Ultrasound sensitization

Abstract

To provide an improved method for MRI-guided ultrasound-enhanced focused ultrasound treatment of malignant tissue.SOLUTION: Disclosed herein is a method of selectively inducing direct cytotoxic effects in malignant tissue of a subject, comprising: a) providing an effective amount of 5-aminolevulinic acid, or pharmaceutically acceptable salts or esters thereof, to the malignant tissue; and b) exposing the tissue to ultrasound energy ("sonicating") using a focusedultrasounddevice at a frequency of from about 0. 1MHz to about 3MHz and at an intensity of ultrasound beam focusing of from about 3W / cm2 to about 100W / cm2.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Field The disclosure provided herein relates generally to medical procedures. More particularly, the disclosure relates to medical procedures using ultrasound and ultrasound sensitizers. [Background technology]

[0002] background Hematoporphyrin and its derivatives were described in 1942 as preferentially accumulating in tumor cells (H. Auler et al., Z. Krebsforsch. (1942) 53:65-68). The fluorescent properties of the compounds are used to identify tumor tissue as surgical aids and diagnostics (R. Vanseviciute et al., Medicina (2014) 50:137-43; J. Zhang et al., Acta Pharm Sinica B (2018) 8:137-46). In 1972, it was demonstrated that hematoporphyrin could be used to selectively sensitize tumor cells to light in the presence of oxygen (the "photodynamic effect"), leading to tumor size reduction, a procedure now known as photodynamic therapy (I. Diamond et al., Lancet (1972) 2:1175-77). In most organisms, hematoporphyrin is converted to heme and related molecules. Hematoporphyrin is thought to accumulate in certain tumor tissues due to iron deficiency or dysfunction of metabolic enzymes (W. Song et al., Anticancer Res (2011) 31:39-46; C.J. Gomer et al., Cancer Res (1979) 39:146-51). This allows for the selective destruction of tumor cells using light, leaving healthy adjacent tissue relatively unaffected. It has been found that when exogenous 5-aminolevulinic acid ("5-ALA") is administered, the photosensitizing porphyrin, protoporphyrin IX, the first molecule involved in the heme pathway, accumulates, enabling tissue photosensitization within one hour (C. Perotti et al., Br J Cancer (2004) 90:1660-65). However, photodynamic therapy is limited by tissue opacity: effectively treating tumors that are not accessible from the body surface or lumen requires surgical access to the tumor site.

[0003] Sonodynamic therapy (SDT) is a method of cell destruction using focused ultrasound (FUS) after sensitization with an ultrasound sensitizer. The mechanism of action has not been conclusively determined but is thought to result from singlet oxygen produced by thermal effects and / or cavitation. Ultrasound can penetrate tissue much farther than light, making noninvasive treatments available to more of the body. Surprisingly, protoporphyrin IX was also discovered to be an effective ultrasound sensitizer (N. Yumita et al., Jpn J Cancer Res (1989) 80(3):219-22), enabling ultrasound-mediated cell destruction under otherwise ineffective conditions. While the mechanism of action is unclear, it has been hypothesized to result from singlet oxygen formation.

[0004] To date, there have been many reported in vitro and in vivo experiments, but no clinical trial results (H. Hirschberg et al., Ther Deliv (2017) 8:331-42). For example, N. Yumita et al. (ibid.) reported that hematoporphyrin (10, 25, or 50 μg / mL) and 1.27, 2.21, or 3.18 W / cm for durations of 15, 30, or 60 seconds were used to treat rheumatoid arthritis. 2 investigated the effects of SDT on mouse sarcoma 180 or rat ascites hepatoma 130 cells in vitro using ultrasound (1.92 MHz) at an intensity of 1.5 W / cm. Hematoporphyrin was applied to the cells 15, 30, or 60 seconds before ultrasound application. Yumita reported that 60 seconds of ultrasound alone damaged a significant number of cells (16% and 17% for sarcoma 180 and ascites hepatoma, respectively) as measured by trypan blue dye exclusion. Higher intensities damaged more cells (2.21 W / cm). 2 : 71% and 75%; 3.18W / cm 2 79% and 86%). When hematoporphyrin was added (50 μg / mL), the 2 or 3.18 W / cm 2 Although the radiation damaged substantially more sarcoma cells (67% and 98%) after exposure to 2.21 and 3.18 W / cm2 At 3.18 W / cm, more AH cells were damaged (95% and 96%). Statistically significant cell damage was also observed at 3.18 W / cm. 2 The use of 25 μg / mL hematoporphyrin with an ultrasound intensity of 10 μg / mL has also been reported (98% and 96%), which was equivalent to cell destruction using 50 μg / mL.

[0005] N. Yumita et al., Cancer Sci (2004) 95:765-69, reported that porfimer sodium (0, 0.5, 1.0, 2.5, or 5 mg / kg, iv 24 hours before ultrasound) was administered with 1, 2, 3, or 5 W / cm 2 reported the treatment of female Sprague Dawley rats bearing mammary tumors (induced by injection of 7,12-dimethylbenz(α)anthracene) with 1.92 MHz ultrasound at an intensity of 1.5 W / cm for 15 minutes. 2 SDT treatment with 1000 W / cm or higher effectively inhibited tumor growth, and higher treatment levels (3 W / cm or higher) 2 5mg / kg or 5W / cm 2 2.5mg / kg) is 3W / cm 2 reported that there was no significant difference between treatment with 2.5 mg / kg and treatment with 2.5 mg / kg.

[0006] W. Song et al. (cited above) reported that the 2Song investigated the response of SAS cells in vitro using 5-ALA (1, 10, or 50 μg / mL) applied 4 hours before 1.05 MHz ultrasound with an intensity of 100 Hz, a pulse repetition frequency of 100 Hz, and a duty cycle of 60% for a duration of 2 minutes. Song reported cell viabilities of 89%, 88%, 75%, and 62% (for 0, 1, 10, and 50 μg / mL 5-ALA); the decrease in viability was statistically significant at 10 and 50 μg / mL. Song also reported that treatment with ultrasound enhanced SAS cell apoptosis, intracellular reactive oxygen species, and lipid peroxidation, and that the addition of 10 μg / mL 5-ALA significantly enhanced these effects.

[0007] S. Suehiro et al., J Neurosurg (2018) 129:1416-28, used a 20% duty cycle for 3 min, 5-ALA (1 mM) and 2 W / cm 2 Ultrasound (3MHz) was used to measure U87 and U251 glioma cells, and U251 Oct-3 / 4 Suehiro reported an in vitro study using glioma stem-like cells, U87 and U251, using ultrasound alone. Oct-3 / 4 reported that ultrasound destroyed tumor cells to a significant extent, but not U251 cells. Ultrasound in combination with 5-ALA (1 mM) was substantially cytotoxic to all tumor cells tested (but not to normal control cells), and was more effective than ultrasound alone in all three cases. Suehiro also reported the combination of 5-ALA (100 mg / kg) and ultrasound (2.2 MHz, 0.5 or 2.0 W / cm), repeated weekly for 3 weeks. 2 Human U87 glioma cells or U251 treated with 1000kJ / cm2 PBS (20% duty cycle for 3 min). Oct-3 / 4 They also used immunodeficient BALB / c nude mice injected with glioma stem-like cells. Suehiro reported that SDT treatment significantly improved survival of the treated mice.

[0008] JY Kou et al., Cell Death Dis (2017) 8:e2558 was administered at an intensity of 0.4 W / cm for a duration of 10 min. 2 reported an in vitro study on human THP-1 monocytes (induced to become macrophages and foam cells) exposed to ultrasound (1.0 MHz) at 30 μg / mL using berberine as an ultrasound sensitizer (other concentrations, intensities, and durations were also tried, but no combinations were). Kou reported that the treatment induced autophagy in macrophages and increased cholesterol efflux.

[0009] M. Nonaka et al., Anticancer Res (2009) 29:943-50 was injected into Wistar rats, and then treated with rose bengal (50 mg / kg, i.v. 10 min before treatment) and ultrasound (25 W / cm 2 C6 rat glioma cells were treated with a 1 MHz transducer (at 1000 kJ / s for 5 min, using a transducer applied directly to the brain surface through a 10 mm craniotomy). Control groups lacking glioma cells were also treated with 0, 10, or 50 mg / kg rose bengal and 25 and 110 W / cm. 2 Nonaka showed that rats with normal brain tissue were exposed to ultrasound at 25W / cm with or without rose bengal. 2 reported that no lesions were observed at 110W / cm 2 Rats with normal brain tissue treated with ultrasound alone for 3 minutes at 110 W / cm showed coagulative necrosis in 2 of 6 rats and in 5 of 6 rats treated for 5 minutes at that intensity. 2 All rats treated with 3 minutes of ultrasound at 110 W / cm showed lesions (5 / 5 for each group). 2 Rats treated with 5 minutes of ultrasound at 10 mg / kg also showed lesions (6 / 7 at 10 mg / kg; 6 / 6 at 50 mg / kg).

[0010] T. Ohmura et al., Anticancer Res (2011) 31:25274-33 were injected into Wistar rats, and 5-ALA (100 mg / kg, orally administered 3 hours before ultrasound treatment) and 10 W / cm using an ultrasound transducer applied directly to the brain surface through a 10 mm craniotomy. 2 C6 rat glioma cells were treated with 1.04 MHz ultrasound for 5 minutes at 10, 15, 20 or 25 W / cm. Rats with normal brain tissue were also treated with 1.04 MHz ultrasound at 10, 15, 20 or 25 W / cm. 2 The patients were also treated with 5 minutes of ultrasound at 15, 20 or 25 W / cm. 2 reported that rats with normal brain tissue that received ultrasound showed necrosis in the ultrasound focal area. Rats with glioma cells treated with SDT showed significantly smaller tumors than rats that received only 5-ALA or ultrasound.

