WO2018144094A1 - Methods for improving tumor chemotherapy - Google Patents

Abstract

Provided are methods of enhancing the efficacy an anti-tumor agents by administering to an individual who has a solid tumor a vasoconstrictor, and subsequently intravenously administering an anti-tumor agent. Performing the method results in an increase in blood flow in the tumor, or a reduction in the volume of the tumor, or an increase in open blood vessels in the tumor, or an increase in survival time of the individual who is treated with the vasoconstrictor and the chemotherapeutic agent, or a combination thereof. Kits that contain distinct formulations are provided. The kit includes a first formulation that is suitable for intravenous administration and contains a vasoconstrictor agent, and a second formulation that is suitable for intravenous administration and contains an anti-tumor agent.

Description

METHODS FOR IMPROVING TUMOR CHEMOTHERAPY

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent application no. 62/416,965, filed on November 3, 2016, the disclosure of which is incorporated herein by reference. FIELD

This disclosure relates generally to enhancing the efficacy of anti-tumor agents by increasing intra-tumoral blood flow.

BACKGROUND

All current cancer therapies including chemotherapy, radiation, and immunotherapy depend on the tumor vasculature as the main access-point to the tumor microenvironment. Applications of intravital microscopy (IVM) in the preclinical setting have revealed key biological processes in cancer therapy including angiogenesis, vascular normalization to therapeutics, and immune cell trafficking. These quantifiable microscopic observations combined with standard pathologic examination of surgically-excised patient tumors are the gold-standard for extrapolating how the vasculature influences tumor biology in humans. A recent report describes the first-in-human IVM platform and successful completion of a feasibility trial in melanoma patients that provided an unprecedented view of tumor vascular function (Fisher, D. T., Muhitch, J. B., Kim, M., Doyen, K. C, Bogner, P. N., Evans, S. S., & Skitzki, J. J. (2016). Intraoperative intravital microscopy permits the study of human tumour vessels. Nature Communications. 2016;7: 10684; doi: 10.1038/ncommsl0684, the disclosure of which is incorporated herein by reference). The results corroborate certain expected tumor vessel characteristics, but also yielded unanticipated findings. In agreement with prior preclinical observations, -50% of blood vessels in melanoma patient tumors appeared patent but exhibited no detectable blood flow. These findings support the prevailing paradigm that vessel dysfunction is a major hurdle to effective delivery of drug and immune therapeutics to tumors. There is accordingly an ongoing and unmet need to address vessel dysfunction in connection with administration of pharmaceutical agents, and particularly anti-cancer agents. The present disclosure is pertinent to this need. SUMMARY

The present disclosure provides methods and kits for improving treatment of solid tumors. The disclosure is based in part on the unexpected discovery that administering a vasoconstriction agent promotes increased blood flow in a solid tumor, in part by improving blood flow through existing vessels in the tumor.

In an aspect the disclosure provides a method of enhancing the efficacy of an antitumor agent by intravenously administering to an individual who has a solid tumor a vasoconstrictor, and subsequently intravenously administering the anti-tumor agent. In certain non-limiting implementations the vasoconstrictor comprises a sympathomimetic drug. In embodiments, the method comprises administering the anti-tumor agent within a period of from 1-10 minutes after administration of the vasoconstrictor. In certain approaches, performing a method of the disclosure results in an increase in blood flow in the tumor, or a reduction in the volume of the tumor, or an increase in open blood vessels in the tumor, or an increase in survival time of the individual who is treated with the vasoconstrictor and the chemotherapeutic agent, or a combination thereof.

In an embodiment, the disclosure provides a kit comprising distinct formulations, wherein a first formulation is suitable for intravenous administration and comprises a vasoconstrictor agent, and a second formulation that is suitable for intravenous administration and comprises an anti-tumor agent.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 provides a protocol flow chart for dynamic control of tumor vessels with saline bolus and phenylephrine in B 16-tumor bearing mice.

Figure 2A shows an implanted window chamber for intravital microscopy (IVM) observation.

Figure 2B shows representative examples of aberrant characteristics of tumor vessels.

Figure 2C is a graph showing average tumor vessel diameters following treatment over a given IVM observation field in an individual B 16-bearing mouse.

Figure 2D is a graph showing average tumor flow velocities within same tumor vessels following treatment.

