UA78186C2 - Use of aplidine for treatment of malignant tumours - Google Patents

Abstract

Aplidine demonstrates considerable promise in phase (I) clinical trials for treatment of malignant tumours, and various dosing regimes are given. Tumor reduction has been observed in several tumour types including renal carcinoma, colorectal cancer, lung carcinoid, medullary thyroid carcinomas and melanoma. It has also been found that aplidine has a role in inhibiting angiogenesis, complementing the anti-tumour activity.

Description

Description of the invention

Malignant tumors include a group of malignant neoplasms that can be divided into two 2 categories: carcinomas, in most cases observed in the clinic, and other, less common malignant tumors, which include leukemia, lymphoma, tumors of the central nervous system and sarcoma.

Carcinomas originate in epithelial tissues, while sarcomas develop from connective tissues and structures derived from the mesoderm. Sarcomas can affect, for example, muscle and bone and are found in bone, bladder, kidney, liver, lung, parotid gland, or spleen. 70 Malignant tumors are characterized by invasiveness and tend to metastasize to new sites. They spread directly into the surrounding tissues and can also be disseminated through the lymphatic and circulatory systems. There are many ways to treat malignant tumors, including surgery and radiation for localized disease and drugs. However, the effectiveness of available treatments for many types of malignant tumors is limited, and new, improved forms of treatment with clinical advantages are needed. This is especially true for patients with advanced and/or metastatic disease. This is also true for patients who have relapsed advanced disease after previous treatment with traditional methods, whose additional treatment with the same methods is in most cases ineffective due to the acquisition of resistance or limitations in the use of these treatment methods due to the toxicity associated with them.

Chemotherapy plays an important role in the treatment of malignancy, as it is necessary for the treatment of advanced malignancies with resected metastases and is often useful in shrinking the tumor before surgery, and numerous anticancer drugs based on different mechanisms of action have been developed.

Dehydrodidemnin B, now known as aplidine, is an object (in UU/091/049851.

Additional information about aplidine can be found, for example, in: p

Yitepo, U., "Ehrioyayop oi tagpe tisgoogdapizte apa ipmeperga(ez: Apiisapseg gdz bot tagipe (9 ogidip), IVS Sopi Oizsom Ogydv iot Mai Mome! Arrgoaspez Mem/ Zoshigsez (Oes 8-9, I opdop) 1994, 19941;

IRaigsio(y, 0. ei aiI., "Sepudgodidetpip V (OOM) a pem/ tagipe dehimey apiisapseg adepi (MOA) m/yyp asiimnu adaiipvi ekhregitepia! (utoig todeiv", 9 MSI-EOVTS Zutr Mem Ogoidz SapsegTNeg (Magsy 12- 15, Atwiegaat) 1996,

Arvy 1111; oh

IzZakKai, KK. ey ai., "Bigisige-asiimyu geiayopeyirv oi (Pe aidetpipv", Ootsgpa! oi Meadisipai Spetisiu "c 1996, 39 (14): 2819); (Ogaiiaiev, 9.1. ei ai., "Apiiirgoiiegaime oePesi oi depudgodidetpip B (008), a derzireriide izoiaiigeid - iot Meadiyetapeap (Shpisaiev), Sapseg |eTsege 1996, 102(1-2): 31 Raigsioi(y, (3. ei aiYi., "Rhesiipisai s spagasiegigayop oi ariidipe (ARO), a pem/ tagipe apiisapseg derzireriide (MAOER)", Rgos Ateg Azzos Sapseg Kev 41997, 38: Arvi 6921; -

IoereprgosK, N. ei aiYi., "p mygo asiimyu oi ariidipe a pem/ tagipe-dehimey apii-sapseg sotrotspa, op itezpIu ehriapiei siopodepis pitap (shtoytsg save apa Paetaiioroieiys rgesigvog seiYiv", Vgyivy docsgpa! oi

Sapseg 1998. 78(6): 739); " du IRaigsio(y, p. ei aiY., "Ariydile (aryydile) iv a opome! tagipe-degimei derzireride m/yy o ip omimo -o apiyitoig asiimpu", Rgos Ateg Azzos Sapseg Kez 1998, 39: Arvi 15511; p Raigsio(f, 0. ei ai., "Rgesiipisai demeiortepi oi ariidipe a pome! tagipe-degimei adepi m/y roiepi :z» apiyitochig asiimyu", 10 MSI-EOVTS Wutr Mem Ogoidz Sapseg Tneg (dype 16-19, Atviegdat) 1998, Abzvi 129;

IMaziregdep, 5.S. ei ai.,, "Suyuyohisyu apa peitsgosuyohisyu oi aryidile, a pem/ tagipe apiisapseg adepi emaichaiei ivipou ip mygo azvzauv", 1017 MSI-EOVTS Zutr Mem Ogydz Sapseg Tneg (dype 16-19, Atviegaat) 1998, Abvi -I 131).

In preclinical studies, aplidine had dose-dependent cytotoxic activity against two epithelial cell lines, ST-1 and ST-2, and the human colon cancer cell line, HT-29. The most proliferative line, ST-2, was the most sensitive to aplidine. In addition, this compound reduced the activity of ornithine decarboxylase in all three cell lines (oro C, Sagsia-Rogo 50, ei aYi. 1997). IN

In a similar study, aplidine at a concentration of 5 mol/l inhibited the growth of breast cancer cell lines,

MOA-MV231 and MSE-7 at 17 and 4795, respectively. A significant increase in the content of spermidine and spermine was observed in the treated cells. Roiiuatipe sopiepiv oii pitap Bgeavi sapseg seiYiv 5B igeaied m/yyp (pe sufoiohis adepiv sPpiogrpepigatipe apa depudgodidetpip V. Sapseg I eChCegv. 113: 141-144, 26

Ehrer 1997). Flow cytometric analysis showed that aplidine did not cause any visible disturbances (F) of the cell cycle (Iegra E, VaiYisopi S, ey a). Sei susie rpazez repibrayope ipaised ru pem/ paishga! tagipe ka sotrotspaz. Appaiv oi Opsoiodu. 7 (Zirri. 1): 82, 1996). In mice, aplidine showed activity against implanted leukemia RZ88 and melanoma B16, with an optimal dose of 1bOmkg/kg. In contrast to didemnin B, 60 aplidine showed activity against subcutaneously implanted carcinomas of the I em/iz lungs (Eaigsioii o, Kipepag

K, ey aiYi. YuOepuagodidetpip V a pem/ tagipe dehimey apiiisapseg adepi m/yyp asiimiu adaipvi ekhregitepia! Shtoig tode! 5. Appaiz oi Opsoiodu. 7 (Zirri. 1): 34, 19961.

Continuous exposure to low concentrations of aplidine inhibited the growth of a number of tumor cell lines, including non-Hodgkin's lymphoma, melanoma and breast cancer, ovarian cancer and non-small cell lung cancer 65. The magnitude of the effect depended on the exposure time and was achievable at non-myelotoxic concentrations.

Cell lines of non-small cell lung cancer, breast cancer, and melanoma were sensitive to continuous exposure to aplidine at concentrations of 0.001 μmol/L. Aplidin had a toxicity similar to that of doxorubicin against clonogenic stem hematopoietic cells (OereprgoskK N, Reieg K, ei aiYi.

Naetayoroieiiis rgesygzog seiIv. Vgyivp doshigpa! oh Sapseg 78: 739-744, Mo. 6, March 19981.

