WO2008033039A1

WO2008033039A1 - Cancer treatment

Info

Publication number: WO2008033039A1
Application number: PCT/NZ2007/000266
Authority: WO
Inventors: Adam Vorn Patterson; William Robert Wilson
Original assignee: Adam Vorn Patterson; William Robert Wilson
Priority date: 2006-09-11
Filing date: 2007-09-11
Publication date: 2008-03-20

Abstract

The invention relates to a combination therapy for cancer treatment. In particular, the invention relates to a method for the treatment of cancer in a warm-blooded animal such as a human, which comprises administering to the animal an effective amount of a compound of Formula (I) wherein: X represents at any available ring position -CONH-, -SO2NH-, -O-, -CH2-, -NHCO- or -NHSO2-; R represents a lower C1-6 alkylene optionally substituted with one or more groups including hydroxyl, amino and N-oxides therefrom or dialkylamino and N-oxides therefrom; Y represents at any available ring position -N-aziridinyl, -N(CH2CH2W)2 or -N(CH2CHMeW)2, where each W is independently selected from halogen or -OSO2Me; Z represents at any available ring position -NO2, -halogen, -CN, -CF3 or -SO2Me; or a pharmaceutically acceptable salt or derivative thereof, before, after or simultaneously with an effective amount of Gemcitabine.

Description

CANCER TREATMENT

FIELD OF THE INVENTION

This invention is directed to methods for treating cancer and to compositions for use therein.

BACKGROUND OF THE INVENTION

Cancer is a significant cause of death, particularly in industrialised nations. While there are a number of anti-cancer therapies now available, there remains a need for new approaches to treating cancer which offer better outcomes for patients. It is towards one such approach that the present invention is directed.

SUMMARY OF THE INVENTION

The present invention is broadly based upon the unexpected and surprising finding that compounds of Formula (I) and their salts as defined in WO 2005/042471 used in combination with chemotherapeutic agent Gemcitabine (Gemzar®) produces significantly better effects than either agent alone.

Therefore, according to a first aspect of the present invention there is provided a method for the production of an anti-cancer effect in a warm-blooded animal such as a human, which comprises administering to said animal an effective amount of a compound of Formula (T)

wherein:

X represents at any available ring position -CONH-, -SO₂NH-, -O-, -CH_2., -NHCO- or -NHSO₂-;

R represents a lower Cl-6 alkylene optionally substituted with one or more groups including hydroxy, amino and N-oxides therefrom or dialkylamino and N-oxides therefrom;

Y represents at any available ring position — N-aziridinyl, -N(CH₂CH₂W)₂ or —

N(CH₂CHMeW)₂, where each W is independently selected from halogen or -OSO₂Me;

Z represents at any available ring position -NO₂, -halogen, -CN, -CF₃ or -SO₂Me; or a pharmaceutically acceptable salt or derivative thereof, before, after or simultaneously with an effective amount of Gemcitabine.

Anti-cancer effects include, but are not limited to, anti-tumor effects, the response rate, the time to disease progression and the survival rate. Anti-tumor effects include but are not limited to, inhibition of tumor growth, tumor growth delay, regression of tumor, shrinkage of tumor, increased time to regrowth of tumor on cessation of treatment and slowing of disease progression.

An "effective amount" includes amounts of die compound which provide an anti-cancer effect on their own as well as amounts of the compound which, while being less than a therapeutic dose for the compound as a monotherapy, do provide an anti-cancer effect when the second compound is administered in combination.

According to a further aspect of the present invention there is provided a method for the treatment of a cancer in a warm-blooded animal such as a human, which comprises administering to said animal an effective amount of a compound of Formula (I) as defined above or a pharmaceutically acceptable salt or derivative thereof, before, after or simultaneously with an effective amount of Gemcitabine.

Preferably, in each such method, the compound of Formula (T) or salt or derivative thereof and Gemcitabine may each be administered together with a pharmaceutically acceptable excipient or carrier.

According to a further aspect of the present invention there is provided a therapeutic combination treatment comprising the administration of an effective amount of a compound of Formula (I) as defined above or a pharmaceutically acceptable salt or derivative thereof, optionally together with a pharmaceutically acceptable excipient or carrier, and the simultaneous, sequential or separate administration of an effective amount of Gemcitabine, optionally together with a pharmaceutically acceptable excipient or carrier, to a warm-blooded animal such as a human in need of such therapeutic treatment.

