RU2801665C2 - Pharmaceutical combination for cancer treatment - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: invention relates to a pharmaceutical combination for the treatment of cancer in a human patient, comprising (A): a compound which is a polyunsaturated fatty acid of the formula COOH-CHOH-(CH₂)₆-(CH=CH-CH₂)₂-(CH₂)₃-CH₃(ABTL0812), its pharmaceutically acceptable salt, or a combination thereof, and (B): a compound which is a chemotherapeutic agent, where compound (B) is paclitaxel and the cancer is lung cancer, preferably non-small cell lung adenocarcinoma; compound (B) is carboplatin and the cancer is endometrial cancer; compound (B) is retinoic acid and the cancer is neuroblastoma; compound (B) is paclitaxel and the cancer is breast cancer, preferably triple negative breast cancer; compound (B) is paclitaxel and carboplatin, and the cancer is lung cancer; compound (B) is paclitaxel and carboplatin and the cancer is squamous cell carcinoma, preferably non-small cell squamous cell lung cancer; compound (B) is paclitaxel and carboplatin, and the cancer is non-small cell lung adenocarcinoma; compound (B) is cisplatin and pemetrexed, the cancer is lung cancer; compound (B) is cisplatin and pemetrexed and the cancer is non-small cell lung adenocarcinoma; compound (B) is paclitaxel and the cancer is endometrial cancer; compound (B) is paclitaxel and carboplatin, and the cancer is endometrial cancer; compound (B) is paclitaxel and gemcitabine, and the cancer is pancreatic cancer; compound (B) is nab-paclitaxel and gemcitabine, and the cancer is pancreatic cancer; compound (B) is doxorubicin and the cancer is breast cancer, preferably triple negative breast cancer; compound (B) is cisplatin and the cancer is neuroblastoma; or Compound (B) is cisplatin and gemcitabine and the cancer is cholangiocarcinoma.

EFFECT: invention provides a significant synergistic effect in the treatment of the above types of cancer with such a pharmaceutical combination.

10 cl, 17 dwg, 7 tbl, 4 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to a pharmaceutical combination containing (A): a polyunsaturated fatty acid and (B): a chemotherapeutic agent compound for simultaneous, separate or sequential use in the treatment of cancer in a human patient.

BACKGROUND OF THE INVENTION

European patent EP 2409963 B1 (application filed by Lipopharma in 2010) describes the use of 1,2-substituted polyunsaturated fatty acid derivatives (called P-PUFAs) for the treatment of cancer.

The described compounds, which are derivatives of fatty acids, have the following formula:

An example of a preferred compound is:

In the article "Erazo, et al.; Clinical Cancer Research; 22(10) May 15, 2016" the above compound (182A1) is described in more detail - in this article, the said compound is called "ABTL0812", and this term is used herein.

According to the description of the article, ABTL0812 induces autophagy-mediated death of cancer cells without activating cellular apoptosis. The article says:

[p. 2515]:

“Most existing cancer treatments activate apoptosis, and chemotherapy resistance is a major problem in cancer (24). Autophagy-mediated cell death has been proposed as an alternative to killing cancer cells without inducing resistance to apoptotic drugs (25).”

[p. 2517]:

“On the other hand, mTORCI activation is often associated with anticancer drug resistance (6). Since ABTL0812 is a highly potent inhibitor of the Akt/mTORC1 axis, its administration in combination with standard chemotherapeutic drugs may be effective in therapy-resistant or apoptotic-resistant tumors.”

At the time of filing of this application - on the website of the applicant of this application (AbilityPharma - www.abilitypharma.com) there was a News section (all authors of this application transferred all rights related to this document to the applicant of this application and priority application EP 17382282.6, filed on May 16, 2017 year, - in other words, the publication discussed below from the web page of the present applicant can be considered as the so-called "disclosure by the inventor" - (i.e. public disclosure of the essence of the invention must be made by the inventor, one or more co-authors or another party, who received information about the essence of the invention directly or indirectly from the inventor or co-author).

A news note dated November 22, 2016 states:

“Catalan biopharmaceutical company Ability Pharmaceuticals, SL, announced today the launch of the first phase 2 clinical trial of a new targeted anti-cancer agent ABTL0812 to evaluate its efficacy and safety in combination with paclitaxel and carboplatin in 80 patients with advanced or recurrent endometrial cancer or squamous cell lung cancer in as first line therapy (…)

In preclinical cancer models, ABTL0812 is effective as a single agent and has an excellent safety profile against a wide range of cancer types: lung, endometrial, pancreatic, and neuroblastoma. In these models, this compound also has a synergistic effect with chemotherapy (taxanes, platinum compounds and gemcitabine) without increasing its toxicity.”

A news note dated December 14, 2016 states:

“In preclinical studies, ABTL0812 has been shown to be effective against pancreatic cancer as a single agent and synergistic effect (8-90-fold) in combination with taxanes, platinum compounds and gemcitabine, with tumor regression occurring without increased toxicity associated with chemotherapy (… )

ABTL0812 is currently being studied in phase 2 as a first-line therapy in combination with chemotherapy in patients with endometrial cancer or squamous cell lung cancer.”

With regard to the use of the ABTL0812 compound in combination with other chemotherapeutic agents, the Erazo article mentioned above and the Applicant's news release (AbilityPharma) do not describe any significant experimental data - ie. claims relating to the combination can be regarded as mere claims that are not supported by any significant verifiable experimental data.

The published news items relate to phase II studies - as is known in the art, the fact that phase II studies have been completed means that phase I studies have been completed, and based on this information, the specialist can only conclude that the results of safety and tolerability in humans, as well as pharmacokinetic studies were positive, ie. it does not provide any information about the possible therapeutic effect in the human patient, in particular about the possible synergistic effect of the combination. After completion of phase II trials and evaluation of the results, the specialist will only know whether the drug is therapeutically effective - at the time of filing of this application, no experimental data obtained from phase II trials related to the present invention have been published.

BRIEF DESCRIPTION OF THE INVENTION

The problem to be solved by the present invention is to provide an improved method for treating cancer.

As discussed above, the compound COOH-CHOH-(CH ₂ ) ₆ -(CH=CH-CH ₂ ) ₂ -(CH ₂ ) ₃ -CH ₃ is referred to herein as ABTL0812.

The working examples herein provide numerous detailed experimental data demonstrating a significant synergistic effect associated with the use of the above-discussed compound ABTL0812 in combination with other chemotherapeutic agents.

As discussed in more detail herein - the experimental data given herein are from conventional in vitro and in vivo experimental tests (e.g. mice) - respectively, based on the experimental data given herein, it seems likely/certain that that synergistic effects associated with the present invention can be obtained in a human cancer patient.

Example 4 herein discusses previous preliminary results from human clinical trials - the results from previous human clinical trials are positive in that these results also indicate that there is also a synergistic effect in humans from ABTL0812. in combination with paclitaxel and carboplatin in patients with advanced endometrial or squamous cell carcinoma.

Based on the information known from the prior art, the person skilled in the art would not be able to predict and at the same time have sufficient reason to expect the achievement of the significant synergistic effects described herein, obtained in the experiments.

According to the above discussion of the disclosure made by the present applicant (AbilityPharma) and provided on the web page, the statements relating to the combination in the information provided on the web page can be considered mere statements that are not supported by any significant verifiable experimental data - it is obvious that on the basis of this information presented on the web page, it is impossible to state with sufficient probability / certainty that the synergistic effects associated with the present invention can be obtained in a human patient with cancer.

Briefly, the working examples in this paper demonstrate, among other things, the following:

Example 1: - Experiments in vitro:

1.1: ABTL0812 and docetaxel have a synergistic effect in vitro against a typical non-small cell lung adenocarcinoma cell line, where ABTL0812 lowered the IC50 of docetaxel by more than 80-fold - i.e. significantly increased the cytotoxicity of docetaxel;

1.2: ABTL0812 and paclitaxel have a synergistic effect in vitro against 4 different lung carcinoma cell lines, where ABTL0812 lowered the IC ₅₀ of paclitaxel in the range of 2 to 7 times depending on the cell line, i.e. increased the cytotoxicity of paclitaxel;

1:3: ABTL0812 and gemcitabine have a synergistic effect in vitro against a typical pancreatic cancer cell line, where ABTL0812 lowered the IC ₅₀ of gemcitabine by 7 times - i.e. significantly increased the cytotoxicity of gemcitabine;

1:4: ABTL0812 and carboplatin have a synergistic effect in vitro against a typical endometrial cancer cell line, where ABTL0812 lowered the IC ₅₀ of carboplatin by 3 times - i.e. increased the cytotoxicity of carboplatin;

1:5: ABTL0812 and retinoic acid have a synergistic effect in vitro against a typical neuroblastoma cancer cell line;

1:6: ABTL0812 and paclitaxel have a synergistic effect in vitro against a typical breast cancer cell line, where ABTL0812 lowered the IC ₅₀ of paclitaxel by 3 times, i.e. increased the cytotoxicity of paclitaxel.

Example 2: - Experiments in vivo:

2.1: In a typical in vivo mouse model, low doses of ABTL0812 enhanced the antitumor activity of docetaxel against lung cancer without adverse toxic effects.

2.2: In a typical in vivo mouse model, treatment with the combination of ABTL0812+ paclitaxel and carboplatin (P/C) showed a significant increase in survival in a squamous cell carcinoma (SCC) model where survival was 75% at 20 days post-treatment compared to 0% survival in groups in which ABTL0812 and vehicle were administered, and a survival rate of 25% in the P/C group.

2.3: In a typical in vivo mouse model, the combination of ABTL0812 + P/C showed a synergistic effect compared with the effect of each drug alone on lung adenocarcinoma, as animals treated with the combination showed a significant reduction in tumor volume compared to control animals and animals treated with chemotherapy;

2.4: In a typical in vivo mouse model, the combination of ABTL0812 + pemetrexed and cisplatin showed a synergistic effect compared with the effect of each chemotherapy regimen alone on adenocarcinoma of the lung, as animals treated with the combination showed a significant reduction in tumor volume compared to control animals and animals treated with chemotherapy;

2.5: In a typical in vivo mouse model, the combination of ABTL0812 + paclitaxel showed a synergistic effect compared with the effect of each drug alone on endometrial cancer, as animals treated with the combination showed a significant reduction in tumor volume compared to controls ;

Figure 2.6: In a typical in vivo mouse model of patient-derived xenografts, the combination of ABTL0812 + P/C showed a synergistic effect on endometrial cancer in terms of a significantly higher reduction in tumor volume compared to P/C, as well as a significant reduction in tumor volume. tumors compared to the vehicle group during the first 47 days;

2.7: In a typical in vivo mouse model, the combination of ABTL0812 + paclitaxel/gemcitabine showed a synergistic effect on pancreatic cancer in terms of significantly higher tumor volume reduction compared to P/Gm alone. P/Gm also showed a greater reduction in tumor volume compared to vehicle;

2.8: In a typical in vivo mouse model, five combinations of ABTL0812 + nab-paclitaxel/gemcitabine showed a synergistic effect on pancreatic cancer in terms of a significantly greater reduction in tumor volume compared to Nab-P/Gm alone. Nab-P/Gm also showed a greater reduction in tumor volume compared to vehicle;

2.9: In a typical in vivo mouse model, the combination of ABTL0812 + cisplatin showed a synergistic effect against neuroblastoma, where the combination of ABTL0812 with cisplatin resulted in stabilization of tumor progression over an extended period of time;

2.10: In a typical in vivo mouse model, the combination of ABTL0812 + doxorubicin showed a synergistic effect on breast cancer, where the combination of ABTL0812 with paclitaxel showed a greater reduction in tumor volume compared to vehicle.

The experimental data provided herein are from conventional in vitro and in vivo (e.g., mice) experimental tests - accordingly, based on the experimental data provided herein, it seems likely/certain that synergistic effects associated with the present invention may be obtained from a human cancer patient.

Examples of chemotherapeutic agents investigated in the working examples herein include:

Taxanes: paclitaxel (taxol), nab-paclitaxel (albumin-bound paclitaxel) and docetaxel;

Platinum-based agents: carboplatin and cisplatin;

Nucleotide analogs and precursor analogs: gemcitabine;

Folate antimetabolites: pemetrexed;

Anthracyclines: doxorubicin;

Accordingly, various groups/classes of chemotherapeutic agents have been studied and all have demonstrated significant synergistic effects when used in combination with ABTL0812.

Based on the experimental data presented herein, it seems likely that the positive synergistic effect described herein can be obtained for most significant chemotherapeutic agents.

As discussed above, most existing cancer treatments activate apoptosis, and all of the aforementioned other chemotherapeutic agents under investigation activate apoptosis.

ABTL0812 induces autophagy-mediated cancer cell death without activating cellular apoptosis.

The experimental data reported herein demonstrate that ABTL0812 in combination with chemotherapeutic agents can unexpectedly increase the rate of apoptosis, although this is not the main mechanism of action of ABTL0812.

Based on the experimental data reported herein, for a chemotherapeutic agent (eg, docetaxel), essentially the following has been demonstrated:

Docetaxel in an amount that provides 100% therapeutic effect => leads to toxicity Y.

Docetaxel in an amount that provides 50% therapeutic effect => leads to a decrease in toxicity.

Docetaxel at 50% therapeutic effect + ABTL0812 at 50% therapeutic effect => results in 100% effect and toxicity remains at the same reduced level.

The inventors of the present invention surprisingly found that, by combining with ABTL0812, the effect of a chemotherapeutic agent (eg, docetaxel) was significantly increased without a significant increase in toxicity.

Connection ABTL0812 structurally and functionally similar to other compounds, which are 1,2-substituted derivatives of polyunsaturated fatty acids (P-PUFA), as described in the above-discussed patent EP 2409963 B1.

Accordingly, based on the available data, it is likely that essentially all compounds that are derivatives of fatty acids, according to EP 2409963 B1 can have a synergistic effect associated with the present invention in combination with a chemotherapeutic agent.

