Hodgkin's lymphoma), sarcoma (including osteosarcoma, chondrosarcoma and fibrosarcoma) Selected from the group of cancers.

  In the treatment of human and non-human animals with various malignancies, no adverse effects were found, and dogs and cats treated with HSP70 were found to live longer than historical controls. In addition, human patients experienced a reduction in cancer-related pain and showed improved stamina and vitality. The treatment also appeared to reduce the rate of disease progression in human subjects.

Example 1
In this example, two breast adenocarcinoma cell lines (MDA MB 231) and (MDA MB 231 T) cells were grown in DMEM 10% FBS and 24 hours with various doses of Hsp70 protein (Novus Fine Chemicals). Treated. A drop of PBS saline solution containing 0.0068 micrograms of Hsp70 was added to each well of a standard 96 well cell culture plate. In addition, the same experiment was performed with 0.1 and 10-fold concentrations of Hsp70.

  After 24 hours exposure, cells were harvested to determine endogenous Hsp70 RNA production. Cellular RNA was extracted with Trizol and RNA quality and quantity were measured with a Nano-drop spectrophotometer (Thermo-Fisher Scientific, Wilmington, DE). To determine the level of Hsp70 mRNA, RT-PCR (real-time PCR) and qPCR were performed according to the manufacturer's protocol (Quigen). The results shown in FIG. 1 show that Hsp70 treatment resulted in a 40-60% reduction in endogenous Hsp70 transcription in treated breast cancer cells. Without being bound by the theory of the present invention, the presence of exogenous Hsp70 is believed to serve to down-regulate endogenous Hsp70 production by the tumor. Since Hsp70 is a protein required for tumor cells, it induces remission.

Example 2
In this example, a 33-year-old man was diagnosed with stage 4 glioblastoma multiforme after a stroke and treated according to the present invention. Following initial diagnosis of the subject, continuous MRI showed a rapidly progressing tumor. Two rounds of surgery and each round of chemotherapy and radiation therapy were unsuccessful to eliminate the cancer. Eight months after the initial diagnosis, the patient was admitted to the hospital as rapid deterioration progressed over 3 to 4 hours, during which time the patient's motor skills, speech ability, and ultimately consciousness were lost. The patient was classified as “coma” upon admission. After several aggressive interventions, the patient was discharged 4 days later, but was cognitively impaired and both gross and fine motor functions were impaired.

  Within 2 days of discharge, patients were treated by sublingual administration of a drop of PBS saline solution containing Hsp70 at a concentration of 0.0068 microgram per drop 4 times a day. Other than discontinuing the antitumor agent (Avastin), no other changes were made to the treatment plan.

  There was significant progress shortly after the start of Hsp70 treatment. Cognitive ability, speaking ability and energy improved, followed by fine motor control (we were able to tie shoelaces and insert contact lenses for the first time in several months), balance and endurance. At 5 weeks, the patient was able to mow the lawn, clean the house and walk alone for more than a mile. MRI taken 2 months after discharge showed no change in tumor size.

Example 3
In this example, peripheral blood mononuclear cells (PBMC) were treated for 24 hours with various doses of Hsp70 (0.1-fold, 1-fold or 10-fold). After extracting total mRNA, the gene expression of PBMC was determined using a Cancer Pathway Finder qPCR array compared to an untreated control. As a result, it was shown that the expression of ARNT and urokinase plasminogen activator SERPINB2 was down-regulated in PBMC treated with Hsp70 compared to the untreated control. It is known in the art that ARNT and / or SERPINB2 expression is associated with tumor growth and invasiveness. Thus, this data supports that Hsp70 is useful for treating cancer in a subject by downregulating the expression of at least two tumor expression genes (eg, ARNT and SERPINB2).

  One of ordinary skill in the art having access to the teachings herein will envision many variations and modifications to the embodiments of the invention described above. Accordingly, only such limitations as set forth in the appended claims shall take precedence.

Claims (11)

  A method of treating cancer in a subject in need of treatment, comprising administering to said subject an effective amount of heat shock protein 70 (Hsp70).   2. The method of claim 1, wherein the subject is a human.   The cancer is a brain tumor (meningioma, glioblastoma multiforme, anaplastic astrocytoma, cerebellar astrocytoma, other high-grade or low-grade astrocytoma, brain stem glioma, Oligodendroglioma, mixed glioma, other glioma, brain neuroblastoma, craniopharyngioma, diencephalon glioma, germinoma, medulloblastoma, ependymoma, choroid plexus tumor, pineal parenchymal tumor , Gangliomas, neuroepithelial tumors, including neuronal or mixed neuroglial tumors), lung tumors (small cell carcinoma, epidermoid carcinoma, adenocarcinoma, large cell carcinoma, carcinoid tumor, bronchial gland tumor, Including mesothelioma, sarcoma or mixed tumor), prostate cancer (including adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, carcinoma of the prostate chamber or carcinoid tumor), breast cancer (including adenocarcinoma or carcinoid tumor), Or stomach cancer, intestinal cancer or colon cancer (adenocarcinoma, invasive ductal carcinoma, invasive lobular carcinoma) Or invasive lobular carcinoma, medullary carcinoma, in situ ductal carcinoma, in situ lobular carcinoma, colloid carcinoma or papillary Paget's disease), skin cancer (melanoma, squamous cell carcinoma, human skin keratinocyte Selected from the group of cancers including tumor progression, basal cell carcinoma, perivascular cell carcinoma and Kaposi's sarcoma), lymphoma (including Hodgkin's disease and non-Hodgkin's lymphoma), sarcoma (including osteosarcoma, chondrosarcoma and fibrosarcoma) The method of claim 1, wherein:   2. The method of claim 1, wherein the cancer is a brain tumor.   2. The method of claim 1, wherein the Hsp70 is administered at a dose of 0.0003 micrograms to 3 micrograms / day. 2. The method of claim 1, wherein the Hsp70 is administered at a dosage of 0.003 microgram to 0.3 microgram / day.   2. The method of claim 1, wherein the Hsp70 is administered by a mode selected from the group consisting of sublingual, buccal, oral liquid, subcutaneous, intramuscular, intradermal or intravenous.   2. The method of claim 1, wherein the Hsp70 is administered sublingually.   2. The method of claim 1, wherein the Hsp70 is administered in the absence of other cancer therapeutics.   2. The method of claim 1, wherein the Hsp70 is administered in the absence of a vaccine.   2. The method of claim 1, wherein the Hsp70 is not complexed with any other molecule.

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