[0011] EJ Jeong et al., Ultrasound Med Biol (2012) 38:2143-50, 5-ALA (60 mg / kg) or Radachlorin (40 mg / kg); and 1.0 MHz, 2.65 W / cm 2 used Sprague Dawley rats inoculated with C6 glioma cells that were treated with 20 minutes of ultrasound (16 minutes of ultrasound, followed by a 3-minute break, followed by another 4 minutes of ultrasound) at 20°C. Jeong reported that rats that underwent SDT exhibited tumors that were significantly smaller in size than the control group.

[0012] Y Li et al., PLoS One (2015) 10:e0132074, treated with 5-ALA (250 mg / kg iv, 8 hours before ultrasound) and 8-minute pulse repetition at 100 Hz, 10% duty time ratio, 1.0 MHz, 2.5 W / cm daily for 10 days. 2Li used rat osteosarcoma UMR-106 cells in BALB / c nude mice treated daily with ultrasound at 100 rpm for 24 hours. He reported that mice receiving ultrasound alone exhibited smaller tumor volumes than mice receiving control or 5-ALA alone, and that mice receiving SDT exhibited tumor volumes that were significantly smaller than all other groups.

[0013] Further advances in sonodynamic therapy have often focused on improving sensitizers, increasing selectivity for malignant tissue, systemic distribution, or sensitivity to ultrasound. Woodburn et al., US20010002251, disclosed the use of texaphyrins as SDT sensitizers. Sanderson et al. disclosed the use of tetrasulfamoyl phthalocyanine and naphthalocyanine derivatives (GB2343186) and tetrasulfamoyl phthalocyanine and naphthalocyanine (GB2343186) as dyes to indicate diseased tissue and as sensitizers for SDT or PDT. See also Alfheim et al., US6498945; Lawandy, US5817048; Iger, WO1998 / 052610; Lewis et al., US20090275548A1; and Wang et al., US20190070296A1. However, to date, sonodynamic therapy has not been approved by any regulatory agency for use in humans. Summary of the Invention

[0014] Quick Overview A safe and effective sonodynamic therapy is provided herein.

[0015] One embodiment is a method of selectively inducing a direct cytotoxic effect in malignant tissue of a subject, the method comprising: administering an effective amount of 5-aminolevulinic acid to the malignant tissue; and injecting the tissue at a concentration of about 3 W / cm. 2 ~about 100W / cm 2The method includes exposing the subject to ultrasonic energy using a focused ultrasound device at a frequency of about 0.1 MHz to about 3 MHz ("ultrasonic treating"), with an ultrasound beam focusing intensity of 0.1 MHz to about 3 MHz.

[0016] Another embodiment is a method of selectively inducing apoptosis in malignant tissue of a subject, the method comprising: administering an effective amount of 5-aminolevulinic acid to the malignant tissue; and injecting the tissue at a concentration of about 3 W / cm. 2 ~about 100W / cm 2 The ultrasonic treatment includes applying ultrasonic energy using a focused ultrasonic device at a frequency of about 0.1 MHz to about 3 MHz with an ultrasonic beam focusing intensity of 100 MHz.

[0017] Another embodiment is a dosage form for use with a FUS device in treating a subject in need of treatment, the dosage form comprising: a container containing an effective amount of 5-aminolevulinic acid sufficient to treat the subject; and a machine-readable identifier readable by the FUS device, wherein the machine-readable identifier identifies the source of the 5-aminolevulinic acid, the amount of 5-aminolevulinic acid, the subject for whom the 5-aminolevulinic acid has been prescribed, the FUS treatment parameters prescribed for the subject, an identification code or serial number, or a combination thereof.

[0018] Another embodiment is a system for treating malignant tissue in a subject, the system comprising: an effective amount of 5-aminolevulinic acid; and a focused ultrasound (FUS) device. [Brief explanation of the drawings]

[0019] [Figure 1]Figure 1 shows the inhibition of tumor growth of C6 glioma tumors implanted in mice resulting from (a) no treatment (solid circles); (b) treatment with 5-ALA only (squares); (c) treatment with focused ultrasound only (triangles); (d) treatment with 5-ALA and focused ultrasound, which limited maximum brain temperature to 32°C (inverted triangles); (e) treatment with 5-ALA and focused ultrasound, which limited maximum brain temperature to 37°C (diamonds); and (f) treatment with 16 cycles (85 seconds on, 60 seconds off each) of 5-ALA and focused ultrasound, which limited maximum brain temperature to 37°C (open circles).

[0020] [Figure 2] Figure 2 shows the survival rate of mice bearing implanted C6 neuroglial tumor cells with (a) no treatment; (b) treatment with 5-ALA only; (c) treatment with focused ultrasound only; (d) treatment with 5-ALA and focused ultrasound, which limited the maximum brain temperature to 32°C; (e) treatment with 5-ALA and focused ultrasound, which limited the maximum brain temperature to 37°C; and (f) treatment with 5-ALA and focused ultrasound at 16 multiple time points, which limited the maximum brain temperature to 37°C. DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed Description One embodiment is a method of selectively inducing a direct cytotoxic effect in malignant tissue of a subject, comprising administering to the malignant tissue an effective amount of 5-aminolevulinic acid; and injecting the tissue at a concentration of about 3 W / cm. 2 ~about 100W / cm 2The method involves exposing tissue to ultrasound energy ("ultrasonic treatment") using a focused ultrasound device at a frequency of about 0.1 MHz to about 3 MHz, with an ultrasound beam focus intensity of 100 Hz. Direct cytotoxic effects include inducing cellular apoptosis, necrosis, and / or physical destruction. An effective amount of 5-ALA can be determined by standard methods. Generally, an effective amount is an amount sufficient to substantially stain the malignant tissue being treated without substantially staining normal tissue or inducing unacceptable levels of toxicity. Without being bound by any particular theory, it is believed that ultrasonic treatment of tissue causes cavitation and microbubbles (whose collapse produces photons with wavelengths of about 300 nm to 700 nm within the tissue), and these photons activate protoporphyrin IX, leading to tissue destruction.

[0022] When a range of values ​​is given herein, it is understood that each intervening value between the upper and lower limits of that range (to one-tenth of the unit of the lower limit unless expressly stated otherwise), as well as any other stated or intervening value in the stated range, is encompassed within the disclosed scope. The upper and lower limits of these narrower ranges may independently be included in the narrower ranges, which are also encompassed within the disclosed scope, subject to any specifically excluded limit in the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0023] All ranges disclosed herein also encompass any and all possible subranges and combinations thereof. Any recited range will be considered fully descriptive and allows for the same range to be divided into at least equal halves, thirds, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily divided into a lower third, middle third, upper third, etc. As will be apparent to one of ordinary skill in the art, all language such as "up to," "at least," "greater than," "less than," etc., refers to a range that includes the recited numbers and can be subsequently divided into subranges as previously described. Finally, as will be apparent to one of ordinary skill in the art, a range includes each individual number. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, etc.

[0024] It is understood that certain features of the disclosure (which are, for clarity, described in the context of separate embodiments) may also be provided in combination in a single embodiment. Conversely, various features of the disclosure (which are, for brevity, described in the context of a single embodiment) may also be provided separately or in any suitable subcombination. All combinations of the embodiments relevant to the disclosure are specifically embraced by the present disclosure and are disclosed herein exactly as if each and every combination were individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein exactly as if each and every such subcombination were individually and explicitly disclosed herein.

[0025] Malignant tissue Malignant tissues are typically tumorous or cancerous, but may generally be any type of tissue capable of absorbing 5-ALA and accumulating protoporphyrin IX, such as benign tumors or other unwanted growths. Focused ultrasound can penetrate intercalated tissues, allowing for the treatment of malignant tissues located in otherwise inaccessible locations. For this reason, the disclosed methods are useful for treating intracranial tumor types, such as glioblastoma multiforme (including low- and high-grade glioblastoma), optic pathway glioma, diffuse intrinsic pontine glioma, astrocytoma, ependymoma, medulloblastoma, oligodendroglioma, hemangioblastoma, rhabdoid tumor, brain metastases from other cancers (including, but not limited to, breast adenocarcinoma, small cell lung cancer, non-small cell lung cancer, squamous cell lung cancer, metastatic malignant melanoma, and prostate cancer), meningioma, primary pituitary malignant tumor, malignant nerve sheath tumor, and neurofibroma. Other malignancies include, but are not limited to, neoplasms, carcinomas, sarcomas, leukemias, lymphomas, etc. Leukemias and lymphomas include, for example, cutaneous T-cell lymphoma (CTCL), non-cutaneous peripheral T-cell lymphoma, human T-cell leukemia virus (HTLV)-associated lymphomas such as adult T-cell leukemia / lymphoma (ATLL), acute lymphocytic leukemia, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, and multiple myeloma. Other tumors include, but are not limited to, solid tumors of childhood, such as brain tumors, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, and soft tissue sarcomas; common solid tumors of adults, such as head and neck cancers (e.g., invasive or metastatic squamous cell carcinoma, salivary gland tumors, nasopharyngeal carcinoma, oral cavity, laryngeal, and esophageal tumors); genitourinary cancers (e.g., urethral, ​​ureteral, renal cell, bladder carcinoma, and bladder carcinoma); These include carcinoma in situ, locally advanced or metastatic carcinoma of the prostate, bladder, kidney, uterus, ovary, testis, uterine, cervical, and uterine carcinoma), rectal and colon cancer; lung cancer (including mesothelioma, small cell lung cancer, non-small cell lung cancer, squamous cell lung carcinoma); breast cancer; gastric, esophageal, and colon carcinoma, bile duct carcinoma, liver carcinoma, and adenocarcinoma of the pancreas; melanoma, invasive basal cell carcinoma, and other skin cancers; stomach cancer, brain cancer, liver cancer, and thyroid cancer.