Accordingly, the first aspect of the invention relates to a pharmaceutical combination comprising:

(A): a compound which is a polyunsaturated fatty acid of the formula COOR ₁ -CHR ₂ -(CH ₂ )a-(CH=CHCH ₂ )b-(CH ₂ )c-CH ₃ , a pharmaceutically acceptable salt thereof, or a combination thereof, Where

(i) a may be any integer between 0 and 7,

(ii) b may be any integer from 2 to 7,

(iii) c may be any integer between 0 and 7,

(iv) R ₁ is H, Na, K, CH ₃ , CH ₃ -CH ₂ or PO(O-CH ₂ -CH ₃ ) ₂ , and

(v) R ₂ is OH, OCH ₃ , O-CH ₃ COOH, CH ₃ , Cl, CH ₂ OH, OPO(O-CH ₂ -CH ₃ ) ₂ , NOH, F, HCOO or N(OCH ₂ CH ₃ ) ₂ ;

And

(B): a compound that is a chemotherapeutic agent,

for simultaneous, separate or sequential use in the treatment of cancer in a human patient.

In this context, the specialist should be clear that the compound (B), which is a chemotherapeutic agent according to the first aspect, is certainly not a compound included in the scope of the Compound (A) according to the first aspect.

In this context, the specialist should be clear that for the combination treatment discussed herein, it is immaterial whether the two compounds (A) and (B) are administered, for example, simultaneously in the form of one composition, or, for example, sequentially in the form of two separate compositions. It is important that an effective amount of the first compound/agent administered is present in the patient's body at the time the second compound/agent is administered.

Accordingly, the term "combination" according to the first aspect refers herein to various combinations of compounds (A) and (B), for example, in one pharmaceutical composition, in a combination mixture consisting of separate pharmaceutical formulations/compositions of active compounds separately, such as "mixture in a vessel", and to the combined use of active ingredients separately, administered sequentially, that is, one after the other for a sufficiently short period of time, such as several hours or days, or simultaneously. The order in which compounds (A) and (B) are applied is not important.

The combination of compounds (A) and (B) can be prepared with the possibility of simultaneous, separate or sequential administration. In particular, if the administration is not simultaneous, then the compounds are administered with a relatively small time interval from each other. In addition, the compounds are administered in the same or different dosage forms or by the same or different routes of administration, for example, one compound may be administered topically and another compound may be administered orally. A combination of two compounds can be entered, for example:

- in the form of a combination included in one medicinal composition, in this case the two compounds are always administered simultaneously;

- in the form of a combination of two dosage forms/compositions, each of which contains one of the substances, which makes possible simultaneous, sequential or separate administration;

For example, compound (A) is administered independently of compound (B) (ie, in two dosage forms), but at the same time.

In another suitable example, compound (A) is administered first, and then compound (B) is administered separately or sequentially - alternatively, compound (B) is administered first, and then compound (A) is administered separately or sequentially.

The term "pharmaceutical", for example in relation to "pharmaceutical composition", should be understood in accordance with the prior art, i. it refers to a preparation/composition that is in a form that effectively provides the biological activity and physiological tolerance of the active ingredients, that is, does not contain additional components that cause unacceptable toxicity in the subject to which the composition is administered. In particular, the term "pharmaceutically acceptable" means state or federal regulatory approval or inclusion in the USP or other generally accepted pharmacopoeia for use in animals, and in particular in humans.

An embodiment of the present invention is described below solely as examples.

The combination of a preferred embodiment described herein with another preferred embodiment described herein is an even more preferred embodiment.

BLUEPRINTS

Figure 1: In vitro synergy of ABTL0812 with docetaxel for human lung adenocarcinoma cell line A549 is shown. Cytotoxicity of ABTL0812, docetaxel and the combination of both drugs. An increase in the cytotoxicity of docetaxel can be observed, since its IC ₅₀ was reduced by 86 times when ABTL0812 was added at a low concentration (about half of the IC ₅₀ ). Results are shown as the average of two independent experiments. See the working example in this document for more information.

Figure 2: In vitro synergy of ABTL0812 with paclitaxel for human lung adenocarcinoma cell lines A549 and H1975 and for human squamous cell lung cancer cell lines H157 and HTB182 is shown. Cytotoxicity of ABTL0812, paclitaxel and the combination of both drugs in all four different lung cancer cell lines. An increase in the cytotoxicity of paclitaxel can be observed for all four cell lines, as its IC ₅₀ decreased in the range of 2 to 7 times, depending on the cell line, when ABTL0812 was added at a low concentration (about half of the IC ₅₀ ). Results are shown as the average of two independent experiments for each cell line. See the working example in this document for more information.

Figure 3: In vitro synergy of ABTL0812 with gemcitabine for the human pancreatic cancer cell line MiaPaca2 is shown. Cytotoxicity of ABTL0812, gemcitabine, and a combination of both drugs. An increase in the cytotoxicity of gemcitabine in the presence of ABTL0812 can be observed, as its IC50 was reduced by a factor of 7 when ABTL0812 was added at a low concentration (approximately half of the IC ₅₀ ). Results are shown as the average of two independent experiments. See the working example in this document for more information.

Figure 4: Shows the in vitro synergy of ABTL0812 with carboplatin against the Ishikawa human endometrial cancer cell line. Cytotoxicity of ABTL0812, carboplatin and combinations of both drugs. An increase in the cytotoxicity of docetaxel can be observed, since its IC ₅₀ was reduced by 3 times when ABTL0812 was added at a low concentration (about half of the IC ₅₀ ). Results are shown as the average of two independent experiments. See the working example in this document for more information.

Figure 5: In vitro synergy of ABTL0812 with retinoic acid for human neuroblastoma cell lines LAI-5S and SK-N-BE(2) is shown. Cytotoxicity of ABTL0812, retinoic acid and a combination of both drugs. An increase in retinoic acid (RA) cytotoxicity can be observed for both cell lines, as cell viability in the presence of RA decreased from 82.8% to 23.8% for LAI-5S cells and from 70.4% to 34.7% for cells SK-N-BE(2) when RK is incubated with ABTL0812 at a low concentration (about half of the IC30). Results are shown as the average of two independent experiments. See the working example in this document for more information.

Figure 6: Shows the in vitro synergy of ABTL0812 with paclitaxel against the triple negative human breast cancer cell line MDA-DB-231. Cytotoxicity of ABTL0812, paclitaxel and the combination of both drugs. An increase in the cytotoxicity of paclitaxel can be observed, since its IC50 was reduced by 2.7 times when ABTL0812 was added at a low concentration (below IC ₅₀ ). Results are shown as the average of two independent experiments. See the working example in this document for more information.

Figure 7: ABTL0812 enhances the therapeutic effect of docetaxel without increasing toxicity in an in vivo A549 human lung adenocarcinoma xenograft model. Left graph: Antitumor effect of the combination of ABTL0812 with docetaxel in the A549 human lung adenocarcinoma xenograft model showing a significant reduction in tumor growth compared to ABTL0812, docetaxel and vehicle groups. P-values were calculated to compare the ABTL0812+docetaxel group with the docetaxel-administered group and the vehicle-administered group. Right graph: total body weight in different treatment groups during the whole period of the experiment. See the working example in this document for more information.

Figure 8: ABTL0812 enhances the therapeutic effect of paclitaxel/carboplatin (P/C) without increasing toxicity in an in vivo H157 human squamous cell lung cancer xenograft model. Kaplan-Meier plot of H157 xenograft after administration of ABTL0812, P/C, ABTL0812 + P/C and vehicle, where ABTL0812 + P/C shows the highest survival rate. See the working example in this document for more information.

Figure 9: ABTL0812 enhances the therapeutic effect of P/C without increasing toxicity in an in vivo H1975 human lung adenocarcinoma xenograft model. Left graph: Antitumor effect of the combination of ABTL0812 with P/C in the A549 human lung adenocarcinoma xenograft model, showing the largest reduction in tumor volume compared to P/C, ABTL0812, or vehicle groups. P-values were calculated to compare the ABTL0812 + P/C group with the P/C group and the vehicle group. Right graph: total body weight in different treatment groups during the whole period of the experiment. See the working example in this document for more information.

Figure 10: ABTL0812 enhances the therapeutic effect of pemetrexed and cisplatin without increasing toxicity in an in vivo A549 human lung adenocarcinoma xenograft model. Left graph: Antitumor effect of the combination of ABTL0812 with pemetrexed and cisplatin, showing a significant reduction in tumor growth compared to pemetrexed and cisplatin and vehicle groups. P values were calculated to compare the ABTL0812 + pemetrexed and cisplatin group with the vehicle group. Right graph: total body weight in different treatment groups during the whole period of the experiment. See the working example in this document for more information.

Figure 11: ABTL0812 enhances the therapeutic effect of paclitaxel without increasing toxicity in the Ishikawa orthotopically implanted human endometrial cancer xenograft model in vivo. Left graph: Animals were sacrificed after three weeks of treatment, tumors were removed and tumor volume was determined. A statistically significant decrease was observed in animals treated with the ABTL0812 + paclitaxel combination compared to control animals treated with vehicle alone. P values were calculated to compare the ABTL0812 + paclitaxel group with the vehicle group. Right graph: total body weight in different treatment groups during the whole period of the experiment. See the working example in this document for more information.

Figure 12: ABTL0812 enhances the therapeutic effect of P/C without increasing toxicity in patient-derived human endometrial cancer xenografts in vivo. Left graph in the figure: A tumor fragment surgically removed from a patient with a serous tumor type defined in histology, stage IIIC2, 100% myometrial invasion and invasion of the pelvic and aortic lymph nodes and lymphovascular invasion, and having mutations in the p53 and PI3KCA genes, was implanted athymic mice. ABTL0812 in combination with P/C significantly reduces tumor size compared to P/C, ABTL0812 and vehicle. The p values are shown in the figure. Right graph: The total body weight of the different treatment groups during the whole period of the experiment is shown below. See the working example in this document for more information.

Figure 13: ABTL0812 enhances the effect of gemcitabine and paclitaxel (Gm/P) without increasing toxicity in the MiaPAca2 human pancreatic cancer xenograft model MiaPAca2 in vivo. Left graph: Antitumor effect of the ABTL0812 + Gem/P combination, showing the largest reduction in tumor volume compared to Gm/P, ABTL0812 or vehicle. P values were calculated to compare the ABTL0812 + gemcitabine and paclitaxel group with the gemcitabine and paclitaxel, gemcitabine and vehicle group. Right graph: total body weight in different treatment groups during the whole period of the experiment. See the working example in this document for more information.

Figure 14: ABTL0812 enhances the effect of gemcitabine (Gm) alone and the effect of gemcitabine and nab-paclitaxel (Gm/Nab-P) without increasing toxicity in two MiaPAca2 human pancreatic cancer xenograft models in vivo. Left graph: Antitumor effect of the ABTL0812+Gm/Nab-P combination, showing the largest reduction in tumor volume compared to Gm/Nab-P, ABTL0812 or vehicle. Right graph: Antitumor effect of ABTL0812 + Gm combination, showing the largest reduction in tumor volume compared to Gm, ABTL0812 or vehicle. P-values were calculated to compare ABTL0812 + Gm/Nab-P with ABTL0812 + Gm and the vehicle group. The total body weight in the various treatment groups during the entire duration of the experiment is shown below. See the working example in this document for more information.

Figure 15: ABTL0812 enhances the therapeutic effect of cisplatin without increasing toxicity in the SH-SY5Y human neuroblastoma xenograft model in vivo. Left graph: Antitumor effect of the combination of ABTL0812 + cisplatin, showing the largest reduction in tumor volume compared to ABTL0812, cisplatin or vehicle. On day 10, animals from the control group were sacrificed, at the same time, half of the animals from the treatment groups were sacrificed, and the rest were examined for a longer period of time. Left graph: Tumor weight after sacrifice after 10 days of treatment (p<0.05 by t-test). See the working example in this document for more information.

Figure 16: ABTL0812 enhances the therapeutic effect of doxorubicin without increasing toxicity in the MDA-DB-231 triple negative human breast cancer xenograft model in vivo. Left graph: Antitumor effect of the combination of ABTL0812 + doxorubicin, showing the largest reduction in tumor volume compared to doxorubicin, ABTL0812 or vehicle. P values were calculated to compare ABTL0812 + doxorubicin with the vehicle group. Right graph: total body weight in different treatment groups during the whole period of the experiment. See the working example in this document for more information.

Figure 17: ABTL0812 enhances the therapeutic effect of gemcitabine and cisplatin (Gm/Cis) without increasing toxicity in an in vivo human cholangiocarcinoma xenograft EGI-1 model. Left graph: Antitumor effect of the combination of ABTL0812 + Gm/Cis showing the largest reduction in tumor volume compared to Gm/Cis, ABTL0812 or vehicle. P values were calculated to compare ABTL0812 + Gm/Cis with the vehicle treated group. Right graph: total body weight in different treatment groups during the whole period of the experiment. See the working example in this document for more information.

DETAILED DESCRIPTION OF THE INVENTION

Compound (A) according to the first aspect

In the preferred implementation

(i) a may be any integer between 5 and 7,

(ii) b may be any integer from 2 to 4,

(iii) c may be any integer from 1 to 5.

Preferably R ₁ may be H, Na, K, CH ₃ , CH ₃ -CH ₂ or PO(O-CH ₂ -CH ₃ ) ₂ ,

Preferably R ₂ may be OH, OCH ₃ , O-CH ₃ COOH, CH ₃ , Cl, CH ₂ OH, OPO(O-CH ₂ -CH ₃ ) ₂ , NOH, F, HCOO or N(OCH ₂ CH ₃ ) ₂ .

In a preferred embodiment, R ₁ is H and R ₂ is OH.

In another preferred embodiment, R ₁ is Na and R ₂ is OH.

Preferably compound (A) is at least one compound selected from the group consisting of:

COOH-CHOH-(CH ₂ ) ₆ -(CH=CH-CH ₂ ) ₂ -(CH ₂ ) ₃ -CH ₃ (ABTL0812),

COOH-CHOH-(CH ₂ ) ₆ -(CH=CH-CH ₂ ) ₃ -CH ₃ (183A1),

COOH-CHOH-(CH ₂ ) ₃ -(CH=CH-CH ₂ ) ₃ -(CH ₂ ) ₃ -CH ₃ (183A2),

COOH-CHOH-(CH ₂ ) ₂ -(CH=CH-CH ₂ ) ₄ -(CH ₂ ) ₃ -CH ₃ (204A1),

COOH-CHOH-(CH ₂ ) ₂ -(CH=CH-CH ₂ ) ₅ -CH ₃ (205A1), and

COOH-CHOH-CH ₂ -(CH=CH-CH ₂ ) ₆ -CH ₃ (226A1).