Such therapeutic treatment includes an anti-cancer effect and an anti-tumor effect.

A combination treatment of the present invention as defined herein may be achieved by way of the simultaneous, sequential or separate administration of the individual components of said treatment. A combination treatment as defined herein may be applied as a sole therapy or may involve surgery or radiotherapy or an additional chemotherapeutic agent in addition to a combination treatment of the invention.

Surgery may comprise the step of partial or complete tumor resection, prior to, during or after the administration of the combination treatment described herein.

The effect of a combination treatment of the present invention is expected to be a synergistic effect. According to the present invention a combination treatment is defined as affording a synergistic effect if the effect is therapeutically superior, as measured by, for example, the extent of the response, the response rate, the time to disease progression or the survival period, to that achievable on dosing one or other of the components of the combination treatment at its conventional dose. For example, the effect of the combination treatment is synergistic if die effect is therapeutically superior to the effect achievable with a compound of Formula (I) or Gemcitabine alone. Further, the effect of the combination treatment is synergistic if a beneficial effect is obtained in a group of patients that does not respond (or responds poorly) to a compound of Formula (I) or Gemcitabine alone. In addition, die effect of the combination treatment is defined as affording a synergistic effect if one of the components is dosed at its conventional dose and the other component(s) is/are dosed at a reduced dose and the therapeutic effect, as measured by, for example, the extent of the response, the response rate, the time to disease progression or the survival period, is equivalent to that achievable on dosing conventional amounts of the components of the combination treatment. In particular, synergy is deemed to be present if the conventional dose of compound of Formula (I) or Gemcitabine may be reduced without detriment to one or more of the extent of the response, the response rate, the time to disease progression and survival data, in particular without detriment to the duration of the response, but with fewer and/or less troublesome side effects than those that occur when conventional doses of each component are used.

Combination treatments of the present invention may be used to treat cancer, particularly a cancer involving a solid tumor. In particular such combination treatments of the invention are expected to slow advantageously the growth of primary and recurrent solid tumors of, for example, the ovary, colon, stomach, brain, thyroid, adrenal, pituitary, pancreas, bladder, breast, prostate, lungs, kidney, liver, head and neck (including esophageal), cervix, endometrium, vulva, skin and connective tissues or bone. More especially combination treatments of the present invention are expected to slow advantageously the growth of tumors in colorectal cancer and in lung cancer, for example mesothelioma and non-small cell lung cancer (NSCLC). More particularly such combination treatments of the invention are expected to inhibit any form of cancer associated with VEGF including leukaemia, multiple myeloma and lymphoma and also, for example, to inhibit the growth of those primary and recurrent solid tumors which are associated with VEGF, especially those tumors which are significantly dependent on VEGF for their growth and spread, including for example, certain tumors of the kidney, ovary, colon (including rectum), brain, thyroid, pancreas, bladder, breast, prostate, lung, vulva, skin and particularly NSCLC.

The therapeutic combination of the invention may be administered in the form of a combination product or a pharmaceutical composition. Therefore, according to one further aspect of the present invention there is provided a combination product comprising a compound of Formula (I) as defined above or a pharmaceutically acceptable salt or derivative thereof, and Gemcitabine.

"Pharmaceutically acceptable" is to be understood as meaning that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary as well as human pharmaceutical use.

Pharmaceutically acceptable derivatives of the compounds of formula (I) are to be understood as including amides and esters, that are pharmaceutically acceptable, as defined herein. Esters include carboxylic acid esters in which the non-carbonyl moiety of the ester grouping is selected from straight or branched chain C,_₆ alkyl, (methyl, n-propyl, n-butyl or t-butyl); or C_3.6 cyclic alkyl (e.g. cyclohexyl), or a chain of from one to three D- or L- aminoacids. Amides include non-substituted and mono- and di-substituted derivatives. Such derivatives may be prepared by techniques known per se in the art of pharmacy.