[0026] In some embodiments, the malignant tissue is glioblastoma multiforme, optic pathway glioma, diffuse intrinsic pontine glioma, astrocytoma, ependymoma, medulloblastoma, oligodendroglioma, hemangioblastoma, rhabdoid tumor, brain metastasis from other cancers (e.g., breast adenocarcinoma, small cell lung cancer, non-small cell lung cancer, squamous cell lung cancer, metastatic malignant melanoma, or prostate cancer), meningioma, primary pituitary malignant tumor, malignant nerve sheath tumor, neurofibroma, cutaneous T-cell lymphoma (CTCL), non-cutaneous peripheral T-cell lymphoma, human T-cell leukemia virus (HTLV)-associated lymphoma, adult T-cell leukemia / lymphoma (ATLL), acute lymphocytic leukemia, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, Leukemia, chronic myeloid leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, soft tissue sarcoma, invasive or metastatic squamous cell carcinoma, salivary gland tumors, nasopharyngeal carcinoma, oral cavity, laryngeal and esophageal tumors, urethral cancer, ureteral cancer, renal cell carcinoma, bladder carcinoma, carcinoma in situ of the bladder, metastatic carcinoma of the prostate, bladder, kidney, uterus, ovary, testis, uterine, cervical, uterine carcinoma, rectal or colon cancer, lung cancer, mesothelioma, small cell lung cancer, non-small cell lung cancer, squamous cell lung carcinoma, breast cancer; gastric cancer, esophageal cancer, and colon carcinoma, bile duct carcinoma, liver carcinoma, adenocarcinoma of the pancreas, melanoma, invasive basal cell carcinoma, other skin cancers, liver cancer, and thyroid cancer. In some embodiments, the malignant tissue is glioblastoma multiforme, astrocytoma, ependymoma, medulloblastoma, oligodendroglioma, hemangioblastoma, or rhabdoid tumor. In some embodiments, the malignant tissue is glioblastoma multiforme.

[0027] 5-aminolevulinic acid 5-ALA can be provided in any pharmaceutically acceptable formulation, and may be provided as the free acid, a pharmaceutically acceptable salt, or a pharmaceutically acceptable ester. One formulation, Gliolan®, is commercially available.

[0028] Salts, esters, amides, prodrugs, and other derivatives of active substances can be prepared using standard techniques known to those skilled in the art of synthetic organic chemistry and described, for example, in March (1992) Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 4th Ed. NY Wiley-Interscience. Pharmaceutically acceptable salts are salts that retain the biological effectiveness and properties of the parent compound and are not biologically or otherwise undesirable. 5-ALA can form acid and / or base salts in the presence of amino and / or carboxyl groups. Many such salts are known in the art, for example, as described in WO 87 / 05297. Pharmaceutically acceptable acid addition salts can be formed using inorganic and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed using inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. Pharmaceutically acceptable esters include those obtained by replacing the hydrogen of an acidic group with an alkyl group, for example, by reacting the acidic group with an alcohol or a haloalkyl group.Examples of esters include, but are not limited to, those in which the hydrogen of a -C(O)OH group is replaced with an alkyl to form -C(O)O-alkyl.

[0029] In some embodiments, 5-ALA is sterilized by gamma irradiation (see US Pat. No. 6,335,465, incorporated herein by reference in its entirety). 5-ALA formulations can be administered orally, intravenously, intrathecally, or intratumorally. In some embodiments, 5-ALA is administered intravenously. In some embodiments, gamma-irradiated 5-ALA is administered intravenously. In some embodiments, 5-ALA is administered at a dosage of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100, 120, 125, 150, 175, 200, 300, 400, 500, 600, 750, or at least about 1000 mg / kg. In some embodiments, 5-ALA is administered at a dosage of about 1000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 180, 175, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, or 20 mg / kg or less. In some embodiments, 5-ALA is administered at a dosage of about 0.5 to about 250 mg / kg. In some embodiments, 5-ALA is administered at a dosage of 1 to 150 mg / kg. In some embodiments, 5-ALA is administered at a dosage of 5 to 90 mg / kg. In some embodiments, 5-ALA is administered at a dosage of 10 to 40 mg / kg.

[0030] The effective amount of 5-ALA required can be determined by standard methods known to those skilled in the art. For example, as described in Example 1 herein, one skilled in the art can implant tumor tissue into mice or other laboratory model subjects and treat the subjects with various amounts of 5-ALA and FUS. As shown in Figure 1, rats were treated with 20 mg / kg of 5-ALA, followed by 20 minutes of 6.9 W / cm 2 , 13.8W / cm 2 , 27.6W / cm 2 , or 55.2 W / cm 2 The result was 13.8W / cm 2 This shows that ultrasound treatment at 27.6 W / cm raised tumor temperature by approximately 2°C to 32°C. 2 Ultrasound treatment at 55.2 W / cm raised tumor temperature to approximately 37°C. 2 The rats treated with ultrasound at 1000 rpm suffered tissue damage of 32 ± 10 mm compared with the untreated control group. 3 The normalized tumor volume of the 5-ALA-only and FUS-only groups was 24±6 mm. 3 The normalized tumor volume was 100 mg / kg to 500 mg / kg. In some embodiments, the effective amount of 5-ALA is 1 mg / kg to 750 mg / kg. In some embodiments, the effective amount of 5-ALA is 10 mg / kg to 750 mg / kg. In some embodiments, the effective amount of 5-ALA is 20 mg / kg to 500 mg / kg. In some embodiments, the effective amount of 5-ALA is 40 mg / kg to 500 mg / kg. In some embodiments, the effective amount of 5-ALA is 10 mg / kg to 40 mg / kg. In some embodiments, the effective amount of 5-ALA is 10 mg / kg to 20 mg / kg.

[0031] In some embodiments, the incubation period includes administering 5-ALA and treating the malignant tissue with ultrasound to allow sufficient time for 5-ALA to be taken up by the malignant tissue and converted to protoporphyrin IX. In some embodiments, the incubation period is at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, or at least about 24 hours. In some embodiments, the incubation period is 72 hours, less than about 72 hours, less than about 60 hours, less than about 48 hours, less than about 36 hours, less than about 24 hours, less than about 22 hours, less than about 20 hours, less than about 18 hours, less than about 16 hours, less than about 15 hours, less than about 14 hours, less than about 13 hours, less than about 12 hours, less than about 11 hours, less than about 10 hours, less than about 9 hours, less than about 8 hours, less than about 7 hours, less than about 6 hours, less than about 5 hours, less than about 4 hours, or less than about 3 hours. In some embodiments, the incubation period is 1 to 72 hours. In some embodiments, the incubation period is 2 to 48 hours. In some embodiments, the incubation period is 3 to 36 hours. In some embodiments, the incubation period is 4 to 24 hours. In some embodiments, the incubation period is 4 to 18 hours. In some embodiments, the incubation period is 4 to 24 hours. In some embodiments, the incubation period is 4 to 18 hours, hi some embodiments, the incubation period is about 6 hours.

[0032] Enhancer In some embodiments, the method further comprises administering a potentiating agent that enhances the therapeutic effect of 5-ALA, for example, by promoting the uptake or accumulation of protoporphyrin IX and / or 5-ALA or by slowing the rate at which protoporphyrin IX and / or 5-ALA are metabolized. The potentiating agent may thereby reduce the amount of 5-ALA required to achieve a given effect, or may enhance the effect achieved from a given amount of 5-ALA, or any combination of effect and amount in between. Suitable potentiating agents include, but are not limited to, methotrexate, doxycycline, minocycline, vitamin D3, and derivatives thereof. See, e.g., D.-F. Yang et al., J Formos Med Assoc (2014) 113(2):88-93; M.-J. Lee et al., PLoS ONE (2017) 12(5):e0178493; and EV Maytin et al., Isr J Chem (2012) 52(8-9):767-75. In some embodiments, the potentiator is selected from the group consisting of methotrexate, doxycycline, minocycline, vitamin D3, and derivatives thereof. In some embodiments, the potentiator is methotrexate. In some embodiments, the potentiator is doxycycline. In some embodiments, the potentiator is minocycline. In some embodiments, the potentiator is vitamin D3. In some embodiments, a combination of two or more potentiators is used. In some embodiments, a combination of two or more of methotrexate, doxycycline, minocycline, and vitamin D3 is used.

[0033] The augmenting agent is administered simultaneously with 5-ALA or at another time prior to ultrasound treatment. The optimal time for administering the augmenting agent may vary depending on the choice of augmenting agent or combination of agents. In some embodiments, the augmenting agent is administered simultaneously with 5-ALA. In some embodiments, the augmenting agent is administered in the same formulation as 5-ALA. In some embodiments, the augmenting agent is administered at different times. In some embodiments, the augmenting agent is administered before administration of 5-ALA. In some embodiments, the augmenting agent is administered at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 3 days, at least about 4 days, at least about 5 days, or at least about 6 days before the first ultrasound treatment. In some embodiments, the enhancement agent is administered 8 days, 7 days, 6 days, 5 days, 4 days, 84 hours, 72 hours, less than about 72 hours, less than about 60 hours, less than about 48 hours, less than about 36 hours, less than about 24 hours, less than about 22 hours, less than about 20 hours, less than about 18 hours, less than about 16 hours, less than about 15 hours, less than about 14 hours, less than about 13 hours, less than about 12 hours, less than about 11 hours, less than about 10 hours, less than about 9 hours, less than about 8 hours, less than about 7 hours, less than about 6 hours, less than about 5 hours, less than about 4 hours, or less than about 3 hours prior to the first ultrasound treatment. In some embodiments, the administration period of the enhancement agent is 1 to 72 hours prior to ultrasound treatment. In some embodiments, the administration period of the enhancement agent is 2 hours to 5 days prior to ultrasound treatment. In some embodiments, the administration period of the augmenting agent is 18 hours to 4 days prior to ultrasound treatment, hi some embodiments, the administration period of the augmenting agent is 24 hours to 4 days prior to ultrasound treatment.In some embodiments, the administration period of the augmenting agent is 24 to 48 hours prior to ultrasound treatment. In some embodiments, the administration period of the augmenting agent is 48 to 96 hours prior to ultrasound treatment. In some embodiments, the administration period of the augmenting agent is 4 to 18 hours prior to ultrasound treatment.