Aplidin showed significant activity against xenografts of malignant human tumors in mice. At the maximum tolerated dose of 2.1 mg/kg, aplidine induced almost complete remissions in some animals with a ratio of the tumor size of treated mice to the size of tumors in control mice (O/C) of 995. At 1.25 mg/kg, significant activity against gastric tumors was observed (0 /K 1495), as well as inhibition of prostate tumor growth (O/K 2590) (Raigsio(f b, OSgapi UM, ei ai. Rgesiipisai demyurtepi oi ariidipe, a pomei tagipe-dehimed adepi m/y roiepi apiyshtoig asiimtsu. Appaivz oi Opsoiodu, 9 (Zy, rri. 2): 34, 1998).

The authors of this invention have developed methods of treating human patients with aplidine that lead to clinical improvement.

Thus, the present invention relates to a method of treating any mammal, especially a person affected by a malignant tumor, which involves administering to the affected individual a therapeutically effective amount of aplidine or its pharmaceutical composition.

The present invention also relates to pharmaceutical preparations containing aplidine as an active ingredient, as well as methods of their preparation.

Examples of pharmaceutical compositions include liquids (solutions, suspensions or emulsions) with a suitable formulation for intravenous administration, and they may contain the pure compound or the compound in combination with any carrier or other pharmaceutically active compounds.

The administration of the compounds or compositions of this invention is based on the Dosing Protocol preferably by intravenous administration. The authors prefer to use an infusion time of up to 72 hours, more preferably 1-24 hours, most preferably about 1, about 3 or about 24 hours. Short infusion times that allow treatment without an overnight hospital stay are particularly desirable. However, the infusion can continue for about 24 hours or even longer if needed. The infusion may i) be carried out at appropriate intervals with patterns varying, as an illustration, once a week, twice a week or more often during the week, with repetition every week with optional breaks usually of one week. Additional guidance is provided later in this text.

The exact dose of this compound will vary according to the particular composition, the method of administration and the particular condition of the host and the tumor to be treated. Other factors such as age, body weight, sex, diet, time of administration, rate of excretion, host condition, drug combinations, response sensitivity, and disease burden must be taken into account. The introduction can be carried out continuously or periodically within the limits of so

Зз5 maximum tolerated dose. uh-

The aplidine compound and compositions of this invention can be used with other drugs to provide combined therapy. Other drugs may form part of the same composition or may be provided as a separate composition for administration at the same time or at a different time.

The identity of other drugs is not specifically limited, and suitable candidates include: "a) drugs with antimitotic effects that specifically affect elements of the cytoskeleton, including -ptv) including microtubule modulators, such as taxane drugs (eg, taxol , paclitaxel, taxotere, docetaxel), podophyllotoxins or Mips alkaloids (vincristine, vinblastine); and?" B) antimetabolite drugs (such as 5-fluorouracil, cytarabine, gemcitabine, purine analogues such as pentostatin, methotrexate); c) alkylating agents or compounds of nitrogen mustard (such as nitrosoureas, cyclophosphamide or ifosfamide); -And I) drugs affecting DNA, such as anthracycline drugs adriamycin, doxorubicin, pharmorubicin or epirubicin; bo f) drugs with target topoisomerases, such as etoposide; - C) hormones and agonists or antagonists of hormones, such as estrogens, anti-estrogens (tamoxifen and related compounds) and androgens, flutamide, leuprorelin, goserelin, cyprotron or octreodide; co 9) drugs that affect signal transduction in tumor cells, including derivatives

F antibodies, such as Herceptin;

MR) alkylating drugs, such as platinum-containing drugs (cisplatin, carboplatin, oxaliplatin, paraplidineatin) or nitrosoureas; i) drugs that potentially affect tumor metastasis, such as inhibitors of metalloproteinases of the extracellular matrix;

Ф) І) gene therapy and antisense agents; ka K) antibody-retaining therapeutic agents;

I) other bisactive compounds of marine origin, especially kahalalide E or ecteinascidins, such as EI-743; in t) protective agents of skeletal muscles, such as carnitine supplements; o) other drugs that eliminate the side effects of aplidine, such as antiemetics; p) more often medicines that allow you to administer aplidine in the recommended dose and cope with toxicity.

The authors of the invention found that aplidine inhibits the expression of the gene (PI T1) encoding the vascular endothelial growth factor receptor (MESRE).

In addition, it was found that aplidine strongly inhibits the production of the MESE protein itself by tumor cells.

The secretion of MESE by a cell mass, in particular, a mass of tumor cells, causes the formation of de pomo blood vessels (angiogenesis), which leads to the formation of new blood vessels that are formed in the direction of this cell mass and form a network of capillaries that is able to supply blood to this cell mass

For its long-term proliferation. It is expected that these effects, in particular, demonstrated elimination of production

MESE by tumor cells will strongly inhibit the ability of tumor cells to continue angiogenesis. In addition, MESE is required directly for some tumor cells from hematopoietic tissue (such as cells

MY AND human leukemia) as a growth factor.

Thus, it can be predicted that aplidine has an inhibitory effect on the vascularization of de pomo 7/0 growing primary tumors or metastases, thus inhibiting the growth of tumors that are known to require vascularization for growth. Aplidin should also be active on hematopoietic tumors.

In addition, it is known that aplidine modulates the function of calcium channels in cells.

Bladder tumors are one of the types of tumors that overexpress the epithelial growth factor (EGF) receptor, which leads to the upregulation of MEGE and the MEGE receptor. Binding of MESE to its 7/5 receptor is believed to stimulate cell growth through transient local changes in calcium ion content in addition to other signal transduction mechanisms. A compound that inhibits the action of MES is expected to inhibit such tumors.

Experimentally, aplidine has been found to have extremely high activity against human bladder cancer (providing complete remissions in some animal models), consistent with this prediction.

Aplidine can be predicted to have broad-spectrum antitumor activity due to its effects on a large number of tumors.

The action on MESE is more narrowly specific, as it includes inhibition of the formation of new blood vessels. In addition to actions on blood vessels, some tumors require MESE directly for cell growth (namely, leukemias, lymphomas, bladder tumors, and ovarian tumors). high school

Thus, the authors developed a method of treating angiogenic conditions, which involves the administration of aplidine.

In particular, the authors developed a method of tumor treatment that depends on the angiogenic process. Further, this i) invention relates to a method of preparing an inhibitor of angiogenesis, cancer invasion or cancer metastasis, which involves mixing an effective dose of aplidine with a pharmaceutically acceptable carrier.

The authors have also developed a method of treatment of disorders related to angiogenesis, such as retinopathy, which are not related to tumors.

In particular, the authors have developed ways to treat disorders of angiogenesis, such as those involving neoplasms, including metastases; eye conditions such as corneal transplant rejection, diabetic -/- de retinopathy, retrolental fibroplasia and neovascular glaucoma; ulcer diseases, such as stomach ulcers; and other conditions, such as infantile hemangiomas, angiofibroma of the nasopharynx and avascular (avascular) bone necrosis; and disorders of the female reproductive system, such as endometriosis. uh-

Responses in cancer patients were observed in clinical trials with aplidine and demonstrated the applicability of this treatment modality.

Phase I clinical trials and pharmacokinetic analysis demonstrate that aplidine provides a positive therapeutic window with manageable toxicity within the range required for clinical efficacy."

In the treatment of cancer patients. This method consists in the introduction of the medicinal product by intravenous infusion within a period of 72 hours. or less at the recommended dose level (RD) with or without combination with other therapeutic agents.

Aplidin is supplied and stored as a sterile lyophilized product consisting of Aplidin | filler in a composition suitable for therapeutic use. -I Solubilized aplidine exhibits significant degradation under heat and light stress testing conditions, and a lyophilized dosage form has been developed, |see UU099/42125)|, incorporated herein by reference. In the currently preferred variant, lyophilization was performed from a solution of aplidine 50 mg/ml in 4095 (v/v) tert-butanol in water for injections (WY), containing 25 mg/ml of Yu-mannite as a bulking agent . It was found that the 5p prototype, containing 500mg of aplidine and 25mg of O-mannitol as a bulking agent per vial, was the optimal composition with respect to solubility, duration of the lyophilization cycle and dose requirements in clinical trials. It was found,

F that the optimal solution for reproduction was a mixture of 15/15/7090 (vol./vol./o06.) Stetarnog EI /ethanol/LI (SEMU).