"Pharmaceutically acceptable salts" of a compound means salts that are pharmaceutically acceptable, as defined herein, and that possess the desired pharmacological activity of the parent compound. Such salts include: acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or formed with organic acids such as acetic acid, methanesulfonic acid, maleic acid, tartaric acid, citric acid and the like; or salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g. an alkali metal ion, an alkaline earth ion, or an aluminium ion; or coordinates with an organic or inorganic base. Acceptable organic bases include ethanolamine, diethanolamine, N-mediylglucamine, triethanolamine and the like. Acceptable inorganic bases include aluminium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and sodium hydroxide.

According to a further aspect of the invention there is provided a pharmaceutical composition which comprises a compound of Formula (I) as defined above or a pharmaceutically acceptable salt or derivative thereof, and Gemcitabine, in association with a pharmaceutically acceptable excipient or carrier.

Kits may also be provided. According to a further aspect of the present invention there is provided a kit comprising a compound of Formula (I) as defined above or a pharmaceutically acceptable salt or derivative thereof, and Gemcitabine.

According to a further aspect of the present invention there is provided a kit comprising: a) a compound of Formula (I) as defined above or a pharmaceutically acceptable salt or derivative thereof in a first unit dosage form; b) Gemcitabine in a second unit dosage form; and c) ^' container means for containing said first and second dosage forms.

In a further aspect of the invention, there is provided the use of a compound of Formula (I) or a pharmaceutically acceptable salt or derivative thereof and Gemcitabine in the preparation of a medicament for producing an anti-cancer effect in a warm-blooded animal such as a human.

In still a further aspect of the invention, there is provided the use of a compound of Formula (I) or a pharmaceutically acceptable salt or derivative thereof and Gemcitabine in the preparation of a medicament for the treatment of cancer in a warm-blooded animal such as a human.

Although the invention is broadly as defined above, it also includes embodiments of which the following description provides examples.

DESCRIPTION OF THE DRAWINGS

The invention will be better understood through reference to the accompanying drawings, in which

Figures 1-5 graphically represent the effect of various treatment regimes (single agent and combinations) in the Panc-1 human pancreatic tumor xenograft model using Gemcitabine and the preferred compound of Formula (I), PR-104, and

Figure 6 is a Kaplan-Meier plot of A2780 xenograft bearing CD-I nude mice treated with

PR-104, Gemcitabine or a combination of PR-104 and Gemcitabine.

DETAILED DESCRIPTION OF THE INVENTION

This invention is primarily based upon the surprising finding of synergism between anticancer agents. One agent is the chemo therapeutic agent Gemcitabine (Gemzar®; chemical name 4-amino-l -[3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-l H- pyrimidin-2-one) which is commercially available from Eli Lilly. The second agent is a compound of Formula (I) as defined and described in PCT/NZ2004/000275 (published as WO 2005/042471), with the compound 2-[2-bromoethyl)-2,4-dinitro-6-[[[2- phosphonooxy)ethyl]amino]-carbonyl]anilino]ethyl methane sulfonate (known as PR-104) being representative.

The agents are administered in combination. It is to be understood that "combination" encompasses the simultaneous or sequential administration of the agents, with "sequential" meaning either agent can be administered before or after the other provided only that the delay in administering die second agent should not be such as to lose the benefit of die combination dierapy.

The agents may also be in any appropriate form for administration. Commonly, die agents will be formulated for parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion) for example as a sterile solution, suspension or emulsion. However, odier formulations are in no way excluded.

In general die compositions described herein may be prepared in a conventional manner using conventional excipients and/ or carriers, including liposomal or albumin carriers.

Where intended for parenteral injection for example, the component agents can be formulated in accordance widi manufacturer's instructions or as described below in the experimental section.

The dosages and schedules of administration of die component agents may be varied according to the particular disease state and overall condition of the patient Administration may be at single-agent dosages (up to 100 mg/m² for Gemcitabine) employed in current clinical practice for eidier agent or for both. More commonly, however, die dose of one or botii agents will be reduced below single-agent clinical practice, bodi to reflect the dierapeutic benefit of die combination and to minimise die potential for toxicity. Any and all such dose combinations can be employed subject to the component agents being present in amounts which combine to produce an anti-cancer effect.

The final dose, and dose scheduling, will be determined by the practitioner treating the particular patient using professional skill and knowledge.

A combination treatment of die present invention is most desirably a sole therapy but is not limited to that - it may in addition involve surgery or radiotherapy or the administration of a chemodierapeutic agent.