[0034] The amount of the enhancer to be administered can be determined by one skilled in the art and generally depends on the enhancer(s) selected or the degree of enhancing effect to be achieved. Suitable methods include, but are not limited to, cell culture assays and / or in vivo experiments using model animals or explanted tissues to measure the degree of cell killing using various amounts of 5-ALA and / or enhancers together with either ultrasound treatment or photodynamic treatment. See, for example, D.-F. Yang et al., J Formos Med Assoc (2014) 113(2):88-93; M.-J. Lee et al., PLoS ONE (2017) 12(5):e0178493; and EV Maytin et al., Isr J Chem (2012) 52(8-9):767-75.

[0035] The amount of potentiator used is less than the amount that would result in unacceptable toxicity, and is great enough to reduce the amount of 5-ALA required to achieve a potentiating effect. The amount or number of malignant tissues or cells killed can be measured using a range of 5-ALA amounts as a baseline for comparison, and then the amount or number of malignant tissues or cells killed can be measured using the same amount of 5-ALA combined with various concentrations or amounts of an enhancer. Alternatively, one skilled in the art can measure the amount of 5-ALA required to produce the same level of killing in the presence of various concentrations or amounts of an enhancer. The amount or number of malignant tissues or cells killed can be measured by cell counting, measuring tumor volume, vital dye exclusion, and other techniques commonly used in medical research. The effect obtained using an enhancer is an increase in effect or a reduction in the dose of 5-ALA of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% from the baseline standard. In some embodiments, the effect achieved with the potentiator is at least a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100, 120, 125, 150, 175, 200, 300, 400, 500% increase in effect from the baseline measure of extent of kill. In some embodiments, the effect achieved with the potentiator is at least a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98% or 99% reduction in the amount of 5-ALA required to achieve the baseline kill rate.

[0036] The amount of potentiator may be greater than, equal to, or less than the amount normally or typically prescribed for use of the potentiator alone. The upper limit is the amount at which unacceptable toxicity occurs, alone or in combination with 5-ALA. The lower limit is the amount required to achieve a measurable enhancing effect, which may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100, 120, 125, 150, 175, or 200% of the typical dose. For example, but not limited to, methotrexate may be administered as a single oral dose of about 7.5 mg to 10 mg 24 to 72 hours prior to ultrasound treatment; doxycycline may be administered at a dose of 100 mg BID starting with a loading dose of 200 mg starting 2 to 4 days prior to ultrasound treatment; minocycline may be administered at a dose of 50 to 100 mg BID starting 2 to 4 days prior to ultrasound treatment; and vitamin D3 may be administered as cholecalciferol at a dose of 10,000 to 100,000 IU / day 2 to 4 days prior to ultrasound treatment.

[0037] Microbubbles Microbubbles (also known as microspheres) are gas-filled spheres with diameters on the order of 1–5 μm. Because their echogenic properties aid in distinguishing fluid-filled blood vessels from surrounding tissue, they are sometimes used as contrast agents in medical ultrasonography. See, for example, PA Dijkmans et al., Eur J Cardiology (2004) 5:245–56. The gas is often air, nitrogen, sulfur hexafluoride, or a perfluorocarbon, such as octafluoropropane. The outer shell of a microbubble is often albumin, galactose, lipid, or polymer. In an ultrasound acoustic field, microbubbles undergo linear oscillations at low powers and nonlinear oscillations at higher powers, leading to lacerations at high powers. The frequency at which a microbubble resonates is primarily determined by the choice of gas in the core and the mechanical properties of the outer shell. Mixtures of two or more different types of microbubbles may be used. In practicing the methods of the present disclosure, microbubbles can be used to cause cavitation (and consequently, death of target cells) at lower acoustic powers than would otherwise be obtainable, hi some embodiments, an effective amount of microbubbles is administered to malignant tissue.

[0038] An effective dose of microbubbles is an amount sufficient to enhance the direct cytotoxic effect of 5-ALA or FUS on malignant tissue by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100, 120, 125, 150, 175, 200, 300, 400, or 500% above baseline criteria for extent of killing. Alternatively, an effective amount of microbubbles may be expressed as an amount sufficient to reduce the 5-ALA dose or FUS dose by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% from a baseline level.

[0039] Microbubbles may be prepared by methods known in the art or obtained from commercial sources. Suitable microbubbles include, but are not limited to, high-contrast ultrasound microbubbles, such as Definity® perflutren lipid microbubbles (Lantheus Medical Imaging, N. Billerica, MA), Levovist® lipid / galactose microspheres (Schering), Optison® microbubbles (GE Healthcare), and Lumason® microbubbles (Bracco Imaging, Monroe Township, NJ). In some embodiments, the microbubbles are high-contrast ultrasound microbubbles. In some embodiments, the microbubbles comprise sulfur hexafluoride or a perfluorocarbon. In some embodiments, the perfluorocarbon is octafluoropropane or perfluorohexane. In some embodiments, the microbubbles comprise air or nitrogen. In some embodiments, the microbubble shell comprises albumin. In some embodiments, the microbubbles are Definity® perflutren lipid microbubbles, Levovist® lipid / galactose microspheres, Optison® microbubbles, or Lumason® microbubbles.

[0040] Microbubbles can be administered with 5-ALA and / or a potentiating agent depending on the half-life of the microbubbles in the subject's system. Generally, many microbubble agents have a very short half-life in the human circulation, and therefore are typically administered immediately before ultrasound treatment. The amount and method of administration are similar to those used by those skilled in the art when administering microbubbles for contrast-enhanced ultrasound ultrasonography. The amount administered is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 140, 150, 160, 180, 200, 250, 300, 350, or 400% of the amount used or recommended for use as a contrast-enhanced ultrasound ultrasonography agent. The amount administered is no more than 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 75, 70, 60, 50, 45, 40, 35, 30, 25, or 20% of the amount used or recommended for use as an ultrasound imaging agent in contrast-enhanced ultrasound.

[0041] focused ultrasound Malignant tissue is exposed to focused ultrasound energy ("ultrasonic treatment") using a focused ultrasound (FUS) device. Suitable devices include the Exablate® Model 4000 Type-2 system (Insightec, Dallas, TX). The Type-2 has a dedicated 1000-element transducer that can operate in an ultrasound treatment mode (i.e., focused ultrasound pressure wave delivery mode) using a short-duration duty cycle to create "burst ultrasound treatment" at low power. This burst ultrasound treatment mode, when used in conjunction with microbubbles at energy levels much lower than ultrasound-induced cavitation, allows the device to induce stable cavitation. Microbubble oscillation induces temporary, reversible, well-targeted, yet stable blood-brain barrier disruption. A notable feature of the Exablate® device is its ability to monitor acoustic feedback in real time to ensure safe and effective BBB disruption. The Exablate® device is a magnetic resonance guided focused ultrasound (MRgFUS) device, and as such, it utilizes real-time MR imaging to assess and monitor procedural safety.

[0042] The ultrasonic frequency may be at least about 0.1 MHz, at least about 0.2 MHz, at least about 0.25 MHz, at least about 0.3 MHz, at least about 0.4 MHz, at least about 0.45 MHz, at least about 0.5 MHz, at least about 0.55 MHz, at least about 0.6 MHz, at least about 0.65 MHz, at least about 0.7 MHz, at least about 0.75 MHz, at least about 0.8 MHz, at least about 0.85 MHz, at least about 0.9 MHz, at least about 0.95 MHz, at least about 1 MHz, at least about 1.1 MHz, Hz, at least about 1.5 MHz, at least about 2.0 MHz, at least about 2.1 MHz, at least about 2.2 MHz, at least about 2.3 MHz, at least about 2.4 MHz, at least about 2.5 MHz, at least about 2.75 MHz, at least about 3.0 MHz, at least about 3.5 MHz, at least about 4.0 MHz, at least about 4.5 MHz, at least about 5.0 MHz, at least about 6.0 MHz, at least about 7.0 MHz, at least about 8.0 MHz, at least about 9.0 MHz, or at least about 10.0 MHz. The ultrasonic frequency is about 20 MHz or less, about 15 MHz or less, about 10 MHz or less, about 9.0 MHz or less, about 8.0 MHz or less, about 7.0 MHz or less, about 6.0 MHz or less, about 5.0 MHz or less, about 4.0 MHz or less, about 3.0 MHz or less, about 2.8 MHz or less, about 2.6 MHz or less, about 2.5 MHz or less, about 2.4 MHz or less, about 2.3 MHz or less, about 2.2 MHz or less, about 2.1 MHz or less, or about 2.0 MHz or less.