Both the reconstituted product and dilutions (up to 1:100v/06) of the reconstituted product with normal saline appeared to be stable for at least 24 hours after preparation. Shelf life data available so far indicate that this formulation is stable for at least 1 year when stored at 42°C in the dark. iF) Preparation of the infusion solution is also performed under aseptic conditions by taking the volume of reconstituted solution corresponding to the dose calculated for each patient and slowly introducing the necessary volume of the reconstituted solution into an infusion bag or glass containing between 100 and 1000 ml of 0.995 sodium chloride , after which everything is homogenized by slow manual shaking. Aplidin infusion solution should be administered intravenously, as quickly as possible, within 48 hours after preparation. PVC and polyethylene infusion systems, as well as clear glass systems, are preferred materials for containers and tubing.

Administration is carried out in the form of cycles, in the preferred method of application, intravenous infusion of aplidine is given to patients in the first week of each cycle, patients are allowed to return to a normal state b5 during the rest of the cycle. The preferred duration of each cycle is 3 or 4 weeks; if necessary, multiple cycles can be performed. The drug can also be administered on each of the first days of each cycle. Dose administration delays and/or dose reductions and corrections of the administration scheme are carried out as necessary, depending on the tolerance of the procedures by the individual patient, in particular, dose reductions are recommended for patients with higher plasma levels of transaminases or alkaline phosphatase of the liver or bilirubin compared to normal levels.

The recommended dose (RD) is the highest dose that can be safely administered to a patient, yielding tolerable, controllable, and reversible toxicity according to the general toxicity criteria established by

by the US National Cancer Institute, with no more than two out of six patients exhibiting any dose-limiting toxicity (I t). 70 The main guidelines for the therapy of malignant tumors often recommend the introduction of chemotherapeutic agents at the highest safe dose, at which the toxicity is such that it can be controlled, to obtain the maximum effectiveness .A., Sapseg: Rgipsirievg apia Rgasiise oi Opso|iodu,

Z'Ya ey., 1989, MOripsoyi, Rpyadei!rnpia).

BIT for aplidine using this method of treatment was determined in clinical trials. These 75 trials established the recommended dose level for different dosing protocols.

Aplidin can be safely administered at dose levels corresponding to the recommended dose (RD) or at a lower level.

Infusion is currently the preferred procedure, and typical administration schedules include the following: a 24-hour infusion once a week for a number of weeks, such as three weeks, followed by one week off; 24-hour infusion every two weeks; 1-hour infusion once a week for 3 weeks every 4 weeks; infusion once a day, for example, 1 hour x 5 days every 3 weeks; and infusion, for example, From an hour every other week. Ge

In particular, the intravenous infusion can be carried out as a 24-hour infusion once a week for 3 weeks every 4 weeks. Additional data are given in examples 3, 4, 11 and 12. It appears that the recommended dose of 375 Ωkg/m "/week x3 is appropriate. This protocol has been corrected and patients will now be treated using another administration schedule that seems feasible: 3 -hourly infusion every 2 weeks without rest. See Example 12. In a 24-hour infusion trial every two weeks, patients receive an infusion of 7O0Omkg/m2/week 2. See Examples 6, 14, and 18. Patients included in the trial with 1 hour ./week x3 every 4 weeks, receiving doses up to 200 µg/m2/week x3 weeks. See Examples 13 and 17. Another protocol involving patients with a daily 1-hour infusion x5 days every 3 weeks gives patients a dose of 1200 µg/ m2/day x 5 days. When using aplidine in combination with other therapeutic agents, it may be necessary to adjust the doses of both agents.

Previously, the major biological responses reported for aplidine administration were observed in animals or in vivo models, which are well known to be imprecise with respect to their applicability in predicting response in human patients, or in human patients under experimental conditions where an effective, safe method of treatment was not available (or the dose used was a toxic dose, significantly increased "

Regarding the recommended dose or the administration scheme was inappropriate). in c In clinical trials using the method of this invention, the corresponding levels in plasma were reached in . patients with RD and, most importantly, the reactions that are objectively measured in response demonstrated and? evidence of clinical benefit to patients.

Definitions for patient toxicity are borrowed from the WHO (World Health Organization) and these responses are defined according to the WHO response criteria. -I Objective reactions were obtained in patients with advanced and/or metastatic cancer that did not respond to previous treatment methods, which included the reactions described in the examples. In particular, treatment using this method has shown responses in cancer patients with advanced and/or metastatic disease who have developed advanced disease after an earlier 5-hour treatment with conventional standard therapies. So, a preferred method of the present invention includes the identification of cancer patients who

F were treated for cancer, in particular, patients receiving chemotherapy and their treatment with aplidine.

Fig. 1 shows a decrease in the expression of YANI observed using microarrays, confirmed by OT-PCR analysis.

Figure 2 shows a decrease in mRNA 1-1 induced by aplidine in MY T-4 cells.

Fig.Za and ZB show the autocrine loop of MESE-P-1 in MY T-4 cells and the effect of aplidine.

F) Fig. 4 shows that aplidine inhibits the secretion of MESE from MY T-4 cells. ka Fig. 5 shows aplidine-induced inhibition of MESE secretion.

Fig. b6 shows a strong decrease in MESE mRNA levels in MY T-4 cells. in Fig. 7 shows that aplidine does not reduce the activity of the MESE promoter transfected into MY T-4 cells.

Fig.8 shows that aplidine does not inhibit the binding of the transcription factors NIB-1 and AP-1 with their consensus DNA sequences present in the MESR promoter.

Fig.9 shows that aplidine does not inhibit the binding of the transcription factor NIB-1 with its consensus DNA sequences present in the MESR promoter. 65 Figures 10a and 106 show that MESE added to the culture medium of MY T-1 cells slightly reduced the activity at low concentrations of aplidine, while there was no effect at high concentrations.

Fig. 11 shows that aplidine is able to reduce the secretion of MESE from the ISKOM-1 line of human ovarian cancer.

Fig. 12 shows that aplidine also reduces the levels of MESE mRNA in the ISKOM-1 line of human ovarian cancer.

Fig.13 shows that aplidine does not affect the promoter activity of MESE, measured using the system of Deporter genes of luciferase/hepia.

Fig. 14 shows the dependence of ALSHS on the dose.

Figures 15a and 156 show activity in medullary thyroid cancer: CEA levels.

Examples of the invention

Example 1 70 Characteristics of gene expression in leukemic cells of MY T-4 treated with the compound aplidine obtained from marine organisms

Early changes in gene expression induced by aplidine in MY T-4 cells were assessed using cDNA expression matrices (Ayaz Nytap Sapseg, Siopiesp). MY T-4 cells were treated for 1 hour with 5095 growth inhibitory concentrations of aplidine, and total RNA was isolated at 0, 1, 6, and 24 hours 75 after drug washout. Filters were hybridized with an equal amount of Z2p-labeled cDNA. Analysis of the results was carried out using the ATI A5 IMACE 1.0 software. Changes in gene expression greater than 2-fold were taken as significant changes in RNA expression and then confirmed using PCR.

A noticeable time-dependent decrease in the expression of MESB-KI1 (T-1) was observed and it was confirmed at the level of

RNA with the help of PCR and at the protein level with the help of Western blotting.