Surgery may comprise the step of partial or complete tumor resection, prior to, during or after the administration of die combination treatment of the present invention. Chemotherapeutic agents for optional use with the combination treatment of the present invention may include, for example, the following categories of therapeutic agent:

(i) antiproliferative/antineoplastic drugs and combinations thereof as used in medical oncology (for example carboplatin and cisplatin);

(ϋ) cytostatic agents, for example inhibitors of growth factor function such as growth factor antibodies, growth factor receptor antibodies (for example the anti-erbB2 antibody trastuzumab and the anti-erbBl antibody cetuximab), Class I receptor tyrosine kinase inhibitors (for example inhibitors of the epidermal growth factor family), Class II receptor tyrosine kinase inhibitors (for example inhibitors of the insulin growth factor family such as IGFl receptor inhibitors as described, for example, by Chakravarti et al., Cancer Research, 2002, 62: 200-207), serine/threonine kinase inhibitors, farnesyl transferase inhibitors and platelet- derived growth factor inhibitors;

(iii) antiangiogenic agents such as those which inhibit the effects of vascular endothelial growth factor (for example the anti-vascular endothelial cell growth factor antibody bevacizumab and VEGF receptor tyrosine kinase inhibitors such as 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(l -methylpiperidin-4- ylmethoxy)quinazoline (ZD6474; Example 2 within WO 01/32651), 4-(4-fluoro- 2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-l-ylpropoxy)quinazoune (AZD2171; within WO 00/47212), vatalanib (PTK787; WO 98/35985) and SUl 1248 (WO 01/60814));

(iv) vascular damaging agents such as the compounds disclosed in International Patent Applications WO -99/02166, WO 00/40529, WO 00/41669, WO 01/92224, WO 02/04434 and WO 02/08213;

(v) biological response modifiers (for example interferon); and

(vi) a bisphosphonate such as tiludronic acid, ibandronic acid, incadronic acid, risedronic acid, zoledronic acid, clodronic acid, neridronic acid, pamidronic acid and alendronic acid.

Radiotherapy may be administered according to the known practices in clinical radiotherapy. The dosages of ionising radiation will be those known for use in clinical radiotherapy. The radiation therapy used will include for example the use of γ-rays, X-rays, and/or the directed delivery of radiation from radioisotopes. Other forms of DNA damaging factors are also included in the present invention such as microwaves and UV- irradiation. For example X-rays may be dosed in daily doses of 1.8-2.0Gy, 5 days a week for 5-6 weeks.

Normally a total fractionated dose will lie in the range 45-60Gy. Single larger doses, for example 5-1 OGy may be administered as part of a course of radiotherapy. Single doses may be administered intraoperatively. Hyperfractionated radiotherapy may be used whereby small doses of X-rays are administered regularly over a period of time, for example O.lGy per hour over a number of days. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and on the uptake by cells.

The invention will now be illustrated widi reference to the synergistic interaction between Gemcitabine and PR-104 in the experimental section which follows.

EXPERIMENTAL

PART l

MATERIALS AND METHODS Reagents and Compounds

PR- 104 (Lot#MPD-A- 124(1)) was received from Proacta via Albany Molecular Research, Inc. and stored at -2O⁰C until use. PR- 104 was solubilized in a PBS solution containing one equivalent sodium bicarbonate at appropriate concentrations to achieve proper doses. PR-104 was prepared fresh and sterile filtered prior to injections. Gemcitabine (Lot#8SE30M, Eli Lilly) was received from Eli Lilly, reconstituted according to manufacturer's guidelines, and diluted in saline to working concentrations. Sodium bicarbonate (Lot#045K0187) was received from Sigma (St Louis, MO), and PBS (Lot# 1289515) was obtained from Gibco (Carlsbad, CA).

Cell Culture

The Panc-1 tumor cell line was received from American Type Tissue Collection (ATCC, Manassas, Va). Cultures were maintained in DMEM supplemented with 10% fetal bovine serum, and 5% CO₂ atmosphere. The cultures were expanded in T225 tissue culture flasks until the appropriate number of cells could be harvested for inoculation.

Animals

ICR SCID mice, IciΥac:ICR-Prkde^sad' were supplied by Taconic (Hudson, NY). Mice were received at five weeks of age and were acclimated seven days prior to handling. Animals are housed in an ammonia-free environment in individually isolated cages. All procedures were carried out under the institutional guidelines of the TGen Drug Development Services Institutional Animal Care and Use Committee.