Most preferably, compound (A) is COOH-CHOH-(CH ₂ ) ₆ -(CH=CH-CH ₂ ) ₂ -(CH ₂ ) ₃ -CH ₃ (ABTL0812).

A pharmaceutically acceptable salt of compound (A) refers to any pharmaceutically acceptable salt of compound (A). Many pharmaceutically acceptable salts are known in the art. Examples of pharmaceutically acceptable salts include, but are not limited to, sodium (Na) salts, potassium salts, acetates, sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexine-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, gamma-hydroxybutyrates, glycolates, tartrates, alkane sulfonates (e.g. methanesulfonate or mesylate), propanesulfonates, naphthalene-1 -sulfonates, naphthalene-2-sulfonates and mandelates. In a particular embodiment, the salt of compound (A) is the sodium salt.

In this context, one skilled in the art will appreciate that a preferred formula for compound (A), such as, for example, ABTL0812, includes a salt thereof, for example, where said compound (A) is described herein as COOH-CHOH-( CH ₂ ) ₆ -(CH=CH-CH ₂ ) ₂ -(CH ₂ ) ₃ -CH ₃ (ABTL0812), this description also applies to the ABTL0812 salt.

Preferably compound (A) is the sodium salt of COOH-CHOH-(CH ₂ ) ₆ -(CH=CH-CH ₂ ) ₂ -(CH ₂ ) ₃ -CH ₃ (ABTL0812).

Compound (B) according to the first aspect

Preferably, compound (B) is at least one chemotherapeutic agent selected from the group consisting of:

a bifunctional alkylating agent (preferably cyclophosphamide, mechlorethamine, chlorambucil or melphalan);

a monofunctional alkylating agent (preferably dacarbazine (DTIC), nitrosoureas or temozolomide);

an anthracycline (preferably daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone or valrubicin);

a taxane (preferably paclitaxel, docetaxel, nab-paclitaxel or taxotere);

epothilone (preferably patupilone, sagopilone or ixabepilone);

a deacetylase inhibitor (preferably vorinostat or romidepsin);

a topoisomerase I inhibitor (preferably irinotecan or topotecan);

a topoisomerase II inhibitor (preferably etoposide, teniposide or tafluposide);

a kinase inhibitor (preferably bortezomib, erlotinib, gefitinib, imatinib, vemurafenib or vismodegib);

a nucleotide analog and/or a precursor analog (preferably azacitidine, azathioprine, capecitabine, cytarabine, doxyfluridine, fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, or thioguanine);

a peptide antibiotic (preferably bleomycin or actinomycin);

a platinum-based agent (preferably carboplatin, cisplatin or oxaliplatin);

a retinoid (preferably tretinoin, alitretinoin or bexarotene); And

vinca alkaloid and its derivatives (preferably vinblastine, vincristine, vindesine or vinorelbine).

In this context, it should be understood that for any of the preferred examples of compound (B) listed, the most preferred compound (A) is COOH-CHOH-(CH ₂ ) ₆ -(CH=CH-CH ₂ ) ₂ -(CH ₂ ) ₃ -CH ₃ (ABTL0812).

More preferably, compound (B) is at least one compound which is a chemotherapeutic agent selected from the group consisting of:

cyclophosphamide;

melphalan;

docetaxel;

paclitaxel;

nab-paclitaxel;

carboplatin;

cisplatin;

oxaliplatin;

methotrexate;

pemetrexed;

azathioprine;

capecitabine;

fluorouracil;

mercaptopurine;

gemcitabine;

bleomycin;

actinomycin;

vincristine;

vinblastine;

vinorelbine;

retinoic acid;

temozolomide;

daunorubicin;

doxorubicin;

irinotecan; And

topotecan.

Even more preferably, compound (B) is at least one compound which is a chemotherapeutic agent selected from the group consisting of:

docetaxel;

paclitaxel;

nab-paclitaxel;

carboplatin;

cisplatin;

oxaliplatin;

methotrexate;

pemetrexed;

gemcitabine;

bleomycin;

retinoic acid;

temozolomide;

doxorubicin;

irinotecan; And

topotecan.

The compound (B) according to the first aspect may preferably include two or more different chemotherapeutic agents (in particular, if the compound (A) is COOH-CHOH-(CH ₂ ) ₆ -(CH=CH-CH ₂ ) ₂ -(CH ₂ ) ₃ -CH ₃ (ABTL0812)) - while preferably the compound (B) according to the first aspect includes:

paclitaxel and carboplatin;

paclitaxel and gemcitabine;

nab-paclitaxel and gemcitabine;

gemcitabine and cisplatin;

pemetrexed and cisplatin.

Particularly preferably, compound (A) is ABTL0812 and compound (B) is docetaxel, in particular if the cancer is lung cancer (preferably non-small cell lung adenocarcinoma). (See Examples 1.1 and 2.1 herein for an example of this preferred implementation).

Particularly preferably, compound (A) is ABTL0812 and compound (B) is paclitaxel, in particular if the cancer is lung cancer (non-small cell lung cancer). (See example 1.2 herein for an example of said preferred embodiment).

Particularly preferably, compound (A) is ABTL0812 and compound (B) is gemcitabine, in particular if the cancer is pancreatic cancer. (See, for example, Example 1.3 herein for an example of said preferred embodiment).

Particularly preferably, the compound (A) is ABTL0812 and the compound (B) is carboplatin, in particular if the cancer is an endometrial cancer. (See Example 1.4 herein for an example of this preferred implementation).

Especially preferably the compound (A) is ABTL0812 and the compound (B) is retinoic acid, in particular if the cancer is a neuroblastoma. (See Example 1.5 herein for an example of this preferred implementation).

Particularly preferably, compound (A) is ABTL0812 and compound (B) is paclitaxel, in particular if the cancer is breast cancer (preferably triple negative breast cancer). (See Example 1.6 herein for an example of this preferred implementation).

Particularly preferably, compound (A) is ABTL0812 and compound (B) is paclitaxel and carboplatin, in particular if the cancer is a squamous cell carcinoma (preferably non-small cell squamous cell lung cancer). (See Example 2.2 herein for an example of this preferred implementation).

Particularly preferably, compound (A) is ABTL0812 and compound (B) is paclitaxel and carboplatin, in particular if the cancer is non-small cell lung adenocarcinoma. (See Example 2.3 herein for an example of this preferred implementation).

Particularly preferably, compound (A) is ABTL0812 and compound (B) is pemetrexed and cisplatin, in particular if the cancer is non-small cell lung adenocarcinoma. (See Example 2.4 herein for an example of this preferred implementation).

Particularly preferably, the compound (A) is ABTL0812 and the compound (B) is paclitaxel, in particular if the cancer is endometrial cancer. (See Example 2.5 herein for an example of this preferred implementation).

Particularly preferably, compound (A) is ABTL0812 and compound (B) is paclitaxel and carboplatin, in particular if the cancer is endometrial cancer. (See Example 2.6 herein for an example of this preferred implementation).

Particularly preferably, compound (A) is ABTL0812 and compound (B) is paclitaxel and gemcitabine, in particular if the cancer is pancreatic cancer. (See Example 2.7 herein for an example of this preferred implementation).

Particularly preferably, compound (A) is ABTL0812 and compound (B) is either gemcitabine or nab-paclitaxel and gemcitabine, in particular if the cancer is pancreatic cancer. (See Example 2.8 herein for an example of this preferred implementation).

Especially preferably the compound (A) is ABTL0812 and the compound (B) is cisplatin, in particular if the cancer is a neuroblastoma. (See Example 2.9 herein for an example of this preferred implementation).

Particularly preferably, the compound (A) is ABTL0812 and the compound (B) is doxorubicin, in particular if the cancer is triple negative breast cancer. (See Example 2.10 herein for an example of this preferred implementation).

Particularly preferably, compound (A) is ABTL0812 and compound (B) is gemcitabine and cisplatin, in particular if the cancer is cholangiocarcinoma. (See Example 2.11 herein for an example of this preferred implementation).

Preferably, compound (A) is ABTL0812 and compound (B) is temozolomide and irinotecan, in particular if the cancer is neuroblastoma.

Preferably compound (A) is ABTL0812 and compound (B) is doxorubicin and topotecan, in particular if the cancer is neuroblastoma.

Preferably, the pharmaceutical combination discussed herein is one in which Compound (A) is ABTL0812 and:

Compound (B) is docetaxel and the cancer is lung cancer, preferably non-small cell lung adenocarcinoma;

Compound (B) is paclitaxel and the cancer is lung cancer, preferably non-small cell lung adenocarcinoma;

Compound (B) is gemcitabine and the cancer is pancreatic cancer;

the compound (B) is carboplatin and the cancer is endometrial cancer;

the compound (B) is retinoic acid and the cancer is neuroblastoma;

Compound (B) is paclitaxel and the cancer is breast cancer, preferably triple negative breast cancer;

Compound (B) is paclitaxel and carboplatin and the cancer is squamous cell carcinoma, preferably non-small cell squamous cell lung cancer;

Compound (B) is paclitaxel and carboplatin and the cancer is non-small cell adenocarcinoma of the lung;

Compound (B) is pemetrexed and cisplatin and the cancer is non-small cell adenocarcinoma of the lung;

the compound (B) is paclitaxel and the cancer is endometrial cancer;

the compound (B) is paclitaxel and carboplatin and the cancer is endometrial cancer;

Compound (B) is paclitaxel and gemcitabine and the cancer is pancreatic cancer;

Compound (B) is gemcitabine and the cancer is pancreatic cancer;

Compound (B) is gemcitabine and nab-paclitaxel and the cancer is pancreatic cancer;

- Compound (B) is cisplatin and gemcitabine, and the cancer is neuroblastoma;

Compound (B) is doxorubicin and the cancer is breast cancer, preferably triple negative breast cancer;

Compound (B) is cisplatin and gemcitabine and the cancer is cholangiocarcinoma;

Compound (B) is gemcitabine and the cancer is cholangiocarcinoma;

- compound (B) is topotecan and the cancer is neuroblastoma;

- compound (B) is irinotecan and the cancer is neuroblastoma; or

Compound (B) is temozolomide and the cancer is neuroblastoma.

As discussed above, Example 4 herein discusses previous preliminary results from human clinical trials - the results from previous human clinical trials are positive in that these results also indicate that there is also a synergistic effect in humans. from the use of ABTL0812 in combination with paclitaxel and carboplatin in patients with advanced endometrial or squamous cell carcinoma.

Accordingly, preferably the pharmaceutical combination discussed herein is a combination in which Compound (A) is ABTL0812 and:

Compound (B) is paclitaxel and carboplatin and the cancer is advanced endometrial cancer, preferably advanced endometrial cancer; or

Compound (B) is paclitaxel and carboplatin, and the cancer is squamous cell carcinoma.

Preferably ABTL0812 is administered orally, preferably the dose of ABTL0812 administered is between 1200 mg and 1400 mg.

More preferably, ABTL0812 is administered orally, starting at a dose of 1200 mg to 1400 mg three times a day in combination with chemotherapy.

Cancer

Preferably the cancer is at least one cancer selected from the group consisting of:

lung cancer;

non-small cell lung cancer;

small cell lung cancer;

squamous cell carcinoma;

adenocarcinomas;

endometrial cancer;

pancreatic cancer;

glioblastoma;

breast cancer;

head and neck cancer;

neuroblastomas; And

cholangiocarcinoma.

More preferably, the cancer is at least one cancer selected from the group consisting of:

non-small cell lung cancer;

squamous cell carcinoma;

endometrial cancer;

pancreatic cancer;

glioblastoma;

breast cancer;

neuroblastomas; And

cholangiocarcinoma.

Introduction of compound (A) and/or compound (B):

As discussed above, it is immaterial to the combination treatment discussed herein whether the two compounds (A) and (B) are administered, for example, simultaneously as a single composition, or, for example, sequentially as two separate compositions. It is important that an effective amount of the first compound/agent administered is present in the patient's body at the time the second compound/agent is administered.

Preferably, the pharmaceutical combination discussed herein is a single composition containing both Compound (A) and Compound (B).

Compound (A) (particularly ABTL0812) is preferably administered orally.

The dose of compound (A) (particularly ABTL0812) to be administered is preferably a dose of 200 mg to 6000 mg (preferably 2000 mg), more preferably a dose of 300 mg to 1600 mg, and even more preferably a dose of 450 mg to 1450 mg.

The dose of Compound (A) (particularly ABTL0812) administered is more preferably a total dose of 200 mg to 6000 mg (preferably 2000 mg) per day, more preferably a total dose of 300 mg to 1600 mg per day, and even more preferably a total dose of 450 mg to 1450 mg per day. Preferably the total dose is provided by administration 1 to 5 times a day, more preferably 2 to 4 times a day and most preferably 3 times a day.

Accordingly, if the total dose is, for example, 1200 mg per day, and it is provided by administration 3 times a day, then each of the 3 times can be administered, for example, 400 mg.

With respect to compound (B), the preferred route of administration generally depends on the chemotherapeutic agent in question.

The preferred route for administering the preferred compound (B) is briefly described below:

docetaxel; - preferably administered intravenously as a solution for infusion

paclitaxel; - preferably administered intravenously as a solution for infusion

carboplatin; - preferably administered intravenously as a solution for infusion

cisplatin; - preferably administered intravenously as a solution for infusion

gemcitabine; - preferably administered intravenously as a solution for infusion

nab-paclitaxel (Abraxane®); - preferably administered intravenously as a suspension for infusion

pemetrexed; - preferably administered intravenously as a solution for infusion

doxorubicin; - preferably administered intravenously

Aspects/embodiments of the invention in the so-called claims format:

1. Pharmaceutical combination containing:

(A): a compound which is a polyunsaturated fatty acid of the formula COOR ₁ -CHR ₂ -(CH ₂ )a-(CH=CHCH ₂ )b-(CH ₂ )c-CH ₃ , a pharmaceutically acceptable salt thereof, or a combination thereof, Where

(i) a may be any integer between 0 and 7,

(ii) b may be any integer from 2 to 7,

(iii) c may be any integer between 0 and 7,

(iv) R ₁ is H, Na, K, CH ₃ , CH ₃ -CH ₂ or PO(O-CH ₂ -CH ₃ ) ₂ , and

(v) R ₂ is OH, OCH ₃ , O-CH ₃ COOH, CH ₃ , Cl, CH ₂ OH, OPO(O-CH ₂ -CH ₃ ) ₂ , NOH, F, HCOO or N(OCH ₂ CH ₃ ) ₂ ;

And

(B): a compound that is a chemotherapeutic agent,

for simultaneous, separate or sequential use in the treatment of cancer in a human patient.