Figure 2E shows frames from IVM observation of murine tumor vasculature following intravenous injection of bolus saline and phenylephrine as outlined in Figure 1. Figure 3 A shows images of fluorescent staining of DNA adducts treated with melphalan alone.

Figure 3B shows images of fluorescent staining of DNA adducts treated with melphalan plus phenylephrine stained for DNA adducts.

Figure 4A shows individual tumor volume curves among the treatment groups.

Figure 4B shows mean tumor volume curves among treatment groups.

Figure 4C shows Kaplan-Meier survival curve showing statistically superior survival for the combination group, even when compared to the melphalan alone group.

DETAILED DESCRIPTION

Unless defined otherwise herein, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.

Tumor vasculature has been an active area of investigation for many decades. As tumor vasculature is immature and generally lacks pericytes or smooth muscle components, the present disclosure relates to influencing the systemic circulation which is sensitive to manipulation, and thereby generate changes in tumor vascular characteristics that would improve access of chemotherapeutic agents to intra-tumor vasculature. Thus, the disclosure provides a counterintuitive approach whereby the administration of vasoconstrictors instead opens up tumor vessels and/ or affects vessel diameters. It is believed this effect is achieved by vasoconstriction of the draining venous tributaries downstream from the tumor vessels. In connection with this, and without intending to be constrained by any particular mechanism or theory, it is demonstrated herein that in combination with a fluid bolus, vasoconstriction of tumor draining vessels generates enough resistance to overcome tumor interstitial pressure and the bizarre hemodynamics associated with the tumor microcirculation. This is demonstrated in preclinical animal models examining tumor vasculature in real-time. A representative approach of this disclosure is depicted in Figure 1, using dynamic control of tumor vessels with saline bolus and phenylephrine in B 16-bearing mice, which are also used in non-limiting examples of the invention as described further below.

In an embodiment the disclosure comprises administering a vasoconstrictor and an anti-tumor agent to an individual who has a tumor. Preferably the vasoconstrictor is administered prior to the anti-tumor agent, but concurrent administration is also contemplated. Kits and compositions comprising a vasoconstrictor and an anti-tumor agent are also included.

A "vasoconstrictor" is an agent that produces constriction of at least some blood vessels in an individual to whom the vasoconstrictor is administered. A vasoconstrictor will therefore generally cause an increase in arterial pressure. It is expected that any

vasoconstrictor can be used in methods of this disclosure, but sympathomimetic

vasoconstrictors are preferred. Without intending to be bound by any particular mechanism it is considered that sympathomimetic drugs typically have alpha-adrenoceptor agonist (a- agonist) properties and bind to ai-adrenoceptors on vascular smooth muscle to promote smooth muscle contraction. Specific and non-limiting examples of suitable sympathomimetic drugs for use in methods of this disclosure include phenylephrine, methoxamine,

oxymetazoline, tetrahydralazine, and xylometazoline. In certain aspects the sympathomimetic vasoconstrictor is the only vasoconstrictor administered to the individual. While in certain approaches non-sympathomimetic vasoconstrictors, such as vasopressin and its analogs can be used, the disclosure includes the proviso that in certain embodiments, the vasoconstrictor does not include a non-sympathomimetic vasoconstrictor, and/or does not include

vasopressin or any vasopressin analogs.

Vasoconstrictor compositions of the invention can be administered in any suitable form using any suitable route, including but not necessarily limited to oral and intravenous administrations. In one approach the vasoconstrictor is administered intravenously, and the administration may be in combination or in sequence with administration of a fluid bolus. In embodiments the fluid bolus is administered first, followed by the vasoconstrictor, which may then be followed by administration of the chemotherapeutic agent. In certain approaches the disclosure comprises administration a bolus of an aqueous solution, such as normal saline. The fluid bolus can have a volume that is adapted given such factors as the size of individual being treated, the cancer type, size, volume and number of tumor(s), and other factors that will be apparent by those skilled in the art given the benefit of this disclosure. In certain embodiments the fluid bolus comprises from 10ml - 1000ml fluid. In embodiments the bolus comprises about 500ml of fluid. Administration of the fluid bolus can be combined with, or followed by administration of a vasoconstrictor. In embodiments the vasoconstrictor is administered within 10 seconds to 30 minutes of the administration of the fluid bolus. In embodiments the vasoconstrictor is administered within 30 seconds to 5 minutes of the administration of the fluid bolus. In embodiments the vasoconstrictor is administered within about 1 minute of the administration of the fluid bolus.