Single Agent Maximum Tolerated Dose Determination

Five week old female ICR SCID mice were received from Taconic (Hudson, NY). The mice were placed in individually isolated cages and allowed to acclimate for seven days. Following acclimation, mice were randomized into eight groups of five mice each. On Day 0, mice were weighed and weights recorded. On Day 1, mice were administered PR-104 or Gemcitabine via intraperitoneal (IP) route on Days 1, 5 and 9 (Table 1). Mice were observed daily and weighed twice weekly following initial injections. Combination Dose Toleration Study

Five week old female ICR SCID mice were received From Taconic. The mice were placed in individually isolated cages and allowed to acclimate for seven days. Following acclimation, mice were randomized to five groups of five mice each. On Day 1, mice were weighed prior to dosing. Mice received PR-104 and Gemcitabine intraperitoneally (described in Table 2). The Gemcitabine dose was administered within fifteen minutes of the PR-104 dose. Mice were weighed twice weekly and observed daily for overt signs of toxicity.

Panc-1 Human Pancreatic Tumor Xenograft Model

Five to six week old female ICR SCID mice were received from Taconic. The mice were allowed to acclimate for seven days prior to tumor cell inoculation. Each mouse was inoculated subcutaneously with 0.1ml of a 50% RPMI/ 50% Matrigel™ mixture containing a suspension of tumor cells (10 x 10⁶) cells/mouse).

Thirty-five days following inoculation, tumors were measured and tumor weight calculated using the formula: Tumor weight = (a x b²/2) where 'b' is the smallest diameter and 'a' is the largest diameter. Once the established tumors reached 165mg, the mice were pair- matched into the various treatment groups (Day 1). Body weights were recorded when the mice were pair-matched. In addition, body weight measurements were taken twice weekly thereafter in conjunction with tumor measurements. On Day 1, PR-104 (300 and 500 mg/kg) and Gemcitabine (100 mg/kg) were administered intraperitoneally. In the combination regimen groups, PR-104 was administered within 15 minutes prior to the Gemcitabine dose. Beginning on Day 1, mouse body weights and tumor measurements were monitored twice weekly. When the individual tumor of each mouse reached an approximate end-point of 1500mg, the mouse was sacrificed by asphyxiation with regulated CO₂.

Statistical Analysis

All statistical analysis in the xenograft study was performed with GraphPad Prism® v4 software. Survival fractions were calculated using the Kaplan-Meier method. Survival curves were compared using the log rank test and median survival was calculated and reported. RESULTS Single Agent Maximum Tolerated Dose Determination (Figure 1; Table 1)

On Day 1, PR- 104 was prepared as described at concentrations required to deliver an IP dose of 400, 500, 600, and 700 mg/kg. PR-104 was well tolerated at all dose levels. PR- 104 700 mg/kg exhibited maximum body weight loss of 5.9% recorded on Day 12. Positive weight gain was noted by Day 16. No clinical signs of overt toxicity were observed at any of the dose levels. Gemcitabine was tested intraperitoneally at 80, 100, 120, and 160 mg/kg. All doses were tolerated with no weight loss recorded at any dose levels. No clinical signs of toxicity were observed.

Table 1. PR- 104 and Gemcitabine Maximum Tolerated Dose Determination

PR-104 400 IP q4dx3 77 56 48 11 1 0 tPR-fOO *^■ >/500 /4 -% q4dx3f 5-ϊ 401 ' 4 1», ^v 11 1 o^" %%>»

PR-104 600 IP q4dx3 1 0 06 -1 0 63 0 dPR-104<"' 700 ,IP q4dx3 -20 Us -59 1 2 0 '

Gemcitabine 80 IP q4dx3 5 9 67 6 4 120 0

J_jGemαtabme f 5, V¹⁰P IP q4dx3_s \\ _f40 ^* 4^*2^*, t 8 3 ^* 0

Gemcitabine 5 120 IP q4dx3 29 105 133 17 6 0

"*?^■&'*< Is 4 ^"^* Gemcitabine" _# 5 1.60,, ' IP , q4dx3 ^S S 6 6 3 * ■ 11 9 t. ^"l9-2 I '0