2. Pharmaceutical combination according to claim 1, characterized in that

(i) a may be any integer between 5 and 7,

(ii) b may be any integer from 2 to 4, and

(iii) c may be any integer from 1 to 5.

3. Pharmaceutical combination according to any of the preceding claims, characterized in that R ₁ is H and R ₂ is OH.

4. A pharmaceutical combination according to any of the preceding claims, characterized in that said compound (A) is at least one compound or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

COOH-CHOH-(CH ₂ ) ₆ -(CH=CH-CH ₂ ) ₂ -(CH ₂ ) ₃ -CH ₃ (ABTL0812),

COOH-CHOH-(CH ₂ ) ₆ -(CH=CH-CH ₂ ) ₃ -CH ₃ (183A1),

COOH-CHOH-(CH ₂ ) ₃ -(CH=CH-CH ₂ ) ₃ -(CH ₂ ) ₃ -CH ₃ (183A2),

COOH-CHOH-(CH ₂ ) ₂ -(CH=CH-CH ₂ ) ₄ -(CH ₂ ) ₃ -CH ₃ (204A1),

COOH-CHOH-(CH ₂ ) ₂ -(CH=CH-CH ₂ ) ₅ -CH ₃ (205A1) and

COOH-CHOH-CH ₂ -(CH=CH-CH ₂ ) ₆ -CH ₃ (226A1).

5. Pharmaceutical combination according to any of the preceding claims, characterized in that said compound (A) is COOH-CHOH-(CH ₂ ) ₆ -(CH=CH-CH ₂ ) ₂ -(CH ₂ ) ₃ -CH ₃ ( ABTL0812) or a pharmaceutically acceptable salt thereof.

6. The pharmaceutical combination according to claim 5, characterized in that said compound (A) is the sodium salt of COOH-CHOH-(CH ₂ ) ₆ -(CH=CH-CH ₂ ) ₂ -(CH ₂ ) ₃ -CH ₃ (ABTL0812).

7. Pharmaceutical combination according to any of the preceding claims, characterized in that said cancer is at least one cancer selected from the group consisting of:

lung cancer;

non-small cell lung cancer;

squamous cell carcinoma;

adenocarcinomas;

endometrial cancer;

serous cancer;

pancreatic cancer;

glioblastoma;

resistant and recurrent breast cancer;

head and neck cancer;

neuroblastoma, and

cholangiocarcinoma.

8. Pharmaceutical combination according to any one of the preceding claims, characterized in that said compound (B) is at least one compound which is a chemotherapeutic agent selected from the group consisting of:

docetaxel;

paclitaxel;

carboplatin;

cisplatin;

gemcitabine;

nab-paclitaxel;

retinoic acid;

temozolomide;

irinotecan;

doxorubicin; And

topotecan.

9. Pharmaceutical combination according to claim 8, characterized in that said compound (A) is COOH-CHOH-(CH ₂ ) ₆ -(CH=CH-CH ₂ ) ₂ -(CH ₂ ) ₃ -CH ₃ (ABTL0812 ) or a pharmaceutically acceptable salt thereof.

10. Pharmaceutical combination according to claim 9, characterized in that

Compound (B) is docetaxel and the cancer is lung cancer, preferably non-small cell lung adenocarcinoma;

Compound (B) is paclitaxel and the cancer is lung cancer, preferably non-small cell lung adenocarcinoma;

Compound (B) is gemcitabine and the cancer is pancreatic cancer;

the compound (B) is carboplatin and the cancer is endometrial cell cancer;

the compound (B) is retinoic acid and the cancer is neuroblastoma;

Compound (B) is paclitaxel and the cancer is breast cancer, preferably triple negative breast cancer;

Compound (B) is paclitaxel and carboplatin and the cancer is squamous cell carcinoma, preferably non-small cell squamous cell lung cancer;

Compound (B) is paclitaxel and carboplatin and the cancer is non-small cell adenocarcinoma of the lung;

Compound (B) is pemetrexed and cisplatin and the cancer is non-small cell adenocarcinoma of the lung;

the compound (B) is paclitaxel and the cancer is endometrial cancer;

the compound (B) is paclitaxel and carboplatin and the cancer is endometrial cancer;

Compound (B) is paclitaxel and gemcitabine and the cancer is pancreatic cancer;

Compound (B) is gemcitabine and the cancer is pancreatic cancer;

Compound (B) is gemcitabine and nab-paclitaxel and the cancer is pancreatic cancer;

- Compound (B) is cisplatin and gemcitabine, and the cancer is neuroblastoma;

Compound (B) is doxorubicin and the cancer is breast cancer, preferably triple negative breast cancer; or

Compound (B) is cisplatin and gemcitabine, and the cancer is cholangiocarcinoma.

11. Pharmaceutical combination according to any of the preceding claims, characterized in that said pharmaceutical combination is a single composition containing both compound (A) and compound (B).

12. Pharmaceutical combination according to any one of the preceding claims, characterized in that said compound (A) is administered orally.

13. A pharmaceutical combination according to any one of the preceding claims, characterized in that said compound (A) administration dose is a total dose of 200 mg to 2000 mg per day.

14. Pharmaceutical combination according to any one of paragraphs. 12-13, characterized in that said compound (A) is COOH-CHOH-(CH ₂ ) ₆ -(CH=CH-CH ₂ ) ₂ -(CH ₂ ) ₃ -CH ₃ (ABTL0812) or its pharmaceutically acceptable salt.

15. Pharmaceutical combination according to claim 14, characterized in that

Compound (B) is docetaxel and is administered intravenously as an infusion solution;

Compound (B) is paclitaxel and is administered intravenously as an infusion solution;

Compound (B) is carboplatin and is administered intravenously as an infusion solution;

Compound (B) is cisplatin and is administered intravenously as an infusion solution;

Compound (B) is gemcitabine and is administered intravenously as an infusion solution;

Compound (B) is nab-paclitaxel and is administered intravenously as a suspension for infusion;

Compound (B) is temozolomide and is administered orally (eg in the form of capsules);

Compound (B) is irinotecan and is administered intravenously as an infusion solution;

the compound (B) is doxorubicin and is administered intravenously; or

Compound (B) is topotecan and is administered intravenously as an infusion solution.

EXAMPLES

EXAMPLE 1 ABTL0812 in Combination with Various Chemotherapeutic Agents - In Vitro Studies

1.1:

Viability studies of lung cancer cells using AB TL0812 in combination with docetaxel

Study Link: ABT-EI-

Venue: Protein Kinases & Cell Signaling Group, UAB

GLP Compliance: No

Test Compound: ABTL0812 (batch 006/2010)

Standard compound: docetaxel (Fluka, 01855-5MG-F, lot 1425738V)

System under study: cell line A549 (human lung cancer)

Objective: to assess the effect of the combination of ABTL0812 and docetaxel on cell viability. Docetaxel is a cytotoxic compound used in lung cancer; therefore, a possible therapeutic combination for lung cancer could include the use of ABTL0812 and docetaxel.

Methods: A549 cells were incubated with increasing concentrations of ABTL0812 (3-300 μM), docetaxel (0.01-100 μM), or a combination thereof (below IC ₅₀ , i.e. fixed 20 μM concentration of ABTL0812 and 0.01-100 μM docetaxel ) within three days (EBS (English FBS) 0.5%). Cell viability was assessed in all cases in the MTT study and the IC ₅₀ was calculated for ABTL0812, docetaxel and the combination. Finally, the additivity index (Cl) was calculated to evaluate synergy according to the method of Chou and Talalay (Chou 2006; Chou 2010) as follows: Cl=(D)1/(Dx)1+(D)2/(Dx)2, where the values Cl<1, =1 and >1 indicate synergy, additive effect and antagonism, respectively. In the denominator, (Dx)1 refers to D1 "alone" which inhibits the system by x%, and (Dx)2 refers to D2 "alone" which inhibits the system by x%. In the numerators (D)1 and (D)2 "in combination" also inhibit the system by x%. Results are shown as the average of two independent experiments.

Results: As expected, ABTL0812 and docetaxel were cytotoxic when administered alone. The addition of ABTL0812 at a low concentration (20 μM, equivalent to IC ₁₀ ) significantly increased the cytotoxicity of docetaxel. The IC ₅₀ of docetaxel in the presence of ABTL0812 decreased >80-fold, i.e. from 1.7 μM to 0.02 μM (see table below and figure 1 herein).

The possible synergy of ABTL0812 with docetaxel was then calculated according to the method of Chou and Talalay (Chou 2006; Chou 2010). The combination of both drugs showed synergy over the entire range of activity, where Cl=0.47 at 50% cell viability. This CI value is an indicator of synergy.

Conclusions: ABTL0812 and docetaxel have a synergistic effect in vitro against the lung adenocarcinoma cell line A549. ABTL0812 at a suboptimal concentration (20 μM) lowers the IC ₅₀ of docetaxel by more than 80 times. These results open the possibility of using a combination of both drugs in vivo in lung cancer. Docetaxel is the drug of choice for several stages of lung cancer; thus, the combination with ABTL0812 has a potential beneficial effect due to the presence of synergy between both drugs, which enhances their cytotoxic effects on lung cancer cells.

1.2:

Viability studies of lung cancer cells using AB TL0812 in combination with docetaxel

Study link: LN3-T30 and ABT-EI-048

Venue: Protein Kinases & Cell Signaling Group, UAB

NLP Compliance: No

Test Compound: ABTL0812 (batch 002/2012)

Standard compound: paclitaxel (SelleckChem, S1150-10MG, Lot 09)

System under study: A549 cell line (human lung cancer) with KRAS mutation; H157 (non-small cell squamous cell lung cancer) with a PTEN mutation; HTB182: human non-small cell lung cancer, and H1975: human non-small cell lung adenocarcinoma with PI3KCA mutation

Objective: to evaluate the effect of the combination of ABTL0812 and paclitaxel on cell viability. Paclitaxel is a cytotoxic compound used in lung cancer; therefore, a possible therapeutic combination for lung cancer could include the use of ABTL0812 and paclitaxel.

Methods: A549 cells were incubated with increasing concentrations of ABTL0812 (3-300 μM), paclitaxel (0.003-1 μM), or a combination thereof using ABTL0812 at a fixed concentration below the IC ₅₀ and 0.003-1 μM paclitaxel for 72 hours in DMEM containing 0 .1% EBS. H157, H1957 and HTB-812 cells were incubated with increasing concentrations of paclitaxel (0.001-10 μM) alone and in combination with ABTL0812 at a fixed concentration below the IC ₅₀ for 48 hours in DMEM containing 0.1% FBS. Cell viability was assessed in all cases in the MTT study and the IC ₅₀ was calculated for paclitaxel and the combination. The concentrations of combinations for different cell lines were:

A549 cells: IC ₅₀ ABTL0812 = 49 μM. Paclitaxel was combined with 10 (IC10), 20 (IC25) and 30 μM (IC35) ABTL0812

H157 cells: IC ₅₀ ABTL0812 = 23 μM. Paclitaxel was combined with 10 (IC20) and 15 (IC35) μM ABTL0812

H1957 cells: IC ₅₀ ABTL0812 = 43 μM. Paclitaxel was combined with 10 (IC10) and 20 (IC20) μM ABTL0812

HTB-812 cells: IC ₅₀ ABTL0812 = 29 μM. Paclitaxel was combined with 10 (IC20) and 15 (IC35) μM ABTL0812.

Finally, the additivity index (Cl) was calculated as described in the previous section for the IC ₅₀ values of paclitaxel.

Results: ABTL0812 and paclitaxel were cytotoxic to all four lung cancer cell lines. For the lung cancer cell line A549, when used alone, the addition of 15, 20, or 30 μM ABTL0812 increased the cytotoxicity of paclitaxel. The IC ₅₀ of the combination was lower than either drug alone and a 2-fold (15 μM and 20 μM) or 7-fold (30 μM) reduction in the IC ₅₀ of paclitaxel was observed. This decrease in the IC ₅₀ of paclitaxel was synergistic and the CIs were 0.34, 0.28 and 0.22 for 15, 20 and 30 μM ABTL0812, respectively. With respect to H157 cells, the observed synergy was not as pronounced as for A549 cells, and the cytotoxicity of paclitaxel increased only in combination with 15 μM ABTL0812 (IC35), where the decrease in the IC ₅₀ of paclitaxel was from 4.19 to 2.39 μM, or 1 .75 times, which corresponds to synergy with CI 0.7. In the case of H1957 cells, when 10 μM (IC10) ABTL0812 was added, there was no synergy and IC ₅₀ values did not change (3.68 and 3.47 μM), but when 20 μM (IC20) ABTL0812 was added, a decrease in IC ₅₀ value of 6.5 was observed. times, or from 3.68 to 0.56 μM. This decrease in the IC ₅₀ of paclitaxel in relation to H1957 cells was synergistic, CI was 0.3. Finally, with respect to HTB-812 cells, good synergy was shown at both concentrations studied, and with the addition of 10 μM (IC20) ABTL0812 a 3-fold decrease or from 2.71 to 0.81 μM was observed, and with the addition of 20 μM (IC35) ABTL0812 was observed to decrease by 3.6 times or from 2.71 to 0.75 μM. This decrease in the IC ₅₀ of paclitaxel against HTB-812 cells was synergistic, Cl were 0.4 and 0.5, respectively. See figure 2 for more information.

Conclusions: ABTL0812 and paclitaxel have a synergistic effect in vitro against all four studied lung cancer cell lines, regardless of their mutational status. Suboptimal concentrations of ABTL0812 lowered the IC ₅₀ of paclitaxel. These results open the possibility of using a combination of both drugs in vivo in lung cancer.

1.3:

Pancreatic cancer cell viability study using ABTL0812 alone or in combination with gemcitabine

Study link: LN1-T56-T58

Venue: Protein Kinases & Cell Signaling Group, UAB

NLP Compliance: No

Test compounds: ABTL0812 (batch 006/2010)

Standard compound: gemcitabine (Sigma, G6423, lot 041M4727V)

System under study: MiaPaca2 (human pancreatic cancer)

Objectives: To investigate the possible synergy of adding ABTL0812 to gemcitabine in relation to the MiaPaca2 pancreatic cancer cell line. Gemcitabine is considered the standard treatment for most types of pancreatic cancer. In many cases, mainly in advanced pancreatic cancer, it is administered in combination with other drugs (Ghaneh and Neoptolemos 2007); thus, it is interesting to know if there is any additive effect for both drugs.