Example 2

Correlation of the selective antitumor activity of the compound aplidine obtained from marine organisms using different model systems

Various model systems were evaluated to provide a basis for further clinical investigation. Selective antitumor activity was observed against two histologically different solid tumors: gastric carcinoma and human prostate cancer. Strong ip miigo activity against primary gastric tumor samples or gastric tumor cells Ne746T is proved by IC 50 values of 146 and 450 pM, respectively. Less strong, but no less selective ISeo-activity of Z,4NM, was determined against tumor cells PO-3 of the prostate gland. IP migo activity was evaluated in nude rodents using subcutaneously (c) implanted tumor fragments or hollow fibers (HE) containing tumor cells. (Te) 7 so -

Tumor Line Scheme all-model Animal (Dose | Activity

Mo Mo Mon Mon today so 0.62 and 7 s 1.05. " inf. -I Optimal activities were observed in such xenograft tumors of the stomach (17-2096) and prostate gland (25-3890) after intraperitoneal (ir) administration. Further studies led to the use of rats for intravenous infusions (im). With this by changing the scheme with a 24-hour infusion gave similar activity against tumor cells of the stomach in NE (2095) and tumor cells of the prostate gland in NE (31965).

Cytotoxicity was also detected using a 5-day infusion schedule against prostate tumor cells in HE (3395). Advanced ip mimo scores not only show that there is a strong relative

F correlation with the cytotoxicity profile of ip mygo, but also a strong correlation with ip mimo models used to characterize the tumor selectivity that identified aplidine as a candidate for clinical development. 5B Example C

Phase I trials! and a pharmacokinetic study of aplidine given as a 24-hour infusion (F. once a week, in patients with advanced solid tumors ka Studies of ip mimo found that the activity of ip mimo increased by prolonging the duration of the infusion. In this study, treatment was carried out with 16 patients Patient characteristics: median age 55 years, 60 median RB5 1, male/female ratio 11/5, tumor types as follows: head and neck 5, kidney 2, colon

C, rectal 2, sarcoma 1 and melanoma 3, all had prior chemotherapy (HT lines with median 2).

Aplidin was administered as a 24-hour infusion at the following dose levels (01): 133 (Z patient), 266 (Z patients), 532 (Z patients), 1000 (Z patients), 2000 (Z patients), and 3000 (1 patient ) μg/m? per week x3 every 28 days. 65 Dose-limiting toxicities (0IT) were not observed. Only mild non-hematological toxicities were reported, consisting of stage (5) 1 nausea, stage (b) 1 mucosal inflammation, stage (b) 1 asthenia

(b) 1. Phlebitis of the shoulder where the drug was administered was present in all patients and was concentration-dependent.

Pharmacokinetic analysis was performed in all patients and showed plasma levels at dose levels (01) of 100 Ωkg/m/week. and 200 Ωkg/m2/week, equivalent to the active IP mygo concentration (1Tng/ml). At the same dose (01)

332mcg/mo/week, clinical improvement was observed in a patient previously receiving chemotherapy with advanced melanoma.

Example 4

A Phase I Trial and Pharmacokinetic (PK) Study of Aplidine (API) Using a 24-Hour Once-Weekly Administration Schedule

To date, 20 patients (median age 58 years, median ESOS 1) with advanced, previously treated solid tumors have been treated in a phase I trial using a 24-hour aplidine regimen (ARI) once weekly x3 followed by 1 week rest, the following dose levels were tested: 133 (3 patients), 266 (3 patients), 532 (3 patients), 1000 (3 patients), 2000 (Z patients), 4500 (Z patients) and 3750 (3 patients) μg/m "/week. When administered in the form of 60 cycles (180 infusions), all 19 patients were evaluated for toxicity. The maximum tolerated dose was 4500 μg/mo/week x3 with stage (5) C muscle toxicity (biopsy showed muscle atrophy type II), IBS 04 and transaminitis OZ and dose-limiting toxicity in 2 or 4 patients. Discomfort 52 was observed in most patients and vomiting 2/3 at 2000μg/m2?, phlebitis was present in all, but dependent on concentration. All patients took samples for LC analysis (pharmacokinetics analysis) with the help of I C/M5/M5 (LC/MS/MS) . Preliminary data indicate appreciable tissue distribution, long elimination (Its 1/2 10-24 hours) and plasma levels of "ng/ml (value, active ip migo). One patient with advanced melanoma resistant to DTIC (dacarbazine)/ interferon, had clinical improvement that was sustained over 230 weeks. This once-weekly infusion study demonstrates the feasibility and activity of an aplidine regimen (ARI) with doses "densely" administered. As expected, neuromuscular toxicity is sch o Such, which limits the dose The possible recommended dose has been estimated as 3750mcg/m² per week x3.

Example 5 i)

Clinical pharmacokinetics (PC) of aplidine (ARI) in patients with solid tumors and non-Hodgkin's lymphomas

Four IV regimens are being evaluated in a Phase 1 trial: a 24-hour infusion once a week, a 24-hour infusion every two weeks, and a 1-hour infusion on 5 consecutive days every 3 weeks.

Aplidin concentrations in the blood are analyzed by liquid chromatography-tandem mass spectrometry. Initial results show accumulation in blood cells, and pharmacokinetics in plasma (PC) is characterized by extensive distribution (the volume of distribution usually exceeds 200 l/m2) and plasma half-lives of the order of 10-24 hours. The open two-compartment model is suitable for most profiles after a 3-5 24-hour infusion. For 1-hour infusions, the three-compartment model provides a better fit in most cases. It is known that the obtained plasma levels are active IP. Evaluation of additional patients and comparison of pharmacokinetics of blood cells and plasma is carried out.

Example 6 "

Preliminary results of the research phase | and a pharmacokinetic study of aplidine given as a 470 24-hour infusion every 2 weeks in patients with solid tumors and non-Hodgkin's lymphoma - c Aplidine is given as a 24-hour infusion every 2 weeks. The initial dose was 20 Ωkg/m 2/day and "» dose escalation occurs to 400, 800, 1600 and 3200 Ωkg/m 2/day. A total of 18 patients were studied " (males/females: 7/11, median age 52, OM5 0/1 : 10/8). So far, no dose-limiting toxicity has been observed.

Among evaluable patients, toxicity was grade (c) I-II mild nausea/vomiting, stage (0) asthenia, and spasms occurring during or immediately after infusion. Neuromuscular toxicity was not reported at the doses evaluated. One patient with advanced lung cancer and documented

Go) with the progression of the tumor at a dose of 160Omg/m 2 developed hemolytic anemia and thrombocytopenia, which were considered unlikely to be related to the drug used in the study. Primary pharmacokinetic analysis showed that the drug is widely distributed and blood concentrations were determined. To

Ho) 20 most plasma concentration profiles are well suited by a 2-compartment model. The final half-life from plasma is usually within 10-24 hours. Clinical improvement was observed in m, a patient with non-Hodgkin's lymphoma. Dose escalation is being investigated to determine the dose-limiting toxicity and the dose that will be the recommended dose in phase II trials.

Example 7

Characterization of gene expression in human MY T-4 leukemic cells treated with marine

GF) of organisms with the compound aplidine

In this study, the authors of the invention evaluated the early changes in gene expression induced by aplidine in MY T-4 cells using cDNA expression matrices (AMaz Nytap Sapseg, SiIopiesp). The cells are MINE! T-4 was treated for 1 hour with 5095 growth-inhibitory concentrations of aplidine, and total RNA 60 was isolated at 0, 1, b, and 24 hours after drug washout. Filters were hybridized with equal amounts of ZgR-labeled cDNA. Analysis of the results was carried out using ATI AZ software

IMASE 1.0. Changes in gene expression greater than 2 times were taken as significant changes in RNA expression and were then confirmed using PCR. A noticeable time-dependent decrease in the expression of MESB-K1 (yJ-1) was observed and it was confirmed at the RNA level with the help of PCR and at the protein level with the help of Western blotting. whether the negative regulation of Y-1 is involved in the cytotoxic and antitumor effect of aplidine is currently being investigated. At this time, we are also characterizing the expression of other genes that are apparently negatively regulated after exposure to aplidine.