Combination Dose Toleration Study (Figure 2; Table 2)

Doses of 400^'and 600 mg/kg of PR-104 were chosen to be administered in combination with Gemcitabine at doses of 100 and 160 mg/kg. The PR-104 400 mg/kg and Gemcitabine 100 mg/kg combination was well-tolerated with a maximum weight loss of 4.9% recorded at Day 8. Weight was regained by Day 19 with animals showing no obvious signs of toxicity. PR-104 given at 400 mg/kg in conjunction with a Gemcitabine dose of 160 mg/kg produced a sustained weight loss at or above 7.0% with maintained weight gain of 3.8% by Day 19. Though moderate weight loss was observed, animals preserved normal activity with no other clinical signs of toxicity. The combination regimen of PR-104 600 mg/kg with Gemcitabine 100 mg/kg displayed weight loss of 9.3% four days following initial injections and mice continued to lose substantial weight over the dosing period with a maximum weight loss of 17.2% noted on Day 8. One mouse showed additional weight loss as compared to other mice in the cohort and was found dead on Day 10. Animals still maintained normal motility and activity. Evidence of emaciation was noted but not confirmed by gross necropsy. Weight gain was sustained by Day 19. The PR-104 600 mg/kg / Gemcitabine 160 mg/kg combination displayed the most weight loss as compared to the other cohorts with a 19.0% weight loss at Day 12. All mice survived treatment with no effects on normal activity; however emaciation was evident but not confirmed by gross necropsy. Other overt signs of toxicity were not observed and a positive weight gain trend was noted.

Table 2. PR- 104 and Gemcitabine Combination Dose Toleration

Panc-l Human Pancreatic Tumor Xenograft Model (Figures 3, 4, 5; Tables 3, 4)

The Panc-l human pancreatic tumor xenograft is a moderate growing tumor; the vehicle control group produced a median survival of 30 days to the 1500mg end-point. The tumor burden added no observable overall effect to the animals' health.

The antitumor activity of PR-104 300 mg/kg as a single agent produced a tumor growth inhibition (TGI) of 51.7% at Day 27 and an overall median survival of 41 days (Min: 34 days, Max: 48 days). The length of survival is significantly different than vehicle control (P<0.001). A maximum weight loss of approximately 1.0% was observed at Day 5 and was promptly recovered in subsequent days. No signs of clinical toxicity were observed.

Single agent PR-104 500 mg/kg generated a TGI of 82.6% (Day 27) and a median survival of 51 days (Min: 44 days, Max: 65 days). The difference in the length of survival as compared to vehicle control is statistically significant (P<0.0001). The maximum weight loss of 3.0% was observed at Day 12 with recovering recorded by Day 15. All animals maintained normal activity and no indications of toxicity were recorded.

The combination regimen of PR-104 300 mg/kg and Gemcitabine 100 mg/kg demonstrated mean tumor shrinkage of 57.5% occurring in 8 of 9 animals. A median survival of 62 days (Min: 55 days, Max: 75 days) was observed through the course of the study. Significant survival difference was confirmed against the single agent components of the group with values of P<0.0001 and P=0.0001, respectively. The treatment regimen was generally well-tolerated with a maximum weight loss of approximately 8.4% which was rapidly regained by Day 15. One death did occur; treatment related death was not confirmed by necropsy. The remaining mice exhibited no additional signs of toxicity and maintained normal activity.

PR-104 500 mg/kg and Gemcitabine 100 mg/kg administered in combination generated tumor shrinkages in 9 of 9 animals with a mean of 69.7%. Median survival was 65 days (Min: 55 days, Max: 90 days) through the course of the study. Statistical analysis against the single agent components confirmed die significant increase in survival with values of P<0.01 and P<0.0001. The animals tolerated the treatment only recording a maximum weight'loss of 8.9%. Subsequently, a weight gain trend was observed, and all weight was regained by Day 23. No overt signs of toxicity were observed and animals sustained normal activity.