Methods: MiaPaca2 cells were seeded in 24-well plates and gemcitabine (0.01-100 μM), ABTL0812 (3-300 μM), or a combination thereof (fixed concentration of ABTL0812 below IC ₅₀ , i.e. 25 μM and 0.01 -100 μM gemcitabine) and left in the incubator for 72 hours (0.5% FBS). Cell viability was studied in the MTT study and several parameters were determined to evaluate possible synergy. First, the IC ₅₀ was calculated for each drug alone or for the combination. The synergy (Cl) was then calculated as described above.

Results: The IC ₅₀ of the combination was lower than either drug alone and a 7-fold reduction in the IC ₅₀ of gemcitabine was observed. It should be noted that ABTL0812 at the concentration selected for the combination experiment had very low potency when used alone (<10% cytotoxicity), however, it potentiated the cytotoxicity of gemcitabine - see table below and figure 3 herein.

The possible synergy of ABTL0812 with gemcitabine was then calculated according to the method of Chou and Talalay (Chou 2006; Chou 2010) for variable ratio combinations. The combination of both drugs had synergy over the entire range of activity, where Cl=0.65 at 50% cell viability. This Cl value is an indicator of synergy.

Conclusions: ABTL0812 and gemcitabine have synergistic effects in vitro against the MiaPaca2 pancreatic cancer cell line. ABTL0812 at a suboptimal concentration (25 μM) lowers the IC ₅₀ of gemcitabine by 7-fold. These results open the possibility of using a combination of both drugs in vivo.

1.4:

Endometrial cancer cell viability study using ABTL0812 alone or in combination with carboplatin

Study link: ABT-EI-065

Venue: Protein Kinases & Cell Signaling Group, UAB

NLP Compliance: No

Test compounds: ABTL0812 (batch 006/2010)

Standard compound: carboplatin (Sigma, C2538)

Test System: Ishikawa Cells (Human Endometrial Cancer)

Objectives: To investigate the possible synergy of the addition of ABTL0812 to carboplatin in the Ishikawa endometrial cancer cell line. Carboplatin is considered the standard treatment for most types of endometrial cancer. Thus, it is interesting to know if there is any additive effect for both drugs.

Methods: Ishikawa cells were seeded in 24-well plates and carboplatin was added at increasing concentrations (1-300 μM) in the presence of 4 μM ABTL0812 (equivalent to IC10) for 48 hours (0.5% FBS). Cell viability was studied in the MTT study and several parameters were determined to evaluate possible synergy. First, the IC ₅₀ was calculated for each drug alone or for the combination.

Results: The IC ₅₀ of the combination was lower than either drug alone and a 3-fold reduction in the IC ₅₀ of carboplatin was observed. Of note, ABTL0812 at the concentration selected for the combination experiment had very low potency when used alone (<10% cytotoxicity), however, it potentiated the cytotoxicity of gemcitabine - see Figure 4 herein.

Conclusions: ABTL0812 and carboplatin have synergistic effects in vitro against the Ishikawa endometrial cancer cell line. ABTL0812 at a suboptimal concentration (4 μM) lowers the IC ₅₀ of carboplatin by 3 times. These results open the possibility of using a combination of both drugs in vivo.

1.5:

Neuroblastoma cell viability study using ABTL0812 alone or in combination with retinoic acid

Study link: ABT-EI-055

Location: Childhood Cancer Translational Research Laboratory (VHIR)

NLP Compliance: No

Test compounds: ABTL0812 (batch 006/2010)

Standard Compound: Retinoic Acid (Sigma, R2625)

Test system: SK-N-BE (2): human neuroblastoma cell line, and LA1-5S: clonal subline of the LA-N-1 neuroblastoma cell line

Objectives: to evaluate the effect of the combination of ABTL0812 and retinoic acid (RA) on cell viability in neuroblastoma cell lines SK-N-BE(2) and LA1-5S.

Methods: LA1-5S and SK-N-BE(2) cells were incubated with ABTL0812 at a fixed concentration below the IC ₅₀ (30 μM), increasing concentrations of retinoic acid at 10 μM, 20 μM and 30 μM, or a combination thereof. 10 μM retinoic acid is the pharmacological dose that was administered orally in phase I trials in patients with neuroblastoma (Villablanca et al. 1995). Cells were treated for 24 hours in IMDM with 0.5% FBS. Cell viability was assessed in all cases in the study using crystal violet. Different doses were evaluated in six replications and the results are shown as the average of two independent experiments. Statistical analysis was performed based on the T-test using GraphPad Prism® 5.0 software (* p<0.05; ** p<0.01; *** p<0.001).

Results: ABTL0812 had moderate cytotoxicity against neuroblastoma cell lines LA1-5S and SK-N-BE (2) when used as a single agent at low concentrations; the effectiveness of retinoic acid at concentrations of 10 μM and 20 μM was even lower. The combination of 30 μM ABTL0812 and retinoic acid resulted in high cytotoxicity against both neuroblastoma cell lines. The proportion of dead/non-proliferating cells for any of the combinations was higher than for each drug alone, suggesting a synergistic effect. The increase in cell death was statistically significant at all concentrations (*** p<0.001). See figure 5 in this document for more information.

Conclusions: The combination of ABTL0812 and RK has a strong synergistic effect that enhances their in vitro cytotoxic activity against neuroblastoma cell lines SK-N-BE(2) and LA1-5S. RK is commonly used in clinics to control neuroblastoma at the stage of minimal residual disease, thus, the data obtained are a stimulus for further study of this combination for the control of neuroblastoma.

1.6:

Breast cancer cell viability study using ABTL0812 alone or in combination with paclitaxel

Study Link: Pending Review

Place of study: Targets lab, UdG

NLP Compliance: No

Test compounds: ABTL0812 (batch 006/2010)

Standard compound: Paclitaxel (SelleckChem, S1150)

System under study: MDB-DA-231 (triple negative human breast cancer)

Objectives: To investigate the possible synergy of the addition of ABTL0812 to paclitaxel for the triple negative breast cancer cell line MDB-DA-231.

Methods: MDB-DA-231 cells were seeded in 24-well plates and paclitaxel was added at increasing concentrations (1-100 nM) in the presence of 5, 10 and 20 μM ABTL0812 (all doses below IC25) for 48 hours (0.5% EBS). Cell viability was studied in the MTT study and several parameters were determined to evaluate possible synergy. First, the IC ₅₀ was calculated for each drug alone or for the combination.

Results: The IC ₅₀ of the combination was lower than either drug alone and a 2.7-fold reduction in the IC ₅₀ of paclitaxel was observed. Of note, ABTL0812 at the concentration selected for the combination experiment had very low potency when used alone (<10% cytotoxicity), however, it potentiated paclitaxel cytotoxicity and strong synergy was shown with Cl values of 0.3. 0.2 and 0.16 for 5, 10 and 20 μM ABTL0812, respectively, see table below and Figure 6 herein.

Conclusions: ABTL0812 and carboplatin have synergistic effects in vitro against the Ishikawa endometrial cancer cell line. ABTL0812 at a suboptimal concentration (4 μM) lowers the IC ₅₀ of carboplatin by 3 times. These results open the possibility of using a combination of both drugs in vivo.

EXAMPLE 2: ABTL0812 in combination with various chemotherapeutic agents - In vivo studies

2.1:

A549 xenograft in mice, combination study with docetaxel

Study link: ALM-IDIBAPS

Place of study: Molecular and Translational Oncology Research Group. IDIBAPS, Hospital Clinic Barcelona.

NLP Compliance: No

Test Compound: ABTL0812 (batch 002/2012)

Standard compound: docetaxel.

System under study: male nu/nu mice

Objective: To study the antitumor activity of ABTL0812 alone and in combination with docetaxel, the standard drug for the treatment of NSCLC.

Methods: Mice were injected with 5×10 ⁶ A549 cells in each flank to initiate tumor formation. After 20 days, when the tumor volume was about 50 mm ³ , the animals were randomly assigned to equal groups and various treatments were started. 30 mg/kg/day ABTL0812 was administered orally 5 times a week. 5 mg/kg docetaxel was administered intraperitoneally once a week (Coxon et al. 2012). Tumor volume and body weight were monitored 3 times a week.

Results: ABTL0812 significantly reduced tumor volume compared to control animals (ANOVA followed by t-test). The efficacy of ABTL0812 was in fact similar to that observed with docetaxel treatment. Interestingly, ABTL0812 enhanced the antitumor effect of docetaxel. In statistical analysis, this combination therapy was shown to significantly improve tumor growth suppression compared to docetaxel used alone (p<0.001 by t-test). In addition, no reduction in body weight or haematological parameters (not shown) was observed in any of the treated groups, including the groups in which ABTL0812 was co-administered with docetaxel, suggesting that this combination had no toxic effects.

Regarding the antitumor effect of the combination of ABTL0812 and docetaxel in A549 lung cancer xenograft models, all treatments significantly reduced tumor volume compared to control at the time of sacrifice (*, ANOVA followed by t-test). In addition, the combination of 30 mg/kg ABTL0812 + docetaxel was significantly more effective than docetaxel treatment alone (**p<0.01, t-test). On the other hand, no effect on body weight was observed with any of the treatments, either with drugs alone or in combination. See Figure 7 herein for more information.

Conclusion: ABTL0812 reduces tumor growth in A549 cell-derived lung cancer xenograft models. In this model, ABTL0812 has similar SOC efficacy to docetaxel. ABTL0812 and docetaxel as used alone therapeutic agents similarly reduced tumor volume in an A549 cell-derived lung cancer xenograft model. ABTL0812 enhances the antitumor activity of docetaxel in the absence of a toxic effect. These results suggest that combination therapy of ABTL0812 with docetaxel may be of clinical interest in the treatment of lung cancer.

2.2:

Efficacy of ABTL0812 in combination with paclitaxel and carboplatin against human squamous NSCLC (H157) xenograft in mice

Study link: ALM-IDIBAPS

Place of study: Molecular and Translational Oncology Research Group. IDIBAPS, Hospital Clinic Barcelona.

NLP Compliance: No

Test compound: ABTL0812 (batch K102E)

System under study: male nu/nu mice

Objective: Study of survival rates with ABTL0812 alone and in combination with paclitaxel and carboplatin (P/C) and Kaplan-Meier analysis in a human squamous NSCLC xenograft model

Methods: Mice were injected with 5×10 ⁶ H157 cells in one flank to start tumor formation. When the tumor volume was about 100 mm ³ , the animals were randomly assigned to equal groups (n=8 per group) and various treatments were started. Various conditions were studied including vehicle, ABTL0812 120mg/kg po daily, carboplatin 15mg/kg + paclitaxel 50mg/kg ip, and an additional group using a combination of these two regimens. ABTL0812 was always administered two days prior to the first P/C injection and two days after administration, maintaining a regimen of 4 doses of ABTL0812 and one dose of P/C per week. Tumor volume was monitored 3 times a week. When constructing the Kaplan-Meier plot, the final criteria for exclusion of animals from the study was the tumor volume >1000 mm ³ or other indicators of animal welfare, confirmed by the ethical committee. Treatment in the various groups continued until all animals in each group had tumor volumes of 1000 mm ³ or well-being endpoints, except for the ABTL0812 + P/C group, where mice had to be euthanized before reaching tumor volume 1000 mm ³ to complete the experiment procedure.

Results: Treatment with ABTL0812 in combination with R/S was shown to be the most effective treatment modality in the Kaplan-Meier analysis. As can be seen in figure 8, the combination of ABTL0812 + P/C is the most effective treatment in terms of survival rate and has a significant advantage over other groups. At day 20 post-treatment, ABTL0812 + P/C treatment showed a 75% survival compared to 0% for the ABTL0812 and P/C groups and 20% for the vehicle-administered group, in the absence of any significant signs of toxicity .

Conclusion: The final evaluation criteria were established on the basis of various indicators of animal welfare and were decisive for the final evaluation. When the health status of the animals was stable, achievement of a tumor volume of 1000 mm ³ was chosen as the final evaluation criterion. Under these conditions, the combination treatment of ABTL0812 + P/C showed a significant increase in the survival rate measured in the Kaplan-Meier analysis in the H157 squamous cell NSCLC xenograft model, where the survival rate was 75% at 20 days after treatment compared with 0% survival in the groups in which were administered ABTL0812 and vehicle, and 25% survival in the P/C group.

2.3. Efficacy of ABTL0812 in combination with paclitaxel and carboplatin against human NSCLC (H1975) adenocarcinoma xenograft in mice

Study link: ABT-EI-049

Place of study: Molecular and Translational Oncology Research Group. IDIBAPS, Hospital Clinic Barcelona.

NLP Compliance: No

Test Compound: ABTL0812 (batch 002/2012)

Reference Compounds: Paclitaxel (Selleckchem, cat. no. S1150), carboplatin (Sigma Aldrich, cat. no. C2538)

System under study: male nu/nu mice

Objective: To study the antitumor activity of ABTL0812 alone and in combination with paclitaxel and carboplatin in human lung adenocarcinoma xenograft. The combination of paclitaxel and carboplatin is one of the standard therapies for the treatment of NSCLC.

Ways. The H1975 cell line was grown conventionally in DMEM with 10% EBS and cells in the exponential growth phase were harvested and counted for tumor inoculation. Mice were injected in one side with 2.5×10 ⁶ H1957 cells suspended in 50 μl of EBS-free growth medium and 50 μl of Matrigel (Corning Cat. No. 354234). Tumor volume was monitored 3 days per week, and when tumors reached 100 mm ³ (50 to 150 mm ³ ), animals were divided into four equal treatment groups having a similar average tumor volume per group, excluding tumors less than 50 mm ³ or larger than 150 mm 3 to minimize the error.