Quantitative changes in gene expression induced by aplidine in MY T-4 cells

Weight 05005 o vv i 1 dyaRk 00300 incl. 1110 by 75 The decrease in expression of 1-17 observed using microarrays confirmed

RT-PCR analysis, see Fig.1.

Using RNase protection, the authors quantified the decrease in 4-1 mRNA induced by 20 nM aplidine in MY T-4 cells, see Fig. 2.

Fig. ZA and 3B show the MESB-R-I autocrine loop in MY T-4 cells and the effect of aplidine in the autocrine loop

MESE-H1.

Aplidin blocks the secretion of MESE from MY T-4 cells, see Fig. 4. Cells were treated for 1 hour with 200 nM aplidine. MESE, secreted into the medium, was measured with the help of EGISA at the end of treatment and after 6 and 24 hours of incubation in a drug-free medium.

Aplidin-induced inhibition of MESE secretion is concentration-dependent and is observed already at 5nM, see Fig. 5.

With the use of protection against RNases, a strong decrease in the mRNA levels of MESE was observed in MY T-4 (o) cells after 2 ONM aplidine, see Fig. b

Aplidin does not reduce the activity of the MESE promoter transfected into MY T-4 cells, see Fig. 7. Cells were transfected with the MESE promoter (spanning the first 1000 bases to the left of the start site associated with the luciferase reporter gene) and a control plasmid containing the hepia reporter gene. Then the cells were treated with different concentrations of aplidine and luciferase activity was measured after 24 hours and compared with hepiNa activity. "-

Aplidin does not inhibit the binding of transcription factors NIB-1 and AR-1 to their consensus DNA sequences present in the MESE promoter, see Fig. 8. Nuclear extracts were incubated with different concentrations of aplidine and labeled oligonucleotides for 60 minutes. M binding of NIE-1 or AP-1 was observed using the gel retention assay.

Aplidin does not inhibit the binding of the NIB-1 transcription factor to its consensus DNA sequences present in the MESE promoter, see Fig. 9. Nuclear extracts obtained from cells treated with different concentrations of aplidine were incubated with labeled oligonucleotides for 60 minutes. Binding of NIB-1 was observed using the gel retention assay. -at

MESE (1 ng/ml), added to the culture medium of MY T-4 cells cultivated in 1095 FTS, slightly reduced the activity of low concentrations of aplidin, while at high concentrations there was no effect, see :z» Fig. 1ba, 105.

Aplidin is also able to reduce the secretion of MESE from the IZKOM-1 human ovarian cancer cell line, see Fig. 11. 415 Aplidin reduces MESRE mRNA levels also in human ovarian cancer line ISKOM-1, see Fig. 12. -1 Aplidin does not affect the activity of the MESE promoter measured using the luciferase/hepiPa reporter gene system, see Fig. 13. (ee) Example 8 - Aplidin inhibits the secretion of MESE and the MESEL/ESBE-K1 autocrine loop in a human leukemic cell line

Aplidine was found to induce strong apoptosis in the MY T-4 human leukemic cell line. In the same (o) cell line, analysis using microarrays revealed changes in the expression of various genes in the early periods

F time after processing. Among them, the authors found that MESB-K1 (1-1) was negatively regulated by drug treatment and its negative regulation was confirmed by northern and western blotting analysis. Additional studies showed that treatment of the same cell system with this compound resulted in a strong decrease in secretion

MESS in the environment. The decrease in the secretion of MESE was associated with a decrease in the mRNA encoding MESE in MY T-4 cells treated with aplidine. Trying to find out the mechanism by which aplidine inhibits (F) secretion of MESE, the authors of the invention found that this compound does not change the half-life of MESE mRNA. d Similarly, using electromobility shift analysis, the authors found that aplidine did not change the ability of the two transcription factors NIBR-1 and AR-1 to bind to their corresponding (consensus) sequence, because it is present in the MESE promoter, and did not reduce the transcription of MESE when using promoter structures

MESE-luciferase in transient transfection experiments. Decreased secretion by aplidine was associated with increased intracellular accumulation of MESE, suggesting with high confidence that this compound could act through inhibition of MESE secretion. Simultaneous treatment of MY T-4 cells with low concentrations of aplidine and MECE partially reduced the activity of aplidine, suggesting that this drug could partially exert its activity in this cell system by inhibiting the MECE/L/ESB-KI1 autocrine loop.

Example 9

Safety characteristics of miigo aplidin, a marine natural product with chemotherapeutic potential

When using the cytotoxicity analysis of the mouse SePPieg9b (MT5, Rgoteda), aplidine shows little toxicity to the liver (AMI-12) or to the heart (HO c2 (2-1); GO 50 1mM). In contrast, aplidine is highly toxic to skeletal muscle (18) and kidney (MRK-52E) cells (GO 5o 0.1nM) with intermediate toxicity to myelogenic stem cells (ROS-P1T, GO 5o 0.mM), respectively close to the clinical toxicity data. In fact, the dose-limiting toxicity in humans is skeletal muscle atrophy.

Aplidin shows neurotoxicity at higher concentrations of ip mygo. Using a 7/0 fluorescent viability dye (ethidium and calcein AM homodimer, Moiesciag Rhobrez) together with immunochemistry, the authors observed that approximately 1 μM aplidine was toxic to brain cells (both neurons and astrocytes) and motor cells (positive in choline acetyltransferase) of neurons in the spinal cord, but positive for substance P of sensory neurons. The sensitivity of motor neurons may help explain the type II muscle atrophy observed (as predicted) in a small group of /5 patients where the AOS concentration of the drug is increased due to reduced excretion.

Example 10

A phase I trial of aplidine in a b-day bolus regimen every 3 weeks in patients with solid tumors and lymphomas

The objective was to determine the maximum tolerated dose, dose-limiting toxicity (OT), pharmacokinetics (PK) and dose recommended for phase 2 trials that can be administered in a schedule of 1-hour intravenous (IM) infusion x 5 days every 3 weeks. Patients with solid tumors and low or intermediate stage non-Hodgkin's lymphomas were selected. The initial daily dose of aplidine was 8 Ωkg/m2. Groups of 3 patients were treated at each level with escalating doses according to toxicity. 20 patients were treated with b dose levels in the range of 8Omkg-720mkg/m2, 1 patient is currently on a dose of 9bOmkg/m2. A total of 48 cycles were administered. Non-haematological toxicities were grade 1 and 2 (5) with fatigue reported in all patients. at

Stage (0) 1 allergic reactions were documented in 7 patients. Other toxicities included nausea, anorexia, diarrhea, and anxiety. There were no hematological toxicities. LC-analysis was performed in the treatment of course 1. Concentrations of aplidine were analyzed by C (liquid chromatography) with tandem mass spectrography.