Table 3. PR-104 and Gemcitabine vs Panc-1 Xenograft: Median Survival

2 PR-104 300 IP q4dx3 42.9 ± 1.5 41 0 P<0.001' P>0.05^b

Gemcitabine 100 IP q4dx3 44.4 ±2.8 P<0.001-

Note: Survival curves are compared utilizing the log rank test

¹ Compared to Control b Compared to Gemcitabine 100 mg/kg

^■ Compared to Single agent PR-104 300 mg/kg

^Λ Compared to Single agent PR-104 500 mg/kg

Table 4. PR-104 and Gemcitabine vs Panc-1 Xenograft: TGI

DISCUSSION

MTD determination, combination dose toleration assessment, and studied efficacy of PR- 104 when administered as a single agent and in combination with the standard-of-care agent, Gemcitabine was completed. The initial single agent MTD evaluation of PR-104 and Gemcitabine did not identify a maximum tolerated dose of either agent at the doses and schedule tested (Table 1). However, safety of each compound was recognized and doses were selected for use in the combination dose toleration study. Two doses of PR- 104 (400 and 600 mg/kg) and Gemcitabine (100 and 160 mg/kg) were selected to be coadministered to determine any negative effects of the combination regimens. The combination of PR-104 400 mg/kg and both doses of Gemcitabine were well-tolerated with moderate weight loss. No negative clinical signs were observed and these combinations were identified as being safe. The PR-104 600 mg/kg and Gemcitabine combinations exhibited a more robust interaction. One death, possibly due treatment, occurred in the PR-104 600 mg/kg / Gemcitabine 100 mg/kg and a maximum weight loss of 19.0% was seen in the PR-104 600 mg/kg / Gemcitabine 160 mg/kg combination group. These combination dose levels of PR-104 and Gemcitabine can be defined as immediately above the maximum doses that can be administered utilizing the q4dx3 schedule.

Efficacy was evaluated by determining the in-study tumor growth inhibition (TGI) and the overall increase of survival to the end-point (1500mg). TGI was determined at Day 27, the final day that all animals still remained in their respective groups. PR-104 displayed impressive activity as a monotherapy agent using sub-maximum tolerated doses. Both the 300 and 500 mg/kg doses of PR-104 notably inhibited the growth of tumor compared to vehicle control (TGI=51.7% and 82.6%, respectively). Each single agent dose significantly increased survival with median survival times of 41 (P<0.001) and 51 days (P<0.0001), respectively. PR-104 500 mg/kg showed activity equally that of single agent Gemcitabine (TGI=75.3%, Median survival — 41 days) in which both compounds were not administered at their respective MTDs on the q4dx3 schedule.

The combination regimens of PR-104 and Gemcitabine exhibited positive trends consistent with supra-additive (synergistic) interactions. Log-rank analysis of the survival fractions confirmed significant increase of both treatment regimens when compared to their respective single agent counterpart (Table 3, Figure 4). The PR-104 300 mg/kg/Gemcitabine 100 mg/kg gtoup experienced one death. This animal's outcome was not consistent with the other group mates; however, cause of death was not determined by necropsy. The treatment given in both groups resulted in tumour shrinkages by Day 27 in 100% of living animals which was not observed in the single agent components of each regimen (Table 4).

Overall, PR-104 displayed impressive activity as a monotherapy agent and more so in a combination capacity. Both combination regimens tested can be considered generally nontoxic while demonstrating synergistic tumor response.

PART 2

AIM: To assess the interaction of Gemcitabine (Gemzar®) with PR-104 in A2780 xenografts EXPERIMENTAL PROTOCOL As for Part 1 except as noted below. Tumor Inoculations

96 female CD-I nude Subcutaneous A2780 1 7 x 10⁷ 100 μl

Treatment size: Mean diameter 7.0-9.9mm (average: 8.0mm); volume 118-440mm³; average 236+72mm³ (mean ± S.D.)

Drug administration schedule

Saline 002 ml/g q4dx3 4 "

Gemzar 237 q4dx3

End-point: After treatment, tumor size and body weights were measured regularly and mice were culled either when the average diameter of the tumor reached 15 mm (end- point), the tumor ulcerated or when the body weight change reached -15%. Experiment was ended and all remaining mice culled 120 days after treatment.

Analysis: End-points will be expressed as TTE₅₀, Median RTV⁴ and plotted in Kaplan- Meier Plots and analysed by Log Rank P statistical test. Weight loss nadir will be compared between schedules.