The following treatment groups were used:

- Vehicle treatment group (n=7): 200 µl water + 5% glycerol administered orally four days a week and two IPs. injections of 100 µl of saline solution once a week

- ABTL0812 (n=9): administered orally 200 μl 120 mg/kg ABTL0812 resuspended in water +5% glycerol 5 times a week

- Paclitaxel/carboplatin (n=9): administered i.p. 100 µl 15 mg/kg paclitaxel i.p. 100 µl 5 mg/kg carboplatin once a week

- ABTL0812 + paclitaxel/carboplatin (n=9): administered orally 200 µl 120 mg/kg ABTL0812 four days a week and ip. 100 µl 15 mg/kg paclitaxel i.p. 100 µl 5 mg/kg carboplatin once a week

Results: ABTL0812, when administered in combination with paclitaxel and carboplatin, showed the best in vivo antitumor effect in xenografts derived from H1957 cells. The administration of paclitaxel and carboplatin reduced tumor volume compared to the vehicle group, administration of ABTL0812 alone showed a similar reduction in tumor volume with an improved trend, and the triple combination of ABTL0812 + paclitaxel and carboplatin showed the largest reduction in tumor volume with significant differences. In addition, during the first week in the group treated with the triple combination, there was a slight decrease in body weight, which then stabilized until the end of the experiment, (weight loss or hematological parameters (not shown) was not observed in any of the groups in treated, including those treated with ABTL0812 co-administered with docetaxel, suggesting that this combination had no toxic effects See Figure 9 herein for more information.

Conclusion: Orally administered ABTL0812 reduces tumor growth in H1975 cell-derived lung cancer xenograft models. In this model, ABTL0812 has a similar SOC to paclitaxel + carboplatin. In addition, ABTL0812 enhances the antitumor activity of paclitaxel/carboplatin in the absence of a toxic effect. These results suggest that combination therapy of ABTL0812 with paclitaxel/carboplatin may be of clinical interest in the treatment of lung cancer.

2.4. Efficacy of ABTL0812 in combination with pemetrexed and cisplatin against human NSCLC (A549) adenocarcinoma xenograft in mice

Study Link: ABT-EI-052

Venue: Protein Kinases & Cell Signaling Group, UAB

NLP Compliance: No

Test compound: ABTL0812 (batch K102E)

Standard compound: pemetrexed (Sigma Aldrich, cat. no. PHR1596) and cisplatin (Sigma Aldrich, cat. no. P4394)

System under study: male nu/nu mice

Objective: The aim of this study was to evaluate the antitumor efficacy of ABTL0812 alone or in combination with standard chemotherapy using pemetrexed and cisplatin in the treatment in a subcutaneous lung cancer xenograft model in immunosuppressed nude mice implanted with A549 human lung adenocarcinoma cells.

Methods: The A549 cell line was grown conventionally in DMEM with 10% FBS and exponential growth phase cells were harvested and counted for tumor inoculation. 50 female athymic mice, 8 weeks old, were injected in one flank with 5×10 ⁶ MiaPaca2 cells suspended in 50 µl of EBS-free growth medium and 50 µl of Matrigel (Corning cat. no. 354234). Tumor volume was monitored 3 days per week, and when tumors reached 100 mm ³ (50 to 150 mm ³ ), animals were divided into three equal groups having a similar average tumor volume per group, excluding tumors smaller than 50 mm ³ or larger than 150 mm ³ to minimize the error.

The following treatment groups were used:

- Vehicle group (n=7): 200 μl water + 5% glycerol orally administered 4 times per week i.p. twice a week 200 µl saline buffer (chemo vehicle)

- Pemetrexed/cisplatin (n=20): administered i.p. twice a week 100 µl of 100 mg/kg pemetrexed i.p. once a week 100 µl 2 mg/kg cisplatin

- ABTL0812 + pemetrexed/cisplatin (n=20): 200 µl of 120 mg/kg of ABTL0812 resuspended in water + 5% glycerol was orally administered 4 times a week and ip. 100 µl 100 mg/kg pemetrexed i.p. once a week 100 µl 2 mg/kg cisplatin

Results: As can be seen in Figure 1, ABTL0812, when administered in combination with pemetrexed and cisplatin, showed a strong antitumor effect and enhanced therapeutic effect of pemetrexed/cisplatin, thus allowing a significant reduction in tumor volume compared to the pemetrexed/cisplatin group. cisplatin. ABTL0812 + pemetrexed/cisplatin showed significant tumor reduction compared to pemetrexed/cisplatin starting on day 33 of treatment, the difference being more significant on subsequent days until the last day of treatment on day 41. Both groups, pemetrexed/cisplatin and ABTL0812 + pemetrexed/cisplatin, showed a significant reduction in tumor volume compared to the vehicle group from day 22 of treatment until the last day of treatment on day 41.

In terms of treatment-induced toxicity, Figure 2 shows the change in total body weight over time for all four groups, which was monitored over 41 days of treatment. Although no signs of toxicity or clinicopathological symptoms were observed, there was a slight decrease during the first week of treatment in the ABTL0812 + pemetrexed/cisplatin group compared to the rest due to reduced food intake on chemotherapy days. No additional clinical pathological signs were observed in any of the groups. See Figure 10 herein for more information.

Conclusion: As described above, ABTL0812 reduces tumor growth in A549 cell-derived lung cancer xenograft models. In this model, ABTL0812 enhances the antitumor activity of pemetrexed and cisplatin in the absence of a toxic effect. Therapy with pemetrexed and cisplatin is the most commonly used first-line therapy for the treatment of patients with lung adenocarcinoma, thus these results suggest that combination therapy of ABTL0812 with pemetrexed and cisplatin may be of clinical interest in the treatment of patients with lung cancer.

2.5:

Efficacy Study of ABTL0812 in Combination with Paclitaxel in an Orthotopic Mouse Endometrial Cancer Model

Place of study: Xenopat (Barcelona, Spain)

NLP Research: No

Test Compounds: ABTL0812 (Batch 001R/2014), Paclitaxel (Teva)

System under study: Nude-Foxn1nu female nude mice

Objective: to evaluate the antitumor efficacy of orally administered ABTL0812 in combination with i. p. paclitaxel in the Ishikawa orthotopic model of endometrial cancer.

Methods: Female mice were orthotopically implanted in the uterus with a 3 mm ³ tumor fragment derived from the Ishikawa cell line. Before starting drug treatment, all animals were weighed and the volume of the tumor was assessed by palpation. Mice were assigned to groups using a randomized block design based on tumor volume values. Paclitaxel was administered i.p. every 7 days (15 mg/kg). ABTL0812 was administered orally via gavage according to a 5 day dosing schedule with 2 days off (120 mg/kg/day). In general, the animals were divided into four groups, as shown in the table below.

Tumor volume was estimated by the formula V=π/6×L×W ² where L is the long axis and W is the short axis of the tumor, respectively. During conventional monitoring, the animals were tested for the effect of tumor growth and treatment on normal behaviors such as mobility, visual feed assessment, weight gain/loss (body weight was measured twice a week during drug administration), eye/hair blanching, and any other deviations. Death and observed clinical signs were noted using the number of animals from each subgroup.

Given that the size of orthotopic tumors can only be measured after sacrifice, a subset of animals (n=2-3 per group) were sacrificed after one week of treatment to determine the early impact on tumor growth. Most animals were sacrificed after 3 weeks of treatment (n=5-7 per group).

Results: A group of mice (n=36) were orthotopically implanted with a 3 mm ³ tumor fragment derived from the Ishikawa cell line. During the entire period of experimental treatment, no effect on the behavior of animals was noted. Animals treated with the paclitaxel combination showed reduced tumor weight gain and some weight loss, which was partially compensated at the end of the treatment period. Given that this decrease in body weight compared to the control group did not reach 10%, it was not considered as associated with toxicity.

Animals were sacrificed after drug administration for one or three weeks and tumor volume was determined as indicated in the methods. Animals treated for one week showed no difference. However, in animals treated for three weeks with the combination of paclitaxel + ABTL0812, an additive effect was observed - see Figure 11 herein.

Conclusions: The combination of ABTL0812 + paclitaxel showed a synergistic effect compared to the effect of each drug alone, as animals treated with the combination showed a significant reduction in tumor volume compared to control animals. At the same time, some decrease in body weight was observed in animals treated with the combination. However, this effect was not considered toxic.

2.6:

Efficacy study of ABTL0812 in combination with paclitaxel and carboplatin in a human endometrial cancer xenograft (PDX) model in mice

Study link: ABT-EI-043

Place of study: VHIR

NLP Compliance: No

Test compound: ABTL0812 (batch K102E=MEI-014-15)

Standard compounds: paclitaxel (Selleckchem, cat. no. S1150), carboplatin (Sigma Aldrich, cat. no. C2538)

System under study: Nude-Foxn1nu female nude mice

Objective: To study the antitumor activity of ABTL0812 alone and in combination with paclitaxel and carboplatin (P/C) in a model of PDX implanted subcutaneously in nude mice.

Methods: A tumor surgically removed from a patient with histologically defined serous tumor type, stage IIIC2, 100% myometrial invasion and pelvic and aortic lymph node invasion and lymphovascular invasion, having p53 and PI3KCA gene mutations, was implanted in one flank of several nude mice . After the tumors had grown to 100 mm ³ , tumors were removed from mice, cut into 2 mm fragments, and re-implanted in one flank of 40 mice. When the mean tumor volume reached 100 mm ³ , mice were randomly assigned to the following dose groups: vehicle (n=11); ABTL0812: 120 mg/kg po 5 times a week (n=10); paclitaxel/carboplatin (R/C): R: 50 mg/kg/C: 15 mg/kg ip (n=12); and ABTL0812 + P/C: ABTL0812 (n=10) was always administered two days prior to the first P/C administration and two days after administration, maintaining a regimen of 4 doses of ABTL0812 and one dose of P/C per week; the doses were the same as when administered separately.

The effectiveness of therapy was determined by the effect of treatment on tumor growth, which was measured by its volume. The health status of the animals and drug-induced toxicity were determined by the animal's body weight during the study. Tumor size change and tumor weight were assessed in a two-way ANOVA (daily analysis).

In order to mimic the design of the phase II clinical trial, where long-term administration of ABTL0812 is expected after P/C cycles, P/C administration was stopped in the P/C and ABTL0812 + P/C groups and long-term administration of ABTL0812 was maintained. Tumor volume was measured after cessation of P/C administration for another 17 days.

Results: A population of 40 mice were randomly assigned to four treatment groups when the tumor size reached approximately 100 mm3. As shown in Figure 12 herein, at 47 days post-treatment, ABTL0812 + P/C showed superior efficacy and a statistically significant reduction in tumor growth compared to vehicle, ABTL0812 alone, and P/C alone. The improvement in therapeutic effect was not associated with an increase in signs of toxicity, as evidenced by the absence of a significant decrease in total body weight. ABTL0812 alone showed the same efficacy as P/C without any sign of significant toxicity.

On day 47, P/C administration was discontinued while long-term administration of ABTL0812 was continued 5 days a week to determine whether long-term administration of ABTL0812 could prevent or delay tumor recurrence. While in the P/C group, in which the treatment of mice was stopped, the tumor continued to grow at a similar or slightly higher rate, in the ABTL0812 + P/C group, in which the administration of ABTL0812 was continued, the relative growth rate not only did not increase, but also showed signs of remission. In the ABTL0812 group, which continued administration of ABTL0812 throughout the experiment, tumor growth suppression very similar to P/C was shown while maintaining a constant level of growth, indicating no evidence of a resistance process that would increase the relative growth rate.

Conclusion: The combination of ABTL0812 + P/C showed a synergistic effect in terms of a significantly higher reduction in tumor volume compared to the administration of P/C alone, as well as a significant reduction in tumor volume compared to the vehicle group during the first 47 days. ABTL0812 administered as monotherapy has been shown to be as effective in reducing tumor growth as P/C. Partial weight loss was observed only in the ABTL0812 group on day 15, and this weight loss was compensated over the next four days, and the weight remained stable until the end of the experiment. None of the other groups showed any effects that could be classified as toxic.

To simulate a phase II human clinical trial where it is planned to administer ABTL0812 in combination with R/C as first-line therapy and then administer ABTL0812 long-term after cycles of chemotherapy, P/C was stopped on day 47 and ABTL0812 continued. While tumor growth was not reduced in the P/C and ABL0812 group, long-term administration of ABTL0812 was effective in preventing tumor recurrence after P/C treatment in this human endometrial PDX model, showing signs of remission 10 days after cessation of chemotherapy.

2.7:

Efficacy study of ABTL0812 administered in combination with paclitaxel/gemcitabine (P/Gm) in the Miapaca-2 human pancreatic cancer xenograft model implanted in mice

Study Link: ABT_EI_001XP

Research location: Ability Virtual Lab - UAB

NLP Compliance: No

Test Compound: ABTL0812 (Batch K102E = MEI-014-15)

Standard compound: paclitaxel/gemcitabine (P/Gm)

System under study: Nude-Foxn1nu female nude mice

Objective: Evaluate the antitumor efficacy of ABTL0812 administered orally in combination with P/Gm in the MiaPaCa2 pancreatic cancer xenograft model in mice.

P/Gm administered by i.p. was used as a positive control, vehicle, ABTL0812 and the combination of ABTL0812 + P/Gm were also tested. Efficacy was assessed by tumor growth, and tolerability and toxicity of the compound were assessed by changes in body weight of animals.

Methods: Female nude mice (n=9 per group) were injected subcutaneously with 0.1 ml of MiaPaca2 cells (5×10 ⁶ cells/ml in serum-free DMEM at a ratio of 1:1 with Matrigel) in one side. Tumor volume (TV) was _measured by the formula length x width ² × ^1/2 three times a week. When the average tumor volume reached 100 mm ³ , mice were randomly divided into groups and administered the following doses: vehicle; ABTL0812, 120 mg/kg 5 times a week; P/Gm 15 mg/kg and 60 mg/kg twice a week, and a combination of ABTL0812 + P/Gm, where ABTL0812 was administered four times a week (days 1, 2, 4 and 5), always two days before the introduction of P /Gm (days 3 and 6) for a total of 4 weeks.

The effectiveness of therapy was determined by the effect of treatment on tumor growth, which was measured by its volume. The health status of the animals and drug-induced toxicity were determined by the animal's body weight during the study. Tumor size change was assessed by two-way ANOVA (daily analysis) and Student's t-test on days when significant differences were observed.