The results suggest a linear LC of doses with high interpatient variability. There was complete clearance from the body. (about 0.Zvl/min. and the median 1/2 was 14.2 hours. Potentially therapeutic plasma concentrations (21 μg/ml) were reached. No objective reactions in response were documented. 1 patient with colon cancer was stable for 9 months and 1 patient with renal cell carcinoma had a mixed response. No dose-limiting toxicity was documented as a result. Extended study continues at 96 Ωkg/m 2. so

Example 11

Zo Clinical trial of phase I and pharmacokinetic study of aplidine, a new marine didemnin, which in administered as a 24-hour infusion once a week

Phase I trials were performed using a 24-hour infusion once a week x3 followed by 1 week of rest. 32 patients (median age 58 years, median ESOO 1) with advanced, previously treated solid tumors were treated. They received 64 courses (median/patient 2 (1-6)) at 8 dose levels: 133 (3 patients), 266 (3 patients), 532 (3 patients), 1000 (3 patients), 2000 (3 patients), 3,000 (3 patients) with 4,500 (4 patients) and 375 µg/m"/week (10 patients). 2 of the evaluated patients had a limiting dose: with" toxicity (017) at 4,500 µg/m"/week. : reversible neuromuscular toxicity stage (C) 4 (biopsy-obstructive muscle atrophy type II) and SC increase (4 (1 patient) and transient transaminitis 53 (1 patient). Other toxicities included discomfort C 1-2 ( most patients at 3000 mcg/m/week), muscle spasms, -And vomiting (31-2 (susceptible to antiemetics) and injection site reaction (very common and concentration-dependent). All patients were sampled for analysis LC according to method I C/M5/M5 (LC/MS/MS). The pharmacokinetics of CO are linear and the profiles correspond to the open model with 2 compartments. The drug has extensive - tissue distribution (Me5-611/l), high clearance (0, 47 l/min.) and its 1/2 el imination 18.8 hours. Levels that are maintained for 5 years in plasma » ng/ml (active ip migo) were obtained at doses of » 30 bomkg/m2. One patient with DTI/interferon-resistant advanced melanoma had some clinical improvement that was sustained over 230 weeks.

F Four additional patients had small responses or stable disease at 24 months. Thus, the dose-limiting toxicities (01 T) of aplidine administered by a once-a-week infusion regimen were reversible muscle toxicity and transaminemt, which showed a MTO (maximum tolerated dose) of 4 BOOmkg/m2/week. The recommended dose for future trials is 375 Ωkg/m 2/week. xZ administered through a central venous catheter is possible and associated with low toxicity. Example 12

A Phase I Clinical Trial and Pharmacokinetic Study of Aplidin, a Novel Marine Didemnin Administered as a Once-Weekly 24-Hour Infusion in Patient Characteristics

Number of patients. 35 Type of tumor

Gender (male/female) 23/12 Colon 12

Median age, years (range) 56.5 (29-74) Kidneys b

Execution of ESOS Head and neck 5 b5

Status (0) 12 Melanoma 4 1 18 Stomach 2 2 5 Breast 1

Earlier radiotherapy of soft tissue sarcoma 1

Previously chemotherapy

There were none 4 Lymphoma 1 70 1 course 9 Thyroid gland 1 2 courses 12 Cancer of unknown origin 1

From the 10th course

Areas of the disease 1 14 2 12

With d)

Increasing doses (of patients with PE POT o 0103018 0110031 yurvv cm o

F

With I oats 0020161 so

The worst toxicities per patient "- 0

Nudoa 62 1 111111 ev

Astnya 02 1 011 « et 2 Reakiyametsyavted i? 011113 z s mez 83111111 sh Zbilshenyastk 5/1 ' Tvanemnkh 00681012 et 15 Allertyad/ 83111111 -I o Antitumor activity

Patient Me008 - (Madrid) We observed clinical improvement and reduction of the tumor of a previously heavily treated, unmeasurable metastatic melanoma. A biopsy of one of the metastatic lesions on page 50 did not show the presence of residual tumor tissue.

Patient Me032 - (Madrid) Renal carcinoma: reduction of the tumor 2095. 4; Patient Me034 - (Madrid) Medullary carcinoma of the thyroid gland. Clinical improvement and 5O in lung lymphangitis. Reduction of CEA marker (carcinoembryonic antigen?).

Pharmacokinetics

Aplidine concentrations in plasma were determined by liquid chromatography/tandem mass spectrography with a threshold of quantification of 0.25 ng/ml and a wide linear range up to 16.00 ng/ml.

A total of 15 samples were taken up to 24 hours after the end of the infusion. eat) Linear pharmacokinetics depending on the dose.

High median CI (clearance) (quartiles) 0.47(0.40-0.56) l/min) and interpatient variability (coefficient of variation of clearance (CI), 45965)

Intermediate - long half-life (I 1/2) (median (quartiles) 18.8 (15.3-25.4) hours).

Wide distribution with supraphysiological volume of distribution (Mev) (median (quartiles) 611 (434-733)L).

Accumulation in blood cells (2-8 times higher than in plasma). 65 Profiles correspond to the open model with 2 compartments.

Fig. 14 shows the dependence of ALSHS on the dose.

Conclusions

Dose-limiting toxicity (OT) of aplidine administered using this regimen was reversible muscle toxicity and transaminitis observed at the maximum tolerated dose (MT) of aBObmkg/m week. x3.

The recommended dose for future trials is 375 Ωkg/m g/week. x3 is possible and is associated with mild toxicity (mainly mild asthenia).

Phlebitis in the shoulder at the infusion site was present in all patients, depended on the concentration and could be avoided by administration through a central venous catheter.

Hematological toxicity was not observed.

Pharmacokinetics (PK) is characterized by a linear dependence on the dose, relatively prolonged presence of the compound in the body and extensive distribution. Potentially active plasma levels were reached from 2000 μg/m2.

An additional phase I trial investigating a 33-hour IM infusion every other week is ongoing.

The initial dose is 3000 μg/m2 every 2 weeks. 12 Example 13

Test phase | aplidine administered as a 1-hour intravenous infusion once weekly in patients with advanced solid tumors and lymphomas

Adult patients with an advanced disease, P "3, and the corresponding organ function are considered acceptable; patients receive aplidine once weekly xZ/every 4 weeks. 24 patients with solid tumors were registered: median (t) age 55 years, median ESOS-1, 15 of 24 patients were treated with -22 cycles of treatment. Seven levels of doses (01) from 13Zmkg/m g/week. up to 270 Ohmkg/m2/week. were evaluated: 102 infusions were evaluated for toxicity (oh). Hematological toxicity was not reported, vomiting requiring prophylaxis was observed with 8OOmkg/m"/week. At 27OOmkg/m"/week. (4 patients) 1 patient had C3 hyperbilirubinemia, which was considered dose-limiting, and therefore the dose level of 2700 μg/m"/week was studied in more detail. All patients were sampled for pharmacokinetic analysis (PC) (GS/E5BI-M5B/ M5 - PH/EBI-MS/MS); kinetics were linear, and9) median MevZOVl/M, clearance (CI) is high with t-0.bOl/month and median half-life t-14.2h.

Plasma levels of ng/ml 24 hours after infusion are reached from 180 Ωkg/m g/week. Early indications of activity in gastric cancer were noted (1 patient at 1200mcg/m7). A patient with advanced renal cancer, ISE) resistant to MVIMIEM, had an objective response (PE pulmonary metastases and 50 in peritoneal disease) that continued at the dose level (01) 2700mcg/m"/day. Aplidin is apparently clinically applicable at pharmacologically acceptable dose levels. --

Example 14 p

Phase I clinical and pharmacokinetic study of aplidine administered as a 24-hour continuous 3o infusion every other week (d2m/) in patients with solid tumors and lymphoma in

Aplidin was administered to patients with solid tumors/MNI. (non-Hodgkin's lymphoma) in the form of a 24-hour infusion/d2m/; 35 patients: (median age-51, median ESOS-1) with a solid tumor (32 patients) or MNI (C patients). 23 of 35 patients were previously exposed to 53 previous courses of chemotherapy. Nine dose levels were administered: 20 (200-700 Ωkg/m g/week/d42mu) and 65 cycles (120 infusions). Hematological toxicity was not reported. Toxicity consisted of asthenia and vomiting in 9-2 patients and 12-1 patients, respectively. Stage 3 nausea/vomiting (x5ObOMKkg/m 2 ) was effectively treated and then prevented by regimens with 4NTZ3. Cardiac toxicity is not ;" reported At a dose of 500Omkg/m "/week/d2mo, 2 patients experienced short-lived muscle spasms with reversible increases in IBS-MM (53. Among 9 patients receiving bOOOmkg/m g/week, C patients experienced