As with the fluid bolus, dosage of the vasoconstrictor can also be determined based on known factors, such as the size of individual being treated, the cancer type, size, volume and number of tumor(s), and other factors that will be apparent by those skilled in the art given the benefit of this disclosure. In certain embodiments the dosage of the vasoconstrictor can be similar to dosages when the vasoconstrictor is used for a known pharmaceutical purpose, such as to treat sinus pressure, allergic symptoms, etc. In certain non-limiting embodiments the vasoconstrictor is administered i.v. as a bolus comprising from 1-1000 μg of, for example, phenylephrine. In certain approaches the vasoconstrictor is administered i.v. in an amount from 10-500μg. In one embodiment the vasoconstrictor is administered as an approximately 100 μg bolus of phenylephrine i.v., and is administered within about one minute after the administration of the fluid bolus.

In embodiments the disclosure comprises selecting an individual who has been diagnosed with a tumor (which may be a benign or malignant tumor) and based at least in part on the tumor diagnosis, administering sequentially a vasoconstrictor and an anti-tumor agent to the individual. The fluid bolus may also be administered as described above. In certain embodiments administration of the vasoconstrictor is only prescribed because of the presence of the tumor. Stated differently, the individual is not prescribed a vasoconstrictor for any purpose other than as an adjunct to anti-tumor therapy, and has not taken any

vasoconstrictor within a period of time prior to the vasoconstrictor that is administered for the purposes of enhancing the effect of the anti-tumor agent.

The type of cancer that can be treated using approaches of this disclosure is not particularly limited, so long as the individual has a solid tumor. In certain non-limiting examples the individual has been diagnosed with a cancer and a tumor, wherein the cancer is selected from breast cancer, prostate cancer, pancreatic cancer, lung cancer, liver cancer, ovarian cancer, cervical cancer, colon cancer, esophageal cancer, stomach cancer, bladder cancer, brain cancer, testicular cancer, melanoma, fibrosarcoma, angiosarcoma,

adenocarcinoma, rhabdomyosarcoma, and any other form of malignant tumor. In

embodiments the individual is diagnosed with a non-malignant tumor such as Benign

Prostatic Hyperplasia (BPH) and the therapeutic agent is selected accordingly.

The type of cancer anti-tumor agent is not particularly limited so long as it is intended and/or it would be desirable for the agent to access intra-tumoral vessels. In certain embodiments the anti-tumor agent is selected from a biologic agent, such as a therapeutic monoclonal antibody or antigen binding fragment thereof, a polypeptide or peptide, or is a metal-based chemotherapeutic agent such as any platinum-based antineoplastic drug, or is a small molecule chemotherapeutic agent. In embodiments, the anti-cancer agent is any chemotherapeutic agent. In embodiments, the anti-tumor agent is an alkylating agent, an antimetabolite, a natural product such as a plant product, including but not limited to Vinca Alkaloids such as Vincristine, Vinblastine, Taxanes such as Paclitaxel and Docetaxel, or an Etoposide, or a Camptothecin, or an antibiotic, such as Doxorubicin and Bleomycin, or a hormone or another receptor binding agent, or an enzyme inhibitor. In embodiments, the antitumor agent is one that is known to prevent or inhibit the maturation and/or proliferation of neoplasms. The agent may be targeted to the tumor or it may be non-specific. A list of chemotherapeutic agents can be found at, for example, www.cancer.gov/about- cancer/treatment/drugs, from which the list of chemotherapeutic agents is incorporated herein by reference.