Results: The graphs shown in Figure 13 herein show the effect of ABTL0812 administered orally in combination with P/Gm on relative tumor volume and total body weight as a measure of toxicity. ABTL0812 in combination with P/Gm showed the highest efficiency, a significant reduction in tumor volume compared with the introduction of P/Gm alone for the indicated time periods. In addition, for the combined treatment of ABTL0812 + P/Gm, tumor regression was shown in all animals starting from the 10th day of treatment, in contrast to what was observed in the remaining groups. Administration of ABTL0812 alone appears to have a beneficial effect during the first 15 days of treatment, where the tumor volume is similar to that in the vehicle group in subsequent days. The introduction of P/Gm at the indicated doses reduces tumor volume compared to the groups in which the carrier and ABTL0812 were administered, but the differences were not statistically significant for any of the analyzed time periods. On the graph of changes in total body weight, you can see the effect of the combination of ABTL0812 + P/Gm on total body weight compared to the rest of the groups. Although a slight decrease in body weight was observed after the first doses of ABTL0812 + P/Gm, the toxicity caused by the combination was minimal and no acute toxic effects were observed in any of the animals during the experiment.

Conclusion: The combination of ABTL0812 + P/Gm showed a synergistic effect in terms of a significantly higher reduction in tumor volume compared to the administration of P/Gm alone. P/Gm also showed a higher reduction in tumor volume compared to the vehicle and ABTL0812 groups, although it was not statistically significant. ABTL0812 administered as monotherapy showed the same tumor growth curve as the vehicle group. It should be noted that administration of ABTL0812 + P/Gm can induce tumor regression in all animals treated with the combination where the tumor volume remained at about 100 mm ³ up to day 10 of treatment and decreased to less than 100 mm ³ after 10 days of treatment, which indicator of tumor regression. Some reduction in body weight was observed in the ABTL0812 + P/Gm group, although no effects were noted in any of the mice in any of the groups that could be classified as toxic.

2.8:

Efficacy study of ABTL0812 administered in combination with nab-paclitaxel/gemcitabine (Nab-Pac/Gm) in the MicPaca-2 human pancreatic cancer xenograft model implanted in mice

Study Link: ABT-EI-053

Research location: Ability Virtual Lab - UAB

NLP Compliance: No

Test Compound: ABTL0812 (Batch K102E = MEI-014-15)

Reference compound: gemcitabine hydrochloride (Sigma Aldrich G6423). Nab-paclitaxel (Abraxane; ID: 3369272 Celgene)

System under study: Nude-Foxn1nu female nude mice

Objective: The objective of this study was to evaluate the efficacy and safety of ABTL0812 administered orally to enhance the antitumor effect of standard of care (SOC), chemotherapy with gemcitabine and gemcitabine/nab-paclitaxel combination administered intraperitoneally (i.p.) in a cancer cell xenograft model human pancreas MiaPaca2 in vivo implanted in immunosuppressed nude mice. Both chemotherapy options are the most common first-line treatments for advanced pancreatic cancer in humans.

Methods: The MiaPaca2 cell line was grown in the conventional manner and cells in the exponential growth phase were harvested and counted for tumor inoculation. 55 female immunodeficient athymic mice were injected subcutaneously with 5×10 ⁶ MiaPaca2 cells suspended in 50 µl of EBS-free growth medium and 50 µl of Matrigel (Corning cat. No. 354234) in one side. Tumor volume was monitored 3 days per week, and when tumors reached 100 mm ³ (50 to 150 mm ³ ), animals were divided into six equal groups having a similar average tumor volume per group, excluding tumors smaller than 50 mm ³ or larger than 150 mm ³ to minimize the error.

The following treatment groups were used:

- Vehicle group (n=9): Orally administered 200 µl of water + 5% glycerol 5 times a week (vehicle ABTL0812) and ip twice a week 200 µl of saline buffer (vehicle for chemotherapy).

- ABTL0812 (n=9): administered orally 120 mg/kg in 200 μl of distilled water + 5% glycerol 5 times a week.

- Gemcitabine (n=9): administered i.p. 60 mg/kg in 100 µl of sterile water 2 times a week.

- Gemcitabine + nab-paclitaxel (n=8): A fresh preparation of nab-paclitaxel was obtained from the original powder (10% (mol/mol) in 0.9% NaCl solution) and administered i.p. in an amount of 5 mg/kg in 230 µl 2 times a week. Gemcitabine was administered i.p. in the amount of 60 mg/kg in 100 µl of sterile water 2 times a week. It is important to change the injection site to avoid intestinal necrosis and to administer injections of nab-paclitaxel and gemcitabine into the abdominal cavity of mice as far apart as possible.

- Gemcitabine + ABTL0812 (n=10): 120mg/kg ABTL0812 orally in 200µl distilled water+5% glycerol 5 times a week and 60mg/kg gemcitabine i.p. in 100 µl of sterile water 2 times a week. ABTL0812 was always administered prior to chemotherapy to reduce IP-induced stress. injection, which is usually carried out as the final operation of the introduction.

- Gemcitabine + nab-paclitaxel + ABTL0812 (n=8): Orally administered 120 mg/kg ABTL0812 in 200 µl distilled water + 5% glycerol 5 times a week, 60 mg/kg gemcitabine i.p. in 100 µl of sterile water twice a week and 5 mg/kg of nab-paclitaxel i.p. in 230 μl of 0.9% NaCl solution 2 times a week. As in the gemcitabine + nab-paclitaxel group, the injections were administered in different parts of the abdominal cavity. ABTL0812 was always administered prior to chemotherapy to reduce IP-induced stress. injection.

Efficacy of treatment was assessed by measuring tumor volume three days a week. In addition, total body weight was monitored three days a week to study toxicity associated with the treatment method, as well as a visual assessment of signs of clinicopathological symptoms. At the end of the study, mice were sacrificed by carbon dioxide inhalation and cervical dislocation was performed for additional confirmation.

Results: A suboptimal dose of chemotherapeutic agents (according to the literature) was chosen in order to avoid a strong antitumor response that could not be enhanced by ABTL0812, thus reducing the dose of chemotherapeutic agents and therefore reducing adverse events.

14 shows the change in tumor volume in a MiaPaca2 derived xenograft after various chemotherapy regimens, ABTL0812 administration, or a combination of both. Figure 14A shows the change in tumor volume in xenografts after administration of gemcitabine + Nab-Pac compared with administration of gemcitabine + Nab-Pac + ABTL0812, and also with administration of ABTL0812 and vehicle. Compared with the vehicle group, the triple combination of Gem+Nab-Pac+ABTL0812 alone showed a significant reduction in tumor volume starting at day 22 and maintaining these statistically significant differences until the end of the study, where for the final five days of treatment there were showing the largest differences in tumor volume in contrast to the Gem+Nab-Pac group, which did not show significant differences in tumor volume compared to the vehicle group. When comparing Gem+Nab-Pac+ABTL0812 and Gem+Nab-Pac, the triple combination significantly reduced tumor volume on the final day of treatment. It can also be seen that ABTL0812 alone showed a better response in terms of tumor volume change than the Gem+Nab-Pac group during the first 20 days of treatment, with similar tumor volume changes for the remainder of the study, although there were no statistically significant differences for any time point studied.

Figure 14B shows the change in tumor volume in xenografts after administration of gemcitabine compared with administration of gemcitabine + ABTL0812, as well as with administration of ABTL0812 and vehicle. Compared with the vehicle-administered group, the Gem+ABTL0812 dual combination alone showed a significant reduction in tumor volume from day 17 and maintained these statistically significant differences until study completion, in contrast to the gemcitabine-administered group, in which there were no significant differences in tumor volume compared to the vehicle group. When comparing Gem and Gem + ABTL0812, the dual combination significantly reduced tumor volume on the final day of treatment. It can also be seen that ABTL0812 alone showed a better response in terms of tumor volume change than the Gem group throughout the study, although there were no statistically significant differences at any time point studied.

The total body weight of the mice was monitored three times a week throughout the study. Figure 14C shows the change in total body weight for the Gem+Nab-Pac and Gem+Nab-Pac+ABTL0812 groups, as well as the vehicle and ABTL0812 groups, and Figure 14D shows the change in total body weight for the Gem and Gem+ groups. ABTL0812, as well as in groups in which the vehicle and ABTL0812 were administered. None of the groups showed any signs of toxicity in terms of weight loss, all groups experienced weight gain throughout the study, which is indicative of the absence of treatment-related toxicity. No additional clinical pathological signs were observed in any of the groups. See figure 14 for more information.

Conclusion: This study was designed to determine the efficacy of ABTL0812 alone or in combination with SOC chemotherapy in the treatment of advanced pancreatic cancer. Gemcitabine in combination with nab-paclitaxel or gemcitabine administered alone are the treatments of choice for most patients with advanced pancreatic cancer, so the enhancement of both treatments when used in combination with ABTL0812 and the use of suboptimal doses of chemotherapeutic agents, which allowed to reduce unwanted side effects. ABTL0812 enhances both chemotherapy treatments while reducing their toxicity, which is reflected in the highest reduction in tumor volume compared to vehicle and chemotherapy alone. In addition, ABTL0812 administered alone showed similar efficacy compared to chemotherapy, and there were no clinicopathological signs or signs of toxicity as measured by total body weight in all treated groups.

2.9:

Efficacy Study of ABTL0812 in a Neuroblastoma (Cisplatin Receptive) Xenograft Model Alone or in Combination with Cisplatin

Study Link: Journal ABTL0812, pp. 20-36

Place of study: Laboratory for Translational Research on Childhood Cancer at the Research Institute of Val d'Hebron

NLP Compliance: No

Test Compound: ABTL0812 (batch 002/2013)

Standard compound: cisplatin (Sigma, cat. no. C2210000)

Test system: female nu/nu mice, cell line SH-SY5Y

Objective: To determine the efficacy of ABTL0812 against the SH-SY5Y neuroblastoma cell line alone or in combination with cisplatin.

Methods: Immunodeficient NMRI-Foxn1 ^nu /Foxn1 ^nu nude mice were injected subcutaneously with SH-SY5Y cells. This cell line has been genetically modified to express luciferase, which would allow in vivo investigation not only of tumor size, but also of metastasis formation. When the average tumor volume reached 80 mm ³ , mice were randomly assigned to different groups. ABTL0812 was administered orally at a dose of 120 mg/kg per day. Simultaneously, cisplatin, a drug included in the induction phase of chemotherapy in the treatment of neuroblastoma, was used. 2 mg/kg cisplatin was administered i.p. twice a week (Wang et al. 2010). In addition, the effect of combining ABTL0812 with cisplatin at the indicated doses was investigated.

Results: A. Tumor size. In this experiment, ABTL0812 was shown to inhibit tumor progression with similar efficacy to cisplatin. After ten days of treatment, animals in the control group had to be sacrificed due to ethical issues related to the size of the tumors. At this point, half of the animals in the treated groups were sacrificed to measure tumor weight, hematological parameters, and metastasis formation (see below). The choice of animals for killing in these groups was carried out in accordance with the statistical distribution of tumor sizes. Measurement of tumor mass in sacrificed mice supported the observation that the efficacy of ABTL0812 was similar to standard cisplatin treatment. Analysis of tumor volume in the remaining animals in the treated groups (approximately 5 mice per group) showed that the combination of ABTL0812 with cisplatin resulted in long-term stabilization of tumor growth. When tracking body weight, it was shown that treatment with ABTL0812 temporarily causes a slight decrease in body weight (<10%). This effect, however, is compensated after a few days. See Figure 15 herein for more information.

B. Security profile. In a hematological analysis to evaluate the safety of treatments, ABTL0812 showed no effect on blood hematocrit, however, cisplatin caused anemia and reduced the number of white blood cells (see table below). Cisplatin anemia is a common side effect seen in patients treated with this chemotherapy drug (Wood and Hrushesky 1995).

C. Formation of metastases. To study the effect of ABTL0812 on the formation of metastases, the SH-SY5Y cell line transduced with a luciferase reporter vector was used. As described above, mice with tumor xenografts derived from this cell line were treated with ABTL0812, cisplatin, or a combination of both drugs. After ten days of treatment, animals were sacrificed and ex vivo metastases were assessed by monitoring luciferase-expressing cells in the lungs and liver. In these assays, it was shown that ABTL0812, both alone and in combination with cisplatin, inhibited the formation of metastases in these organs. In contrast, cisplatin, when administered alone, did not have a significant effect on the suppression of metastasis formation compared to animals that were injected with the vehicle.

Conclusion: As a single agent, ABTL0812 has comparable efficacy to cisplatin, but has a better safety profile when considering hematological parameters. Interestingly, the combination of ABTL0812 with cisplatin resulted in stabilization of tumor progression over a longer period. In addition, ABTL0812 suppressed spontaneous metastasis formation in a mouse model of neuroblastoma, in contrast to cisplatin. The data obtained further support that ABTL0812 can have improved therapeutic effects compared to existing platinum-based chemotherapy methods.

2.10:

Efficacy of ABTL0812 in combination with doxorubicin against triple-negative human breast cancer xenograft (MDA-MB-231) in mice

Study Link: Pending Review

Research location: Ability laboratory at UAB.

NLP Compliance: No

Test Compound: ABTL0812 (batch 002/2012)

Standard Compound: Doxorubicin (Sigma, cat. no. D1515)

System under study: male nu/nu mice

Objective: To study the antitumor activity of ABTL0812 alone and in combination with doxorubicin against triple-negative human breast cancer xenograft.

Methods: The MDA-DB-231 cell line was grown conventionally in DMEM with 10% FBS and cells in the exponential growth phase were harvested and counted for tumor inoculation. Mice were injected in one side with 2.5×10 ⁶ MDA-DB-231 cells suspended in 50 μl of EBS-free growth medium and 50 μl of Matrigel (Corning cat. no. 354234). Tumor volume was monitored 3 days per week, and when tumors reached 100 mm ³ (50 to 150 mm ³ ), animals were divided into four equal groups having a similar average tumor volume per group, excluding tumors smaller than 50 mm ³ or larger than 150 mm ³ to minimize the error.

The following treatment groups were used:

- Vehicle group (n=7): 200 µl water + 5% glycerol orally four days a week and two ip injections of 100 µl saline once a week

- ABTL0812 (n=9): administered orally 200 μl 120 mg/kg ABTL0812 resuspended in water + 5% glycerol 5 times a week

- Doxorubicin (n=9): 100 µl of 2 mg/kg doxorubicin i.p. once a week

- ABTL0812 + doxorubicin (n=9): 200 µl 120 mg/kg ABTL0812 orally four days a week and 100 µl 2 mg/kg doxorubicin i.p. once a week

Results: ABTL0812, when administered in combination with doxorubicin, showed the best antitumor effect in vivo in xenografts derived from MDA-DB-231 cells. Administration of ABTL0812 alone showed similar tumor volume reduction to doxorubicin alone, but the double combination of ABTL0812 + doxorubicin showed the largest reduction in tumor volume with significant differences at the end of the study. All treatment groups showed a similar change in total body weight, which is indicative of the absence of treatment-related toxicity. See Figure 16 herein for more information.