Increase of IBS-MM and aldolase after the 3rd administration of aplidine. Increases in IBS were stage (1-2) and were not symptomatic in 2 patients, but increase in IBS with stage 53 muscle pain and loss of muscle strength (01 t) was reported in 1 patient. It was reversible and had no sequelae . Muscle biopsy did not reveal significant necrosis of myocytes. Ultrasound electron microscopy did not reveal morphological changes of mitochondria, but showed the loss of thick myosin filaments. At 700Omkg/m"/week/42m/h, 4 patients were studied. at 20 Pharmacokinetic analysis ( And S/E5I-M5/M5) showed that the increase in AOS is linear, with a large M "5-5391 l/m, a high clearance (332 ml/mm.mo) and a long terminal half-life (15-35 hours). Concentrations in plasma concentrations 24 hours after the end of the infusion at doses of x300Omkg/m"/week/42mu are comparable to the effective ip omygo concentrations ("Tng/ml). Activity was observed in the case of MNI. (non-Hodgkin's lymphoma) (1/3 of patients ), medullary thyroid cancer (2/2 patients), kidney cancer (1/5 patients) and 29 neuro endocrine cancer (1 patient). A hypothesis about the mechanism of action and preventive strategies against

GF) muscle toxicity.

Example 15 o Analysis using micromatrices

The MY T-4 human leukemia cell line was used for these experiments. MY T-4 cells were treated with 2O0nM aplidin for 60 minutes for 1 hour. Total RNA was extracted at the end of treatment and after 6 and 24 hours. after extraction in a drug-free environment. 5μg of total RNA was reverse-transcribed to obtain cDNA in the presence of З2Р'dDATP. Equal amounts of radioactive probes were hybridized with Ayaz Nytap Sapseg Misgoagtauz microarrays (Siopiesyp). After washing, the filter was exposed and the results were analyzed using ATI A5 and IMASE software. Only differences in gene expression greater than 2 times were taken into account. RT-PCR and Northern analysis were performed to confirm changes in gene expression detected with microarrays.

The results

Aplidine treatment did not induce significant changes in gene expression as early as 1 h after treatment. After 6 and 24 hours. the expression of an increased number of genes was observed after exposure to a drug-free medium.

At the end of the treatment, the most significant changes were observed in the expression of the early response gene (ETK) and in the expression of the MESB-KI1LT1 gene, which respectively increased and decreased with treatment.

ETC levels returned to normal levels after 24 hours. and 24 h., while YJ-1 levels additionally 7/0 Increased after exposure to drug-free medium.

Aplidin also induced an increase in the levels of B-KAE and ERtv, which is clearly observed due to bhod. after exposure to a drug-free environment.

The changes observed in these genes were confirmed either by RT-PCR or by Northern blot analysis.

Differences in gene expression obtained from analysis using microarrays (not yet confirmed

RT-PCR) was observed for other genes, such as RI K-1.

Example 16

Phase I study of aplidine in a 1-hour infusion schedule once daily x5 every 3 weeks (543) in patients with solid tumors and low- or intermediate-stage non-Hodgkin's lymphoma

The results

27 patients were studied

Which are evaluated for toxicity 24

Outside the study 18

The disease started in 14 Ha

A disease with the development of symptoms 2 o

Death 1

Others (according to the doctor's decision) 1

Characteristics of patients (M-23) (Se) Medenawiu 71111111 patients with 000 mammary carcinomas 11101010 mammary carcinomas 2 kyu | from Mon to Thu « 4 Z is not | in the year 11111111 radiation Tue) 12 p.m

Participating patients/dose level y TO y y y sh tyu 1310003 so z so s 6t 141114 v 14141114 v, yuyu 11 y y o Non-hematological toxicity

Dose level |Fever (Fatigue | Diarrhea |Nausea yu Pishioni chishi chai Mashi a 7 ev tez i tav povia bo 1 p pe a vr rya r v 85 Chat 6 1 1 11 Z shi lishishishe shin shsheshi

Non-hematological toxicity monitored

No, I have neuropathy | neuropathy | (weakness 1 and then in I 6 8 them lym yishyi shi ey ny shi o

Hematological toxicity level 1 of 41 units

Songs she ssh about 5811171

El she shi shi shi shi st Eu now we are and schi

Biochemical toxicity of He)

Non-hematological toxicity controlled by co-

Ethics of social communication PO t z 1

Пе 5 1 « ве нелиши шим шш ши 2 s!

Pharmacokinetics: with» Pharmacokinetics (PC) was characterized by a linear dependence on the dose.

High variability between patients.

High clearance from plasma (median 0.Svl/min.). - I Intermediate - a long period of 1/2, median 14.2 hours.

Therapeutic concentrations in plasma (»Tmkg/μl) were reached. so Conclusions - 01 T (dose-limiting toxicity) has not yet been documented.

Mild drug-related toxicities include fatigue and nausea (easily controlled with antiemetic agents).

Ф Accidental H5K, independent of previous treatment.

There is no clear evidence of neuropathy or myopathy.

No objective documented responses, although there is some evidence of possible antitumor activity: 5B - Two patients with prior chemotherapy and radiation therapy, one had stable disease for more than 6 months (total of 13 cycles) and the other had a 40905 decrease in the size of the tumor. (F) - One patient with renal cell carcinoma and liver metastases had mixed responses (1090-fold decrease in total tumor measurements).

Extended study continues: expansion of studies at this time with a dose of 1200 μg/m 7. in Example 17

Research phase | aplidine given as a 1-hour intravenous infusion once weekly in patients with advanced solid tumors and non-Hodgkin's lymphomas

Administration of the medicinal product

Aplidin was administered as a 1-hour infusion once a week x3 every four weeks. 65 Patient characteristics

Chislolacente 010300 Pypluelim 1

Medana kuroyv 0555 9675) Colorectal clinic B, Felyayun 01

Sttusvyyunani ESO5 0 Letnyaa 5, 61117111 5 uyu i o Ranshekhmotraiy 0000000 Yasna 2, sysloszhmlyuvanya | 117711 87777778 Pancreas)

Increasing the number of patients and increasing the dose 300 01010113 5 va 130015 сч мю111181111 кю о " determination of one cycle: With consecutive infusions once a week with one week of rest. sho, y y (Se)

The worst toxicities per patient so

Chiololacente 00003333 4482 - vomiting 62.00 332 | so from Beashchyamoi 00110118 (on the 11th day) 000000101082 0

Melayaemoi 0000131

Pivyschennyastkoi 00000010 " about Tranemnysyu 00011091 Z - vyvbnoya 00000001 and PR Us tNY EI SNY PRI PEN NELY ST NAI g" Pertnyastoi 00000211 . -I 2) ri t bo Characteristics of the reported limiting dose of toxicity - Patient Mo 28 - (Edinburgh) with adenocarcinoma of the esophagus (without metastases in the liver) who received bo 50.27OOmkg/m2/day, had a delayed recovery of the increase in liver enzymes (AZT O3 ; bilirubin 53; AI R. 3), which eliminates the need for weekly administration of additional doses. 4) Some indications (hints) on activity

Patient Mo16 - (Edinburgh) with gastric adenocarcinoma, initially resistant to FAM (5-fluorouracil, adriamycin and methacin). Slight improvement in the mass of lymph nodes near the reduced curve of the stomach, abdominal trunk and pelvis with aplidine at 1200mcg/m?/g. (360 Ohmkg/m7).