[0043] In the focused ultrasound beam, the focused ultrasound intensity is at least about 1 W / cm 2 , at least about 1.5 W / cm 2 , at least about 2.0 W / cm 2 , at least about 2.5 W / cm 2 , at least about 3.0 W / cm 2 , at least about 3.5 W / cm 2 , at least about 4.0 W / cm 2 , at least about 4.5 W / cm 2 , at least about 5.0 W / cm 2 , at least about 6.0 W / cm2 , at least about 7.0 W / cm 2 , at least about 8.0 W / cm 2 , at least about 9.0 W / cm 2 , at least 10.0 W / cm 2 , at least about 15 W / cm 2 , at least about 20 W / cm 2 , at least about 25 W / cm 2 , at least about 30 W / cm 2 , at least about 35 W / cm 2 , at least about 40 W / cm 2 , at least about 45 W / cm 2 , at least about 50 W / cm 2 , at least about 60 W / cm 2 , at least about 70 W / cm 2 , at least about 75 W / cm 2 ; at least about 80W / cm 2 , at least about 90 W / cm 2 , at least about 100 W / cm 2 , at least about 120 W / cm 2 , at least about 125 W / cm 2 , at least about 130 W / cm 2 , at least about 140 W / cm 2 , at least about 145 W / cm 2 , at least about 150 W / cm 2 , or at least about 200 W / cm 2 With ultrasonic beam focusing, the focused ultrasonic intensity is about 200W / cm 2 Less than 150W / cm 2 Less than 125W / cm 2 Less than 100W / cm 2 Less than 98W / cm 2 Less than 95W / cm 2 Less than 92W / cm 2 Less than 90W / cm 2 Less than 89W / cm 2 Less than 88W / cm 2 Less than 87W / cm 2 Less than 86W / cm 2 Less than 85W / cm 2Less than 84W / cm 2 Less than 83W / cm 2 Less than 82W / cm 2 Less than 81W / cm 2 Less than 80W / cm 2 Less than 75W / cm 2 Less than 70W / cm 2 Less than 68W / cm 2 Less than 67W / cm 2 Less than 65W / cm 2 Less than 66W / cm 2 Less than 64W / cm 2 Less than 63W / cm 2 Less than 62W / cm 2 Less than 61W / cm 2 Less than 60W / cm 2 Less than 58W / cm 2 Less than 55W / cm 2 Less than 54W / cm 2 Less than 53W / cm 2 Less than 52W / cm 2 Less than 51W / cm 2 Less than 50W / cm 2 Less than approx. 45 W / cm 2 Less than 40W / cm 2 Less than 35W / cm 2 Less than or about 30W / cm 2 In some embodiments, the focused ultrasound intensity is less than the spatial-peak temporal-average intensity (I SPTA )

[0044] The FUS energy applied during sonodynamic treatment is generally less than the amount of energy used when using FUS to ablate tissue, and can be further reduced when microbubbles are administered prior to ultrasound treatment. In some embodiments, the applied FUS energy is at least 10, 20, 30, 40, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 1000, 1200, 1400, 1600, 1800, or 2000 Joules. In some embodiments, the applied FUS energy is less than or equal to 5000, 4000, 3000, 2500, 2250, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1250, 1200, 1150, 1100, 1050, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, or 250 J. In some embodiments, the applied FUS energy is between 10 J and 2000 J. In some embodiments, the applied FUS energy is between 20 J and 1500 J. In some embodiments, the applied FUS energy is between 50 J and 1250 J. In some embodiments, the FUS energy applied is between 100 J and 1250 J. In some embodiments, the FUS energy applied is between 250 J and 1250 J. In some embodiments, the FUS energy applied is between 500 J and 1250 J.

[0045] The duration of ultrasound treatment varies depending on the subject, the specific type and stage of the malignant tissue, the location and amount of malignant tissue, and the extent to which the malignant tissue takes up 5-ALA and accumulates protoporphyrin IX. In some embodiments, the malignant tissue is ultrasound-treated at multiple points, e.g., multiple points within a tumor. As used herein, "point" refers to the focus of FUS and points surrounding the tissue affected by FUS. By ultrasound-treating points distributed throughout the malignant tissue, practitioners can achieve a more uniform and consistent effect throughout the tumor volume. This also allows practitioners to use lower power, which reduces the likelihood of temperature (and therefore potential risk to surrounding normal tissue) elevation. In some embodiments, the malignant tissue is ultrasound-treated at individual points, exposing all of the malignant tissue together to FUS. In some embodiments, the points overlap. The points can be ultrasound-treated simultaneously, individually, or in groups. For example, in a treatment involving targeting 16 points, all 16 points may be sonicated simultaneously, or the points may be sonicated sequentially or in random order, or in groups of, for example, two or three, or other sizes. When groups are sonicated, the groups may be physically grouped or distributed in non-contiguous areas. In some embodiments, malignant tissue is sonicated at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, or 30 individual points, or any value between 1 and 30. In some embodiments, malignant tissue is sonicated at 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 or fewer individual points.

[0046] In some embodiments, the ultrasound treatment duration is at least about 20 seconds, at least about 30 seconds, at least about 45 seconds, at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 6 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, at least about 105 minutes, at least about 120 minutes, at least about 135 minutes, at least about 150 minutes, at least about 165 minutes, at least about 180 minutes, at least about 195 minutes, at least about 210 minutes, at least about 230 minutes, at least about 245 minutes, at least about 260 minutes, at least about 275 minutes, at least about 300 minutes, at least about 330 minutes, or at least about 360 minutes. In some embodiments, the ultrasound treatment duration is less than about 360 minutes, less than about 330 minutes, less than about 300 minutes, less than about 290 minutes, less than about 280 minutes, less than about 270 minutes, less than about 260 minutes, less than about 250 minutes, less than about 240 minutes, less than about 230 minutes, less than about 220 minutes, less than about 210 minutes, less than about 200 minutes, less than about 195 minutes, less than about 190 minutes, less than about 185 minutes, less than about 180 minutes, less than about 170 minutes, less than about 160 minutes, less than about 150 minutes, less than about 140 minutes, less than about 130 minutes, less than about 120 minutes, less than about 110 minutes, less than about 100 minutes, less than about 90 minutes, less than about 80 minutes, less than about 70 minutes, less than about 60 minutes, less than about 50 minutes, less than about 40 minutes, less than about 30 minutes, less than about 20 minutes, or less than about 10 minutes.

[0047] Ultrasound treatment can be continuous or periodic. In periodic ultrasound treatment, periods of exposure to focused ultrasound ("ultrasound treatment periods") are interspersed with rest periods without ultrasound treatment. In some embodiments, the ultrasound treatment includes at least one rest period. In certain embodiments, the ultrasound treatment period and rest period are each independently at least about 5 seconds, at least about 10 seconds, at least about 15 seconds, at least about 20 seconds, at least about 25 seconds, at least about 30 seconds, at least about 35 seconds, at least about 40 seconds, at least about 45 seconds, at least about 50 seconds, at least about 55 seconds, at least about 60 seconds, at least about 65 seconds, at least about 70 seconds, at least about 75 seconds, at least about 80 seconds, at least about 85 seconds, at least about 90 seconds, at least about 95 seconds, at least about 100 seconds, at least about 105 seconds, at least about 110 seconds, at least about 115 seconds, at least about 120 seconds, at least about 125 seconds, at least about 130 seconds, at least about 140 seconds, at least about 150 seconds, at least about 160 seconds, at least about 165 seconds, at least about 170 seconds, at least about 175 seconds, or at least about 180 seconds. In certain embodiments, the ultrasound treatment period and rest period are each independently less than about 600 seconds, less than about 500 seconds, less than about 400 seconds, less than about 300 seconds, less than about 250 seconds, less than about 240 seconds, less than about 220 seconds, less than about 200 seconds, less than about 180 seconds, less than about 170 seconds, less than about 160 seconds, less than about 150 seconds, less than about 140 seconds, less than about 130 seconds, less than about 120 seconds, less than about 110 seconds, less than about 100 seconds, less than about 95 seconds, less than about 90 seconds, less than about 85 seconds, less than about 80 seconds, less than about 75 seconds, less than about 70 seconds, less than about 65 seconds, less than about 60 seconds, less than about 55 seconds, or less than about 50 seconds.

[0048] In the disclosed methods, malignant tissue is selectively destroyed without affecting non-malignant tissue present in the ultrasound focus. In some embodiments, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the non-malignant tissue present in the ultrasound focus is damaged. In some embodiments, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, or about 25% of the non-malignant tissue present in the ultrasound focus is damaged. The amount of tissue damage is measured using methods known to those skilled in the art, for example, using MRI. In some embodiments, the temperature of the malignant tissue increases by 15°C or less, 14°C or less, 13°C or less, 12°C or less, 11°C or less, 10°C or less, 9°C or less, 8°C or less, 7°C or less, 6°C or less, 5°C or less, 4°C or less, 3°C or less, 2°C or less, or 1°C or less.

[0049] Ultrasound can be focused on malignant tissue or on a larger volume that includes the malignant tissue. Treatment with 5-ALA renders the malignant tissue more susceptible to FUS, allowing it to be destroyed without undue damage to non-malignant tissue contained within the focus volume. For example, a tumor and its surrounding volume can be sonicated. In addition, one skilled in the art can sonicate entire anatomical regions of the brain, including, but not limited to, the temporal lobe, parietal lobe, frontal lobe, occipital lobe, thalamus, pituitary gland, pons, corpus callosum, basal ganglia, brainstem, entire hemispheres, supratentorial region, and infratentorial region. In addition, one skilled in the art can sonicate part or all of the FLAIR region of the brain (fluid-attenuated inversion recovery—an MRI technique designed to remove signals from fluid in the brain). The methods of the present disclosure can also be used in conjunction with surgical resection of a tumor to treat the resulting tumor cavity and eliminate any malignant cells not removed by the resection.

[0050] In some embodiments, the tumor is located using magnetic resonance imaging (MRI). In some embodiments, the tumor is located using X-ray imaging. In some embodiments, the tumor is sonicated. In some embodiments, the tumor and the volume surrounding the tumor are sonicated. In some embodiments, the tumor and an extended margin up to 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, or 8 cm from the tumor surface are sonicated. In some embodiments, an entire anatomical region of the brain is sonicated. In some embodiments, the temporal lobe, parietal lobe, frontal lobe, occipital lobe, thalamus, pituitary gland, pons, corpus callosum, basal ganglia, brainstem, entire brain hemispheres, supratentorial region, or infratentorial region is sonicated. In some embodiments, a FLAIR field of the brain is sonicated. In some embodiments, more than one anatomical region is sonicated. In some embodiments, the tumor is resected and the tumor cavity is sonicated to eliminate residual malignant tissue or cells. In some embodiments, the tumor cavity is insonified to a depth of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, or 8 cm.