Conclusions: As described above, ABTL0812 reduces tumor growth in breast cancer xenograft models derived from MDA-DB-231 cells. In this model, ABTL0812 enhances the antitumor activity of doxorubicin. Doxorubicin therapy is a traditional treatment option for patients with breast cancer, thus these results suggest that combination therapy of ABTL0812 with doxorubicin may be of clinical interest in the treatment of patients with breast cancer.

2.11:

2.4: Efficacy of ABTL0812 in combination with gemcitabine and cisplatin against human cholangiocarcinoma xenograft (EGI-1) in mice

Study link: ABT-EI

Place of study: Group for the Study of Oven Diseases at the Biodonostia Health Research Institute

NLP Compliance: No

Test Compound: ABTL0812 (batch 002/2012)

Reference compounds: gemcitabine hydrochloride (Sigma Aldrich G6423) and cisplatin (Sigma, cat. no. C2210000)

System under study: male nu/nu mice

Objective: To study the antitumor activity of ABTL0812 alone and in combination with gemcitabine and cisplatin against human cholangiocarcinoma xenograft. The combination of gemcitabine and cisplatin is one of the standard therapies for the treatment of cholangiocarcinoma.

Methods: The EGI-1 cell line was grown conventionally in DMEM with 10% EBS and exponential growth phase cells were harvested and counted for tumor inoculation. Mice were injected in one side with 1×10 ⁶ H1957 cells suspended in 50 μl of EBS-free growth medium and 50 μl of Matrigel (Corning cat. no. 354234). Tumor volume was monitored 3 days per week, and when tumors reached 100 mm ³ (50 to 150 mm ³ ), animals were divided into four equal groups having a similar average tumor volume per group, excluding tumors smaller than 50 mm ³ or larger than 150 mm ³ to minimize the error.

The following treatment groups were used:

- Vehicle treatment group (n=8): 200 μl water + 5% glycerol administered orally four days a week and two IPs. injections of 100 µl of saline solution once a week

- ABTL0812 (n=8): administered orally 200 µl 120 mg/kg ABTL0812 resuspended in water + 5% glycerol 5 times a week

- Gemcitabine/cisplatin (n=8): administered i.p. 100 µl 50 mg/kg gemcitabine i.p. 100 µl 2 mg/kg cisplatin once a week

- ABTL0812 + gemcitabine/cisplatin (n=8): 200 µl 120 mg/kg ABTL0812 orally four days a week, i.p. 100 µl 50 mg/kg gemcitabine i.p. 100 µl 2 mg/kg cisplatin once a week

Results: ABTL0812, when administered in combination with gemcitabine and cisplatin, showed the best in vivo antitumor effect in xenografts derived from EGI-1 cells. The administration of gemcitabine and cisplatin reduced tumor volume compared to the vehicle group, although without any statistically significant difference, in contrast to the administration of ABTL0812 + gemcitabine and cisplatin, which showed a statistically significant reduction in tumor volume compared to the vehicle, however, administration of ABTL0812 alone showed no reduction in tumor volume compared to vehicle until the last day of treatment, when tumor volume was similar to the chemotherapy group. See figure 17 for more information.

Conclusions: As described above, ABTL0812 reduces tumor growth in EGI-1 cell-derived cholangiocarcinoma xenograft models. In this model, ABTL0812 enhances antitumor activity after administration of gemcitabine and cisplatin. Therapy with gemcitabine and cisplatin is a traditional option for the treatment of patients with cholangiocarcinoma, thus these results suggest that combination therapy of ABTL0812 with gemcitabine and cisplatin may be of clinical interest in the treatment of patients with cholangiocarcinoma.

EXAMPLE 3 Toxicity of Combination with Chemotherapy

Information on the toxicity of ABTL0812 in combination with chemotherapeutic agents was obtained during efficacy studies in immunosuppressed mice. Conducted a special toxicological study of the combination ABTL0812 +/- paclitaxel +/- carboplatin.

Study Link: N-02220

Place of study: Vivotecnia (Madrid, Spain)

NLP Research: No

Test Compounds: ABTL0812 (Lot 001R/2014), Paclitaxel (Aurovitas, Lot 68J5041), Carboplatin (Sigma-Aldrich, Lot LSBL7058v)

Test System: 12 week old female CD-1 mice.

Objective: Determination of the toxicological profile of ABTL0812 in combination with carboplatin and paclitaxel after two weeks of dosing.

Methods: Forty-five female CD-1 mice were distributed by body weight stratification into nine experimental groups (A-I) (5 animals per group) in which different treatments were used or standard agents (paclitaxel and carboplatin) were administered on different days. .

The table below summarizes the treatment groups. It should be noted that the administration regimens used and the doses administered were the same as in previous efficacy studies.

The safety assessment was based on observed mortality, local and systemic clinical signs, body weight and food intake obtained throughout the study. In addition, indicators of clinical pathology (biochemistry and hematology) were determined in all animals before sacrifice. At the end of the observation period (one day after the final injection), all surviving animals were sacrificed and macroscopically evaluated after autopsy. In addition, the safety assessment was also based on the mass of individual target organs collected at the time of sacrifice.

Results: Repeated oral administration of study agent and intraperitoneal administration of standard agents did not cause mortality. No local or systemic clinical signs associated with treatment were noted.

During the study, the animals observed insignificant differences in the average increase in body weight. Most animals showed a tendency to decrease in body weight in the first four days of the study, with the exception of animals from group A (control group) and group C (administration of a dose of paclitaxel once a week). However, during the entire study period, most animals did not show statistically significant differences in absolute body weight gain. Only in animals from group F, which were administered an oral dose of ABTL0812 once a day for two 5-day periods and an intraperitoneal dose of paclitaxel and carboplatin once a week (on the same day as the first oral dose in each period), markedly there was a decrease in body weight gain over the entire study period compared to several groups of animals (group A, in which the vehicle was administered, group C, in which paclitaxel was administered, and group E, in which the combination of paclitaxel and carboplatin was administered).

Although statistical analysis was not possible due to the small sample size, animals in Group A were estimated to consume more food than the rest of the animals.

In terms of clinical biochemistry parameters, lower creatinine and triglyceride levels were noted in most groups compared to the control group. On the other hand, there was a statistically significant decrease in hematocrit (group F animals) and platelet levels in the groups treated with the combination of oral dose of ABTL0812 and intraperitoneal dose of paclitaxel and carboplatin (animals in groups F and G) compared with the control group. Although these values were within the normal range, an effect of treatment on clinical pathology parameters cannot be ruled out.

During the macroscopic evaluation at autopsy of all animals euthanized at the end of the treatment period, no significant changes were detected that could be associated with the action of the studied agents. White patches were observed in the liver of the ID32 animal and pigmentation in the pancreas of the ID33 animal. In addition, the absolute and relative organ masses were similar in the treated groups.

The table below summarizes the most relevant safety-related data.

Conclusion: Taking into account the obtained results, we can conclude that under the experimental conditions under study:

- Repeated oral administration of study agent ABTL0812 alone and in combination with a weekly intraperitoneal dose of paclitaxel and carboplatin did not cause mortality and was well tolerated, as neither local nor systemic clinical signs indicative of toxicity were observed in any of the animals during the study.

- In most animals, body weight remained unchanged during the study period. A statistically significant decrease was observed only in animals treated with the combination of study agent and both standard agents (on the first day of each period).

- Feed intake in animals from group A was higher than in almost all animals during the study period.

- Clinical pathology (biochemical and hematological parameters) revealed statistically significant differences in the treated groups compared to the control group. Despite the fact that most of the obtained values were within the normal range in animals of this line and sex, the effect of treatment cannot be excluded. The effect on platelet count is associated with the use of carboplatin, but the additional small decrease in platelet count when the three experimental drugs are combined cannot be neglected.

- No significant effects of treatments according to the results of macroscopic evaluation at the time of autopsy and absolute/relative organ mass were observed.

EXAMPLE 4: ABTL0812 in combination with various chemotherapeutic agents - human data

Figure 4.1: An open-label phase I/II study to evaluate the efficacy and safety of ABTL0812 in combination with paclitaxel and carboplatin in patients with advanced endometrial cancer or squamous cell NSCLC.

A phase I/II clinical trial is being conducted for patients with advanced endometrial cancer or squamous non-small cell lung cancer. It is a multicenter open trial in which ABTL0812 is administered orally starting at a dose of 1300 mg three times a day in combination with chemotherapy.

A. Test objectives

• Phase I Primary Endpoint: Evaluation of the safety and tolerability of ABTL0812 and paclitaxel + carboplatin in patients with advanced or metastatic endometrial cancer or squamous cell NSCLC as first-line therapy

• Phase II Primary Endpoint: Evaluation of ABTL0812 and paclitaxel + carboplatin in patients with advanced or metastatic endometrial cancer or squamous cell NSCLC as first-line therapy

B. Study Design

This study is not randomized and all enrolled patients are treated with ABTL0812 in combination with paclitaxel + carboplatin. This phase is divided into 2 periods:

• Period 1: ABTL0812 is administered in combination with chemotherapy.

• Period 2: After the completion of the administration cycles, SOC ABTL0812 is administered as monotherapy, up to 12 months from the start of period 1.

Conclusion:

Previous preliminary results from human clinical trials are positive in that these results also indicate that there is also a synergistic effect in humans from the use of ABTL0812 in combination with paclitaxel and carboplatin in patients with advanced endometrial cancer or squamous cell carcinoma.

LINKS

1: European patent EP 2409963 B1 (Lipopharma - application filed in 2010)

2: Erazo, et al.; Clinical Cancer Research; 22(10) May 15, 2016

3: News note dated November 22, 2016 - published on the web page of the present applicant (AbilityPharma)

4: News note dated December 14, 2016 - published on the web page of the present applicant (AbilityPharma).

Claims (43)

1. A pharmaceutical combination for the treatment of cancer in a human patient, comprising: (A): a compound which is a polyunsaturated fatty acid of the formula COOH-CHOH-(CH ₂ ) ₆ -(CH=CH-CH ₂ ) ₂ -(CH ₂ ) ₃ -CH ₃ (ABTL0812), a pharmaceutically acceptable salt thereof, or a combination thereof And (B): a compound that is a chemotherapeutic agent, where Compound (B) is paclitaxel and the cancer is lung cancer, preferably non-small cell lung adenocarcinoma; the compound (B) is carboplatin and the cancer is endometrial cancer; the compound (B) is retinoic acid and the cancer is neuroblastoma; Compound (B) is paclitaxel and the cancer is breast cancer, preferably triple negative breast cancer; the compound (B) is paclitaxel and carboplatin and the cancer is lung cancer; Compound (B) is paclitaxel and carboplatin and the cancer is squamous cell carcinoma, preferably non-small cell squamous cell lung cancer; Compound (B) is paclitaxel and carboplatin and the cancer is non-small cell adenocarcinoma of the lung; - Compound (B) is cisplatin and pemetrexed, the cancer is lung cancer; Compound (B) is cisplatin and pemetrexed and the cancer is non-small cell adenocarcinoma of the lung; - Compound (B) is paclitaxel and the cancer is endometrial cancer; the compound (B) is paclitaxel and carboplatin and the cancer is endometrial cancer; Compound (B) is paclitaxel and gemcitabine and the cancer is pancreatic cancer; Compound (B) is nab-paclitaxel and gemcitabine and the cancer is pancreatic cancer; Compound (B) is doxorubicin and the cancer is breast cancer, preferably triple negative breast cancer; - compound (B) is cisplatin and the cancer is neuroblastoma; or Compound (B) is cisplatin and gemcitabine, and the cancer is cholangiocarcinoma. 2. The pharmaceutical combination according to claim 1, characterized in that the compound (A) is the sodium salt of COOH-CHOH-(CH ₂ ) ₆ -(CH=CH-CH ₂ ) ₂ -(CH ₂ ) ₃ -CH ₃ ( ABTL0812). 3. Pharmaceutical combination according to any one of the preceding claims, characterized in that the compound (B) is carboplatin and the cancer is endometrial cancer; Compound (B) is paclitaxel and the cancer is breast cancer, preferably triple negative breast cancer; Compound (B) is paclitaxel and carboplatin and the cancer is squamous cell carcinoma, preferably non-small cell squamous cell lung cancer; Compound (B) is paclitaxel and carboplatin and the cancer is non-small cell adenocarcinoma of the lung; Compound (B) is pemetrexed and cisplatin and the cancer is non-small cell adenocarcinoma of the lung; the compound (B) is paclitaxel and carboplatin and the cancer is endometrial cancer; Compound (B) is paclitaxel and gemcitabine and the cancer is pancreatic cancer; or Compound (B) is cisplatin and gemcitabine, and the cancer is cholangiocarcinoma. 4. Pharmaceutical combination according to any one of the preceding claims, characterized in that Compound (B) is paclitaxel and carboplatin and the cancer is advanced endometrial cancer; or Compound (B) is paclitaxel and carboplatin, and the cancer is squamous cell carcinoma. 5. Pharmaceutical combination according to claim 4, characterized in that said compound (A) is administered orally. 6. Pharmaceutical combination according to claim 5, characterized in that the administered dose of the compound (A) is a dose of 1200 mg to 1400 mg. 7. Pharmaceutical combination according to any one of the preceding claims, characterized in that said pharmaceutical combination is a single composition containing both compound (A) and compound (B). 8. Pharmaceutical combination according to any one of the preceding claims, characterized in that said compound (A) is intended to be administered orally. 9. Pharmaceutical combination according to any one of the preceding claims, characterized in that the administered dose of the compound (A) is a total dose of 200 mg to 6000 mg per day. 10. Pharmaceutical combination according to any one of the preceding claims, characterized in that Compound (B) is paclitaxel and is intended for intravenous administration as an infusion solution; Compound (B) is carboplatin and is intended for intravenous administration as an infusion solution; Compound (B) is cisplatin and is intended for intravenous administration as an infusion solution.