Gee! Patient Mo23 - (Barcelona) with renal carcinoma and pulmonary nodules as the main site of the disease, initially resistant to MVI 4oIEM and to liposomal doxorubicin. Partial remission in pulmonary nodules and clinical improvement with weakening of shortness of breath after 2 infusions of aplidine at a dose of 2700 μg/m g/day. (8100μg/m 2). 60 Patient Mo29 - (Barcelona) with renal carcinoma. Clinical improvement after Z infusions according to the assessment of supraclavicular adenopathy. Expectation of assessment in the 2nd cycle.

Pharmacokinetic data

Linear dependence of RC on dose up to the dose level of 2700μg/m2. c5 Significant variability between patients: SI coefficient of variation, 33905.

Relatively high clearance from plasma (CI): median (quartiles) 329 (288-407 ml/min/m2).

Intermediate and 1/2: median (quartiles) 15.8 (13.3-19.5) hours.

Wide distribution: median (quartiles) Mech 345 (220-398)L/Mm7.

Preliminary compartmental analysis: the profiles are best described by a first-order C Compartment model with a very rapid initial phase (median half-life 0.04h) followed by an intermediate phase (median half-life 1.4h) and a longer terminal phase (median half-life 20 , 7 hours).

Conclusions

This ongoing study shows that aplidine given as a 1-hour infusion for 70 weeks with one week off is clinically feasible up to a dose of ZbOOmkg/m2 every week. Bone marrow toxicity has not been reported. Vomiting can be controlled with prophylactic antiemetics. Muscle toxicity, consisting of muscle weakness and increased SC, was observed in one patient receiving ZbO0Omkg/m2/h. Liver toxicity at the highest dose level was reported in several patients, although OT (dose-limiting toxicity) was reported in one patient. Pharmacokinetic information shows that potentially therapeutic plasma levels are achieved in patients receiving 1200mcg/m2 or more.

The possibility of the MIII dose level, i.e. ZbOOmkg/m2 every week, is being investigated.

Example 18

Phase I clinical trial and pharmacokinetic study of aplidine administered as a 24-hour continuous infusion every other week in patients with solid tumors and non-Hodgkin's lymphoma

Patient characteristics

The number of patients is 00000143 (typically

Median age, years (ranges) 52 (18-71) Pulmonary 6 cm with statusESOS in progress (Colorectal 8 o 6re alone 1111 that 7 Earlier chemotherapy (number of regimens) (Ovarian so

M sleep and major delusions M 00001010 Mesothylum.///- 1oshlna 111 shi « s 70 Increasing the number of patients and increasing the dose 8 z shchi (range) 11111331 v 4 11133 - 111781 vv so m 1lvyu16 1 o - so 5

F

cycle definition: 2 infusions every other week (F) The worst toxicities per patient have)

Cheyuti 1 1z|z|z|6|8|8| 82 vol. 5 Budotayublyut 52532114 022

Pochezhyunyasi 11110121 11111 dotnyasmoi 00001120 11152

Meyazovistasmysto? 00000212 22nd year of the Meyaovyststv" 0221-1912

Muscle weakness C1 (02) -0-1- - - - 1 13) -

Increase in sec ba (from 5vyai 111-111 opr

Twensamnt 25300001 21191111

Pppertnis? 00000001

Neytozhnaya 00000213 (Pain from central catheter 62) - | - 1-12. |.

Characteristics of muscle toxicity (01 t)

Patient Mo27 - Male with medullary thyroid carcinoma, 6000 mcg/m/wk, had 7/0 symptomatic IBS 03 with myalgia (02. Toxicity resolved within 3 weeks after discontinuation of treatment.

The patient (5000 and bObOmkg/m2) experienced small increases in SRK (creatine phosphokinase) (02), consisting of an increase in SRK MM (muscle) without a significant increase in SRK MV (heart). A parallel increase in the level of aldolase was observed. Signs of improvement have been reported with the use of carnitine 75 supplements as muscle protective agents. Muscle biopsies were performed in 2 patients; E/M: partial disappearance of thick myosin filaments.

Pharmacokinetic data

Aplidin appears to have a linear dose-response PK profile (with the inconvenience of small sample sizes).

Relatively high SI. from plasma: the value of the median (quartile) 252 (192-416 ml/min./m 7).

High variability of Si. between patients (coefficient of variation SI. 6290).

Intermediate - long ii 1/2 with the value of the median (quartile) 23.8 (15.7-35,Hod.).

Wide distribution, median (quarters) Me 413 (274-638l/m7).

Preliminary compartmental analysis: the profiles are best described by a first-order model of 2 SM compartments with a very fast initial phase (median half-life 0.6b4h) and a longer terminal phase (median half-life 25.8h).

Relationship of aplidine myotoxicity with pharmacokinetics

Myotoxicity was observed only at high doses and exposures after a 24-hour infusion.

Cmax values after a 1-hour infusion are already higher than Cmax values after a 24-hour (Ce) infusion. In this way, it is possible to exclude the relationship of Smax. co

The values of AHS in patients with myotoxicity are high, but not maximal.

Myotoxicity affected patients with high persistent plasma concentrations of aplidine. Of the patients with obvious "muscle toxicity" there were 154 more in 44 hours. in comparison with the median I 1/2 25, Agree. after a 24-hour infusion. co

Figures 15a and 156 show activity in medullary thyroid cancer: CEA levels.

Conclusions in

Are the drug-induced muscle changes (which would be expected to be the dose-limiting toxicity) reported from the PI dose level and above (at 180µg/m2-5000µg/m?) the dose-limiting toxicity at 6000µg/m? (1 of 9 patients). Antitumor activity was also noted in patients with non-Hodgkin's lymphoma (NHL) and renal carcinoma. With 70 Currently, research is being conducted on the possibility of using 6000-700Omkg/m 2 every other week with the use of carnitine supplements as muscle protective agents.

Claims (16)

;" The formula of the invention - 1. The method of treatment of malignant tumors in humans, which involves the introduction of aplidine by intravenous (ee) infusion at a dose of 1200-7000 μg/m2 of body surface area according to one of the following procedures: - a) 24-hour infusion once a week for three weeks, followed by one week of rest; b) 24-hour infusion once every two weeks; (ee) 50 c) 1-hour infusion once a week for 3 weeks every 4 weeks; F d) 1-hour infusion once a day for five days every three weeks; and e) 3-hour infusion every two weeks. 2. The method according to claim 1, where aplidine is administered in a dose of 3200-5000 μg/m? body surface area. 3. The method according to claim 1 or 2, where aplidine is administered according to procedure a). 4. The method according to claim 3, where aplidine is administered at a dose of 3750 μg/m2 of body surface area. F) 5. The method according to claim 1 or 2, where aplidine is administered according to procedure b). co 6. The method according to claim 5, where aplidine is administered at a dose of 5000 μg/m? body surface area. 7. The method according to claim 5, where aplidine is administered at a dose of 7000 μg/m? body surface area. 6o0 8. The method according to claim 1 or 2, where aplidine is administered according to procedure c). 9. The method according to claim 8, where aplidine is administered at a dose of 3600 μg/m2 of body surface area. 10. The method according to claim 1, where aplidine is administered according to procedure d). 11. The method according to claim 10, where aplidine is administered at a dose of 1200 μg/m? body surface area. 12. The method according to claim 1, where aplidine is administered according to procedure e). for 13. The method according to any of the preceding items, wherein aplidine is administered as part of a combination therapy. 14. The method according to any of the preceding clauses, wherein aplidine is administered in combination with a skeletal muscle protective agent. 15. The method according to claim 14, where the protective agent of skeletal muscles is carnitine. 16. The method according to any of the preceding clauses, wherein the patient has previously received standard treatment for malignant tumors and the tumor is resistant. what about (Se) (ee) "- (ee) - - with . a -I (ee) - so 7 4) ko bo b5