[0051] In some embodiments, the disclosed methods, comprising administration of 5-ALA and ultrasound treatment of malignant tissue, are repeated at treatment intervals of at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 12 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 18 days, at least about 20 days, at least about 21 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 28 days, at least about 30 days, at least about 35 days, at least about 40 days, at least about 45 days, at least about 50 days, at least about 55 days, at least about 60 days, at least about 65 days, at least about 70 days, at least about 75 days, at least about 80 days, at least about 85 days, or at least about 90 days. In some embodiments, the treatment recurrence interval is less than about 120 days, less than about 110 days, less than about 100 days, less than about 90 days, less than about 80 days, less than about 70 days, less than about 60 days, less than about 50 days, less than about 40 days, less than about 30 days, less than about 20 days, less than about 14 days, less than about 10 days, less than about 7 days, less than about 6 days, less than about 5 days, less than about 4 days, less than about 3 days, or less than about 2 days.

[0052] The subject of the disclosure is a mammal, which may be a human, a non-human mammal, such as a companion animal, such as a dog, cat, rat, or livestock, such as a horse, donkey, mule, goat, sheep, pig, or cow, etc. In some embodiments, the subject is a human.

[0053] Another embodiment is a method of selectively inducing apoptosis in malignant tissue of a subject, comprising administering to the malignant tissue an effective amount of 5-aminolevulinic acid and administering to the malignant tissue an amount of about 3 W / cm at a frequency of about 0.1 MHz to about 3 MHz using the methods and parameters described above. 2 ~about 100W / cm 2 and ultrasonically treating the tissue using a focused ultrasound device at an ultrasound beam focusing intensity of 1000 .mu.m.

[0054] Dosage Forms and Systems It is possible that ultrasound treatment may be performed by someone other than the treating physician. To minimize risk and ensure that treatment is performed appropriately, one aspect is a dosage form for tailoring focused ultrasound device operation to the subject being treated. In some embodiments, the 5-ALA formulation is provided in a container including a machine-readable identifier, where the identifier identifies the contents of the container, the origin of the formulation, the amount of the formulation, the subject to whom the formulation is to be administered, the focused ultrasound treatment prescribed for the subject (e.g., specifying the ultrasound frequency, power, energy, duration, or a combination thereof), an identification code or serial number, or a combination thereof. The machine-readable identifier may be encrypted to preserve confidential patient information. In some embodiments, the container is sufficient to contain an effective amount of 5-aminolevulinic acid, an effective amount of the enhancement agent, and / or an effective amount of microbubbles. In some embodiments, the machine-readable identifier is a barcode, a QR code, or an RFID device. In some embodiments, the focused ultrasound device includes a device for reading the machine-readable identifier. In some embodiments, the machine-readable identifier is encrypted. In some embodiments, the FUS device is locked in the absence of the proper machine-readable identifier, hi some embodiments, the FUS device treatment parameters are programmed via the machine-readable identifier.

[0055] Specific Embodiments In some embodiments, a method for selectively inducing a direct cytotoxic effect in malignant tissue of a subject comprises administering to the malignant tissue an effective amount of 5-aminolevulinic acid, or a pharmaceutically acceptable salt or ester thereof; and treating the tissue with a 5-aminolevulinic acid beam at a frequency of about 0.1 MHz to about 3 MHz at a wavelength of about 3 W / cm. 2 ~about 100W / cm 2 and exposing the subject to ultrasound energy using a focused ultrasound device ("ultrasound treating") at an ultrasound beam focusing intensity of .gtoreq..times ...

[0056] In some embodiments, a method of selectively inducing apoptosis in malignant tissue of a subject comprises administering to the malignant tissue an effective amount of 5-aminolevulinic acid, or a pharmaceutically acceptable salt or ester thereof; and treating the tissue with a 5-aminolevulinic acid beam at a frequency of about 0.1 MHz to about 3 MHz at a dose of about 3 W / cm. 2 ~about 100W / cm 2 and exposing the subject to ultrasound energy using a focused ultrasound device ("ultrasound treating") at an ultrasound beam focusing intensity of .gtoreq..times ...

[0057] In some embodiments, the 5-aminolevulinic acid is gamma-irradiated 5-aminolevulinic acid. In certain embodiments, the malignant tissue is exposed to ultrasound for a duration of about 1 minute to about 6 hours. In certain embodiments, the malignant tissue is exposed to ultrasound for a duration of about 1 minute to about 180 minutes.

[0058] In some embodiments, the intensity is about 5 W / cm 2 ~about 80W / cm 2 In some embodiments, the intensity is about 5 W / cm 2 ~approx. 60W / cm 2 In one embodiment, the intensity is about 5 W / cm 2 ~about 50W / cm 2 In some embodiments, the energy applied at the focal point is between 10 J and 2000 J. In some embodiments, the energy applied at the focal point is between 10 J and 2000 J.

[0059] In some embodiments, there is an incubation period between administering 5-aminolevulinic acid and ultrasonically treating the malignant tissue. In some embodiments, the incubation period is about 1 hour to about 72 hours. In some embodiments, the incubation period is about 3 hours.

[0060] In some embodiments, the malignant tissue comprises tumor tissue, which may be glioblastoma multiforme, optic pathway glioma, diffuse intrinsic pontine glioma, astrocytoma, ependymoma, medulloblastoma, oligodendroglioma, hemangioblastoma, rhabdoid tumor, brain metastasis from other cancers (selected from breast adenocarcinoma, small cell lung cancer, non-small cell lung cancer, squamous cell lung cancer, metastatic malignant melanoma, and prostate cancer), meningioma, primary pituitary malignant tumor, malignant nerve sheath tumor, neurofibroma, cutaneous T-cell lymphoma (CTCL), non-cutaneous peripheral T-cell lymphoma, human T-cell leukemia virus (HTLV)-associated lymphoma, adult T-cell leukemia / lymphoma (ATLL), acute lymphocytic leukemia, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, leukemia, chronic myeloid leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumor, soft tissue sarcoma, invasive or metastatic squamous cell carcinoma, salivary gland tumor, nasopharyngeal carcinoma, oral cavity, laryngeal and esophageal tumor, urethral cancer, ureteral cancer, renal cell carcinoma, bladder carcinoma, bladder intraepithelial carcinoma, metastatic carcinoma of the prostate, bladder, kidney, uterus, ovary, testis, uterine, cervical and uterine carcinoma, rectal or colon cancer, lung cancer, mesothelioma, small cell lung cancer, non-small cell lung cancer, squamous cell lung carcinoma, breast cancer, gastric cancer, esophageal cancer, and colon carcinoma, bile duct carcinoma, liver carcinoma, adenocarcinoma of the pancreas, melanoma, invasive basal cell carcinoma, other skin cancers, liver cancer, or thyroid cancer.

[0061] In some embodiments, the tumor tissue comprises a glioblastoma. In some embodiments, the malignant tissue is ultrasound-treated at multiple discrete points. In some embodiments, the malignant tissue is located by magnetic resonance imaging prior to ultrasound treatment.

[0062] In some embodiments, the tissue to be ultrasound-treated includes malignant tissue and non-malignant tissue. In some embodiments, the tissue to be ultrasound-treated includes malignant tissue and the margin surrounding the malignant tissue. In some embodiments, the outer edge of the margin is 0.2 cm to 5 cm from the malignant tissue. In some embodiments, the outer edge of the margin is 1 cm to 3 cm from the malignant tissue. In some embodiments, the tissue to be ultrasound-treated includes the margin surrounding the resection site after malignant tissue resection. In some embodiments, the outer edge of the margin is 0.2 cm to 5 cm from the malignant tissue resection site.

[0063] In some embodiments, the tissue to be ultrasound-treated comprises an entire anatomical region of the brain. In some embodiments, the entire anatomical region of the brain is the temporal lobe, parietal lobe, frontal lobe, occipital lobe, thalamus, pituitary gland, pons, corpus callosum, basal ganglia, brainstem, cerebral hemispheres, supratentorial region, or infratentorial region. In some embodiments, the entire anatomical region of the brain is a FLAIR region of the brain.

[0064] In some embodiments, less than about 25% of the non-malignant tissue within the ultrasound beam focus is damaged. In some embodiments, less than about 10% of the non-malignant tissue within the ultrasound beam focus is damaged. In some embodiments, less than about 5% of the non-malignant tissue within the ultrasound beam focus is damaged. In some embodiments, the temperature of the malignant tissue is increased by about 10°C or less with ultrasound treatment. In some embodiments, the temperature of the malignant tissue is increased by about 5°C or less with ultrasound treatment. In some embodiments, the temperature of the malignant tissue is increased by about 2°C or less with ultrasound treatment.

[0065] In some embodiments, the subject is a human. In some embodiments, 5-aminolevulinic acid is provided to the malignant tissue by oral administration of an oral formulation or intravenous administration of an iv formulation to the subject. In some embodiments, 5-aminolevulinic acid is provided to the malignant tissue by intravenous administration to the subject.

[0066] In some embodiments, the method further comprises providing to the malignant tissue an effective amount of a 5-ALA enhancer. In some embodiments, the enhancer is selected from the group consisting of doxycycline, methotrexate, minocycline, and vitamin D3 or a derivative thereof. In some embodiments, the method further comprises providing to the malignant tissue an effective amount of microbubbles. In some embodiments, the microbubbles are high-contrast ultrasound microbubbles. In some embodiments, the microbubbles comprise sulfur hexafluoride or a perfluorocarbon.