The chemotherapeutic agent in various embodiments is administered subsequent to the vasoconstrictor / aqueous solution bolus. In embodiments the chemotherapeutic agent is administered after a period of time sufficient for the administration of vasoconstrictor to at least initiate an increase of blood flow in blood vessels in a tumor, relative to the blood flow in the blood vessels prior to the administration of the vasoconstrictor, and wherein the increase in blood flow remains present when the chemotherapeutic agent is administered. The period of time that is sufficient for the vasoconstrictor to initiate an increase of blood flow may range based on various factors, such as the type, size, and location and number of tumors. In certain embodiments, the chemotherapeutic agent is administered after a period of from 30 seconds to several hours after the administration of the vasoconstrictor. In embodiments the chemotherapeutic agent is administered after a period of from 1-60 minutes after the administration of the vasoconstrictor. In an embodiment the chemotherapeutic agent is administered within 10 minutes of the administration of the vasoconstrictor. In

embodiments, the chemotherapeutic agent is administered within 5-10, 6-10, 7-10, 8-10, or 9- 10 minutes after the administration of the vasoconstrictor. In embodiments, at least one bolus of saline or another physiologically acceptable aqueous solution is administered, followed by administration of at least one dose of a vasoconstrictor, followed by administration of the chemotherapeutic agent, wherein all of said administrations are performed intravenously. In embodiments, only one saline or similar solution is intravenously administered, and is followed by only one or only two intravenous vasoconstrictor administrations, followed by intravenous administration of the chemotherapeutic agent which can be performed only once, or more than once.

In embodiments, subsequent to the administration of the vasoconstrictor there occurs an increase in blood flow in blood vessels in a tumor of from 1% - 100% relative to the blood flow prior to the administration of the vasoconstrictor. The increase in blood flow can be due to opening of blood vessels in the tumor, and/or by increasing blood flow through open blood vessels in the tumor. Thus, the disclosure includes promoting blood flow in blood vessels in a tumor wherein previous to the administration of the vasoconstrictor there was no blood flow, no detectable blood flow, or less blood flow relative to a control, such as another vessel in the tumor, or relative to blood flow in a normal vessel of similar characteristics, i.e., diameter and bodily location. Blood flow can be determined using any of a variety of techniques know to those skilled in the art, and can be compared to a control, such as a baseline blood flow determination value. Likewise, any other benefit that is correlated with performing a method of this disclosure can be compared to any suitable control, such benefits including but not necessarily limited to changes in tumor volume, and survival time of an individual who is treated according to this disclosure. In an embodiment, the method provides for increased survival time of an individual who is treated with the chemotherapeutic agent but without the vasoconstrictor, or a value determined or inferred from such treatments.

Approaches of this disclosure can be combined with any other medical intervention that would be appropriate for the tumor treatment, including but not necessarily limited to radiation and surgical interventions.

The method of the disclosure can be repeated over a period of time, including but not necessarily limited to a portion of, or the entire time the individual is being treated with the anti-tumor agent.

As illustrated by the examples and figures of this disclosure, in embodiments performance of a method described herein results in one or a combination of: a reduction in tumor volume, an increase in open blood vessels in a tumor and/or an increase in the total blood following through a tumor, or an increased survival time for an individual who has a tumor and is treated according to a method of this disclosure.

In an embodiment the disclosure comprises a kit comprising a chemotherapeutic agent and a vasoconstrictor. In embodiments, the chemotherapeutic agent and the vasoconstrictor are provided in separate formulations suitable for intravenous administration. In

embodiments, the kit provides a separate formulation of an aqueous solution. In

embodiments, the kit contains printed instructions instructing the user to administer the vasoconstrictor and subsequently the chemotherapeutic agent with a specified period of time as described herein.

The following examples are intended to illustrate but not limit the invention.

Examples

Aspects of this disclosure are illustrated in Figures 2A, 2B, 2C and 2D. In particular, Figure 2A shows a photograph of an implanted window chamber for intravital microscopy (IVM) observation which is used to obtain data described here. In this example, 50,000 B16 melanoma cells per 50 μΐ aliquot was inoculated within the dorsal skin fold of B6 mice. Tumors began forming within 5-7 days, and the tumor vasculature is directly observed through the window chamber. Figure 2B shows representative examples of aberrant characteristics of tumor vessels. White arrows highlight nonfunctional vessels within B16- bearing mice. The white arrowhead highlights a tortuous, abnormal tumor vessel branching from a streamlined, normal appearing vessel. Figure 2C provides a graph showing average tumor vessel diameters following treatment over a given IVM observation field in an individual B16-bearing mouse. While the average diameter increased following the saline bolus, there was no consistent pattern in vessel diameter following treatment with