[0067] In some embodiments, the 5-aminolevulinic acid is provided in a container, the container further comprising a machine-readable identifier, wherein the machine-readable identifier identifies the source of the 5-aminolevulinic acid, the amount of 5-aminolevulinic acid, the subject to which the 5-aminolevulinic acid should be administered, the focused ultrasound treatment parameters prescribed for the subject, an identification code or serial number, or a combination thereof. In some embodiments, the focused ultrasound treatment parameters include an ultrasound frequency, power, energy, duration, or a combination thereof. In some embodiments, the focused ultrasound device comprises a scanning element capable of scanning the machine-readable identifier, wherein the treatment parameters of the focused ultrasound device are set corresponding to the machine-readable identifier. In some embodiments, the focused ultrasound device is locked in the absence of the machine-readable identifier.

[0068] In some embodiments, the effective amount of 5-ALA is about 1 mg / kg body weight to 1000 mg / kg body weight. In some embodiments, the effective amount of 5-ALA is about 10 mg / kg body weight to 750 mg / kg body weight. In some embodiments, the effective amount of 5-ALA is about 20 mg / kg body weight to 500 mg / kg body weight.

[0069] In some embodiments, steps a) to b) are repeated at treatment intervals of about 1 day to about 60 days. In some embodiments, the ultrasound exposure time includes at least one rest period. In some embodiments, the ultrasound exposure time includes rest periods of about 10 seconds to about 120 seconds separated by ultrasound treatment periods of about 10 seconds to about 180 seconds. In some embodiments, the ultrasound treatment period is about 60 seconds to about 90 seconds, and the rest periods are about 45 seconds to about 75 seconds.

[0070] In some embodiments, a dosage form for use with a FUS device in treating a subject in need thereof is provided, the dosage form comprising: a container containing an effective amount of 5-aminolevulinic acid sufficient to treat the subject; and a machine-readable identifier readable by the FUS device, wherein the machine-readable identifier identifies the source of the 5-aminolevulinic acid, the amount of 5-aminolevulinic acid, the subject for whom the 5-aminolevulinic acid has been prescribed, the FUS treatment parameters prescribed for the subject, an identification code or serial number, or a combination thereof. In some embodiments, the machine-readable identifier is encrypted and contains information for automatically programming the FUS.

[0071] In some embodiments, a system for treating malignant tissue in a subject is provided, comprising an effective amount of 5-aminolevulinic acid and a focused ultrasound (FUS) device. In some embodiments, the system further comprises an effective amount of an enhancement agent and / or an effective amount of microbubbles. In some embodiments, the system further comprises a container sufficient to contain the effective amount of 5-aminolevulinic acid, the effective amount of enhancement agent, and / or the effective amount of microbubbles; wherein the container comprises a machine-readable identifier readable by the FUS device; wherein the machine-readable identifier identifies the source of the 5-aminolevulinic acid, the amount of 5-aminolevulinic acid, the subject to whom the 5-aminolevulinic acid has been prescribed, the FUS treatment parameters prescribed for the subject, an identification code or serial number, or a combination thereof. In some embodiments, the machine-readable identifier is encrypted and contains information for automatically programming the FUS. In some embodiments, the FUS is locked in the absence of the machine-readable identifier. In some embodiments, the FUS device is a magnetic resonance guided FUS device (MRgFUS). [Example]

[0072] The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention, and should not be considered as limiting the scope of the invention, but merely as being illustrative and representative thereof.

[0073] Example 1 Treatment method Male Wistar rats received 4 × 10 5 Injections of C6 glial tumor cells were given to rats. On the seventh day after injection, 3 hours before treatment, rats were orally administered 5-aminolevulinic acid (20 mg / kg or 0 mg / kg, Levulan®, DUSA Pharmaceuticals, Wilmington, MA).

[0074] Rats were anesthetized and placed in an MRI-guided focused ultrasound system ("FUS", RK300, FUS Instruments, Toronto, Canada) with a 25 mm diameter spherically curved transducer (resonant frequency f = 1.06 MHz, focal number = 0.8). The FUS with the rat was then inserted into a 7 Telsa MRI system (Brucker BioSpec 70 / 30 USR, Bruker Ltd., Milton, Ontario, Canada). MRI was used to precisely target the FUS device at the brain tumor. The rats were then monitored for four spatial-peak temporal-average intensities (I) over a 20-minute period. SPTA )(6.9W / cm 2 , 13.8W / cm 2 , 27.6W / cm 2 , or 55.2 W / cm 2 One group (N=6) was treated with ultrasound at 27.6 W / cm for 20 minutes. 2 The tumors were periodically sonicated at 1000 rpm, divided into 16 85-second sonication sessions separated by 60-second rest periods (multiple-point, or "MP" group). In the MP group, 16 individual points within the tumor were sonicated. Control groups (N=5 each) received 5-ALA without FUS, FUS treatment without 5-ALA, or neither 5-ALA nor FUS treatment. Tumor temperature was monitored during sonication using a magnetic resonance thermometer. Normalized tumor volume was measured by MRI at 7, 14, 21, 28, and 35 days after injection.

[0075] result 13.8W / cm 2 Ultrasound treatment at 27.6 W / cm raised tumor temperature by approximately 2°C to 32°C. 2 Ultrasound treatment at 55.2 W / cm raised tumor temperature to approximately 37°C. 2 The rats treated with ultrasound at 1000 Hz suffered tissue damage and were euthanized. The untreated control group had a 32±10 mm 3 The normalized tumor volume of the 5-ALA-only and FUS-only groups was 24±6 mm. 3The normalized tumor volume of 100 mg / kg / day was 100 mg / kg / day. All experimental groups showed inhibited tumor growth (see Figure 1) and improved survival (see Figure 2). The MP group showed the greatest prolongation of survival. Example 2

[0076] This study was conducted to demonstrate the safety and tolerability of treatment with 5-ALA and MRgFUS.

[0077] Treatment method Human subjects will be selected who meet the following criteria: (a) presence of recurrent glioma (WHO grade 3 or 4); (b) prior treatment using the Stupp protocol; and (c) planned tumor resection. The Stupp protocol consists of daily administration of temozolomide (75 mg / m) over 6 weeks of radiation therapy. 2 Radiotherapy was administered at 2 Gy per 6-week period, Monday through Friday, for a total of 60 Gy, followed by six cycles of temozolomide (150–200 mg / m²) for 5 days of each 28-day cycle after completion of radiotherapy. 2 ) (R. Stupp et al., New Engl J Med (2005) 352:987-96).

[0078] Each subject is treated with intravenous 5-ALA and MRgFUS at the amounts specified in Table 1 below, two to four weeks prior to the subject's planned craniotomy. In each subject, FUS is focused on half of the tumor, including enhancing and non-enhancing areas. Treatment is initially performed with dose level 1, and subjects are examined for dose-limiting toxicity (DLT). If no DLT is observed, the next cohort of subjects is treated with dose level 2. This escalation is repeated for each cohort unless a DLT is observed. If one DLT is observed, that dose level is repeated in the next cohort with an expansion of three additional subjects. If two or more DLTs are observed, the prior dose level is designated as the maximum tolerated dose.

[0079] [Table 1]

[0080] result Tumors are monitored periodically by MRI or X-ray imaging prior to resection. Following tumor resection, the excised tumors are examined for differences between the ultrasound-treated and non-ultrasound-treated halves, including size reduction or histological grade.

[0081] The discussion of general methods presented herein is intended for illustrative purposes only: other alternative methods and means will be apparent to those of skill in the art upon review of this disclosure, and are intended to be within the spirit and scope of this application.

[0082] Throughout this specification, various patents, patent applications, and other types of publications (e.g., journal articles, electronic database entries, etc.) are referenced. The disclosures of all patents, patent applications, and other publications cited herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0083] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their inventors assert, and applicants reserve the right to challenge the accuracy and pertinence of the cited documents. Several sources of information, including journal articles, patent documents, and textbooks, are referenced herein; however, it will be expressly understood that this reference does not constitute an admission that any of these documents form part of the general common knowledge in the art.

Claims

1. 1. A method for selectively inducing a direct cytotoxic effect in malignant tissue of a subject, comprising: a) providing an effective amount of 5-aminolevulinic acid, or a pharmaceutically acceptable salt or ester thereof, to the malignant tissue; and b) applying a pulse of about 3 W / cm to the tissue at a frequency of about 0.1 MHz to about 3 MHz; 2 ~Approx. 100W / cm 2 exposing the subject to ultrasound energy using a focused ultrasound device ("ultrasonic treatment") at an ultrasound beam focusing intensity of A method comprising:

2. 1. A method for selectively inducing apoptosis in malignant tissue of a subject, comprising: a) providing an effective amount of 5-aminolevulinic acid, or a pharmaceutically acceptable salt or ester thereof, to the malignant tissue; and b) applying a pulse of about 3 W / cm to the tissue at a frequency of about 0.1 MHz to about 3 MHz; 2 ~Approx. 100W / cm 2 exposing the subject to ultrasound energy using a focused ultrasound device ("ultrasonic treatment") at an ultrasound beam focusing intensity of A method comprising:

3. 3. The method of claim 1, wherein the 5-aminolevulinic acid comprises gamma-irradiated 5-aminolevulinic acid.

4. 4. The method of claim 1, wherein the malignant tissue is exposed to ultrasound for a duration of from about 1 minute to about 6 hours.

5. 5. The method of any one of claims 1 to 4, wherein the malignant tissue is exposed to ultrasound for a duration of about 1 minute to about 180 minutes.