phenylephrine (either first or second dose). Figure 2D provides a graph showing average tumor flow velocities within same tumor vessels following treatment. Contrary to vessel diameter measurements, consistent and reproducible results were obtained whereby velocity increased following the saline bolus and markedly decreased or even reversed following phenylephrine treatment. However, Figure 2E provides image frames from IVM analysis of tumor vasculature following injection of bolus saline and phenylephrine. The number of patent vessels and blood flow rate increased within 10 minutes of intervention (*= functional vessels). Bar is 100 μπι. Thus, relative to the controls it is apparent that the administration of phenylephrine results in blood flow through vessels through which blood flow was low or was not detectable prior to the phenylephrine administration. Accordingly, this example demonstrates that a vasoconstrictor paradoxically improves blood flow through intra-tumoral blood vessels. The methods of this disclosure are therefore suitable to provide greater access to the intra-tumoral environment for anti-tumor agents that are present in the new blood flow, thereby enhancing efficacy of the anti-tumor agents against the tumor, which is supported by the data in Figure 3 and Figure 4. Figure 3 provides images showing fluorescent staining of DNA adducts. In order to obtain the images, tumors were removed 2 days after treatment perfusion. Figure 3 A shows sections treated with melphalan alone, while Figure 3B shows sections treated with melphalan plus phenylephrine stained for DNA adducts with the MP5/73 fluorescent antibody. There was a significant increase in the number of observed DNA adducts with the combination treatment. The average number of observed DNA adducts per 20x field was 30.3 versus 120.5 for mice treated with melphalan alone versus melphalan plus phenylephrine, respectively (Student's t test p = 0.029).

Figure 4A provides individual tumor volume curves among treatment groups: vehicle (saline control), n = 4; phenylephrine alone, n = 10; melphalan alone, n = 12; and

phenylephrine plus melphalan (combination), n = 12. The combination group had the most number of mice (10/12) with tumors under 50 cubic mm at day 21, with the exception of two outliers. Figure 2B provides mean tumor volume curves among treatment groups. Statistical comparisons are shown to the right of the curves. Average volume between the combination group and the melphalan alone group was not significant, likely due to the presence of the two outliers in the combination group. Figure 3C provides Kaplan-Meier survival curves showing statistically superior survival for the combination group, even when compared to the melphalan alone group. There were 3 complete responses (25%) in the combination group, and no complete responses in any other group. Data were from 3 pooled experiments.

It will be apparent from the foregoing description and figures that the present disclosure provides an unexpected and paradoxical demonstration that administration of a vasoconstrictor as an adjunct to chemotherapy improves not only access of the

chemotherapeutic agent to the tumor microenvironment by way of newly opened blood vessels, it also improves survival.

While the invention has been described through specific embodiments, routine modifications will be apparent to those skilled in the art and such modifications are intended to be within the scope of the present invention.

Claims

What is claimed is:

1. A method of enhancing efficacy of an anti-tumor agent comprising intravenously administering to an individual who has a tumor a vasoconstrictor, and subsequently intravenously administering the anti-tumor agent.

2. The method of claim 1, further comprising administering a bolus of an aqueous solution to the individual prior to administering the vasoconstrictor.

3. The method of claim 1, wherein the vasoconstrictor comprises a sympathomimetic drug.

4. The method of any one of claims 1-3, wherein the anti-tumor agent is administered within a period of from 1-10 minutes subsequent to the administration of the vasoconstrictor.

5. The method of claim 4, wherein subsequent to the administration of the vasoconstrictor and the anti-tumor agent, and optionally the bolus of the aqueous solution, there occurs in the individual at least one of an increase in blood flow in the tumor, a reduction in the volume of the tumor, an increase in open blood vessels in the tumor, or an increase in survival time of the individual.

6. The method of claim 5, wherein the reduction in the tumor volume occurs.

7. The method of claim 5, wherein the increase in blood flow in the tumor occurs.

8. The method of claim 5, wherein the increase in open blood vessels in the tumor occurs.

9. The method of claim 5, wherein the increase in the survival time occurs.

10. A kit comprising distinct formulations, wherein a first formulation is suitable for intravenous administration and comprises a vasoconstrictor agent, and a second formulation that is suitable for intravenous administration and comprises an anti-tumor agent.