6. The intensity is about 5 W / cm 2 ~Approx. 80W / cm 2 The method according to any one of claims 1 to 5, wherein

7. The intensity is about 5 W / cm 2 ~ approx. 60W / cm 2 The method according to any one of claims 1 to 6, wherein

8. 8. The method according to any one of claims 1 to 7, wherein the energy applied at the focal point is between 10J and 2000J.

9. 9. The method according to any one of claims 1 to 8, wherein the energy applied at the focal point is between 10J and 2000J.

10. 10. The method of any one of claims 1 to 9, wherein there is an incubation period between administering the 5-aminolevulinic acid and ultrasonically treating the malignant tissue.

11. 11. The method of claim 10, wherein the incubation period is from about 1 hour to about 72 hours.

12. 12. The method of claim 10 or 11, wherein the incubation period is about 3 hours.

13. The method of any one of claims 1 to 12, wherein the malignant tissue comprises tumor tissue.

14. The tumor tissue is selected from glioblastoma multiforme, optic pathway glioma, diffuse intrinsic pontine glioma, astrocytoma, ependymoma, medulloblastoma, oligodendroglioma, hemangioblastoma, rhabdoid tumor, brain metastasis from other cancers (selected from breast adenocarcinoma, small cell lung cancer, non-small cell lung cancer, squamous cell lung cancer, metastatic malignant melanoma, and prostate cancer), meningioma, primary pituitary malignant tumor, malignant nerve sheath tumor, neurofibroma, cutaneous T-cell lymphoma (CTCL), non-cutaneous peripheral T-cell lymphoma, human T-cell leukemia virus (HTLV)-associated lymphoma, adult T-cell leukemia / lymphoma (ATLL), acute lymphocytic leukemia, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, and chronic myeloid leukemia.

14. The method of claim 13, comprising the treatment of cancers selected from the group consisting of Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, soft tissue sarcoma, invasive or metastatic squamous cell carcinoma, salivary gland tumors, nasopharyngeal carcinoma, oral cavity, laryngeal and esophageal tumors, urethral cancer, ureteral cancer, renal cell carcinoma, bladder carcinoma, bladder intraepithelial carcinoma, metastatic carcinoma of the prostate, bladder, kidney, uterus, ovary, testis, uterine, cervical and uterine carcinoma, rectal or colon cancer, lung cancer, mesothelioma, small cell lung cancer, non-small cell lung cancer, squamous cell lung carcinoma, breast cancer, gastric cancer, esophageal cancer, and colon carcinoma, bile duct carcinoma, liver carcinoma, adenocarcinoma of the pancreas, melanoma, invasive basal cell carcinoma, other skin cancers, liver cancer, or thyroid cancer.

15. The method of any one of claims 1 to 14, wherein the tumor tissue comprises a glioblastoma.

16. The method of any one of claims 1 to 15, wherein the malignant tissue is treated with ultrasound at multiple discrete points.

17. 17. The method of any one of claims 1 to 16, further comprising detecting the malignant tissue by magnetic resonance imaging before the ultrasound treatment.

18. 18. The method of any one of claims 1 to 17, wherein the ultrasound-treated tissue comprises malignant and non-malignant tissue.

19. 19. The method of any one of claims 1 to 18, wherein the ultrasound-treated tissue comprises malignant tissue and margins surrounding the malignant tissue.

20. 20. The method of claim 19, wherein the outer edge of the margin is 0.2 cm to 5 cm from the malignant tissue.

21. 21. The method of claim 19 or 20, wherein the outer edge of the margin is 1 cm to 3 cm from the malignant tissue.

22. 19. The method according to any one of claims 1 to 18, wherein the ultrasound-treated tissue comprises the margins around the excision site after surgical resection of malignant tissue.

23. 23. The method of claim 22, wherein the outer edge of the margin is 0.2 cm to 5 cm from the site of malignant tissue resection.

24. 19. The method of any one of claims 1 to 18, wherein the ultrasound-treated tissue comprises an entire anatomical region of the brain.

25. 31. The method of claim 30, wherein the entire anatomical region of the brain is the temporal lobe, parietal lobe, frontal lobe, occipital lobe, thalamus, pituitary gland, pons, corpus callosum, basal ganglia, brainstem, cerebral hemispheres, supratentorial region, or infratentorial region.

26. 31. The method of claim 30, wherein the entire anatomical region of the brain is a FLAIR region of the brain.

27. 27. The method of any one of claims 1 to 26, wherein less than about 25% of non-malignant tissue within the ultrasound beam focus is damaged.

28. 28. The method of any one of claims 1 to 27, wherein less than about 10% of non-malignant tissue within the ultrasound beam focus is damaged.

29. 29. The method of any one of claims 1 to 28, wherein less than about 5% of non-malignant tissue within the ultrasound beam focus is damaged.

30. 30. The method of any one of claims 1 to 29, wherein the temperature of the malignant tissue is increased by about 10°C or less by ultrasound treatment.

31. 31. The method of any one of claims 1 to 30, wherein the temperature of the malignant tissue is increased by about 5°C or less by ultrasound treatment.

32. 31. The method of any one of claims 1 to 30, wherein the temperature of the malignant tissue is increased by about 2°C or less by ultrasound treatment.

33. 33. The method of any one of claims 1 to 32, wherein the 5-aminolevulinic acid is provided to the malignant tissue by oral administration of an oral formulation or by intravenous administration of an iv formulation to the subject.

34. 34. The method of any one of claims 1 to 33, wherein the 5-aminolevulinic acid is administered by intravenous administration.

35. The method of any one of claims 1 to 34, wherein the subject is a human.

36. 36. The method of any one of claims 1 to 35, further comprising providing to the malignant tissue an effective amount of a 5-ALA potentiating agent.

37. The potentiators are doxycycline, methotrexate, minocycline, and vitamin D 3 or derivatives thereof.

38. 38. The method of any one of claims 1 to 37, further comprising administering an effective amount of microbubbles to malignant tissue.

39. 39. The method of claim 38, wherein the microbubbles are high-contrast ultrasound microbubbles.

40. 40. The method of claim 38 or 39, wherein the microbubbles comprise sulfur hexafluoride or a perfluorocarbon.

41. 41. The method of any one of claims 1 to 40, wherein the 5-aminolevulinic acid is provided in a container, the container further comprising a machine-readable identifier, wherein the machine-readable identifier identifies the source of the 5-aminolevulinic acid, the amount of 5-aminolevulinic acid, the subject to which the 5-aminolevulinic acid is to be administered, the focused ultrasound treatment parameters prescribed for the subject, an identification code or serial number, or a combination thereof.

42. 42. The method of claim 41, wherein the focused ultrasound treatment parameters include ultrasound frequency, power, energy, duration, or a combination thereof.

43. 43. The method of claim 41 or 42, wherein the focused ultrasound device includes a scanning element capable of scanning a machine-readable identifier, and wherein treatment parameters of the focused ultrasound device are set corresponding to the machine-readable identifier.

44. 44. The method of any one of claims 41 to 43, wherein the focused ultrasound device is locked in the absence of a machine readable identifier.

45. 45. The method of any one of claims 1 to 44, wherein the effective amount of 5-ALA is about 1 mg / kg body weight to 1000 mg / kg body weight.

46. 46. ​​The method of any one of claims 1 to 45, wherein the effective amount of 5-ALA is about 10 mg / kg body weight to 750 mg / kg body weight.

47. 47. The method of any one of claims 1 to 46, wherein the effective amount of 5-ALA is about 20 mg / kg body weight to 500 mg / kg body weight.

48. 48. The method of any one of claims 1 to 47, wherein steps a) to b) are repeated at treatment intervals of about 1 day to about 60 days.

49. 49. The method of any one of claims 1 to 48, wherein the ultrasound exposure time includes at least one rest period.

50. 50. The method of any one of claims 1-49, wherein the ultrasound exposure time comprises rest periods of about 10 seconds to about 120 seconds separated by ultrasound treatment periods of about 10 seconds to about 180 seconds.

51. 51. The method of claim 50, wherein the ultrasound treatment period is from about 60 to about 90 seconds, and the rest period is from about 45 to about 75 seconds.

52. 1. A dosage form for use with a FUS device in treating a subject in need thereof, said dosage form comprising: a) a container containing an effective amount of 5-aminolevulinic acid sufficient to treat a subject; and b) a machine-readable identifier that is readable by the FUS device; wherein the machine-readable identifier identifies the source of the 5-aminolevulinic acid, the amount of 5-aminolevulinic acid, the subject for whom the 5-aminolevulinic acid is prescribed, the FUS treatment parameters prescribed for the subject, an identification code or serial number, or a combination thereof.

53. 53. The dosage form of claim 52, wherein the machine-readable identifier is encrypted and contains information for automatically programming the FUS.

54. 1. A system for treating malignant tissue in a subject, comprising: a) an effective amount of 5-aminolevulinic acid; and b) Focused ultrasound (FUS) devices; A system including:

55. c) an effective amount of an enhancer and / or an effective amount of microbubbles; 55. The system of claim 54, further comprising:

56. The system of claim 54 or 55, further comprising a container sufficient to contain an effective amount of 5-aminolevulinic acid, an effective amount of an enhancer, and / or an effective amount of microbubbles; wherein the container comprises a machine-readable identifier readable by the FUS device; and wherein the machine-readable identifier identifies the source of the 5-aminolevulinic acid, the amount of 5-aminolevulinic acid, the subject for whom the 5-aminolevulinic acid has been prescribed, the FUS treatment parameters prescribed for the subject, an identification code or serial number, or a combination thereof.

57. 57. The system of claim 56, wherein the machine-readable identifier is encrypted and includes information for automatically programming a FUS device.

58. 58. The system of any one of claims 54 to 57, wherein the FUS device is locked in the absence of a machine-readable identifier.

59. The system of any one of claims 54 to 58, wherein the FUS device is a magnetic resonance guided FUS device (MRgFUS).

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