RESULTS

Saline 002ml/g - q4d x3 9 0 -06+03 0 6 8 -

PR-104 562 977 q4d x3 7 i c -28±1^:6 0 9 5 15 000896

Gemzar 237 213 q4d x3 9 0 -56+1 5 0 32 33 0 0000217

.^"PR-KH+ 562+237_,- 9,77+213 q4d x|. 8 0 -35±1 0 ' 4

ΪSN29862^' . 33 5 \ 81 5 00000513:

Jp ^•* ^

^A Long Term Control - 1 e. still in the experiment at day 120

^B Against control c Ulcerated tumor (non-drug related cull)

PR-104 treatment: A total dose of 977 mg/kg of PR-104 was administered i.p. (q4dx3), and resulted in a median tumor growth delay of 3.5-days that was not significant (P=0.059). This was independently associated with a 7-day improvement in median survival that was statistically significant as determined by log rank test (P<0.009). A mean body weight loss nadir of -2.8+1.6% was recorded.

Gemzar® (Gemcitabine) treatment: A total dose of 231 mg/kg of Gemcitabine was administered Lp. (q4dx3) and provided a median tumor growth delay of 26-days that was significant (TGI = 433%, P<0.001). This was independendy associated with a 25-day improvement in median survival that was statistically significant as determined by log rank test (P<0.001). A mean body weight loss nadir of -5.6± 1.5% was recorded.

PR-104 + Gemzar® (Gemcitabine) treatment: Co-administration of Gemcitabine + PR-104 (q4dx3) provided a median tumor growth delay of 27.5-days that was significant (TGI = 458%, P<0.001) which was independently associated with a 73.5-day improvement in median survival that was statistically significant as determined by log rank test (P<0.001). 4/8 (50%) of animals experienced complete tumor regression without regrowth in 90-days, indicative of tumor eradication (Le. cure). A mean body weight loss nadir of -3.5± 1.0% was recorded, indicating good tolerance. End-points plotted in a Kaplan-Meier graph are shown in Figure 6.

CONCLUSION

Gemcitabine was active against the A2780 xenograft model as a single agent, producing a significant tumor growth delay (TGI = 433%, P<0.001). However, while Gemcitabine increased median survival by 25-days it failed to provide any (0%) long term controls ("cures") in this model. The addition of PR-104, despite modest and non-significant single-agent antitumor activity (TGI = 58%, P=0.059), provided a large gain in therapeutic activity with 50% long term survivors (median survival 73.5-days; log rank P<0.001). This is an unexpectedly large gain in therapeutic activity and is indicative of a synergistic interaction between these two agents.

SUMMARY

These results clearly demonstrate a synergistic interaction between PR-104 and Gemcitabine across xenograft models. The combination represents a significant advance over single agent treatment.

While the present invention is broadly as described above, those persons skilled in the art will appreciate that the specific description is illustrative only and that variations may be made without departing from the scope of the invention. By way of example, combinations of Gemcitabine with compounds of Formula (I) other than PR- 104 are contemplated, as are variations in the dosing regimes specifically described.

Claims

1. A method for the production of an anti-cancer effect in a warm-blooded animal such as a human, which comprises administering to said animal an effective amount of a compound of Formula (I)

wherein:

X represents at any available ring position -CONH-, -SO₂NH-, -O-, -CH₂-, - NHCO- Or -NHSO₂-;

R represents a lower Cl -6 alkylene optionally substituted with one or more groups including hydroxyl, amino and N-oxides therefrom or dialkylamino and N-oxides therefrom;

Y represents at any available ring position -N-aziridinyl, -N(CH₂CH₂W)₂ or - N(CH₂CHMeW)₂, where each W is independently selected from halogen or -OSO₂Me; Z represents at any available ring position -NO₂, -halogen, -CN, -CF₃ or -SO₂Me; or a pharmaceutically acceptable salt or derivative thereof, before, after or simultaneously with an effective amount of Gemcitabine.

2. The method of claim 1 in which both the compound of Formula (I) or salt or derivative thereof and Gemcitabine are administered together with a pharmaceutically acceptable excipient or carrier.

3. The method of claim 1 in which the compound of Formula (I) is 2[(2-bromoethyl)- 2,4-dirύtro-6-[[[2-(phosphonooxy)ethyl]amino]-carbonyl]anilino]ethyl methanesulfonate.

4. A method for the treatment of a cancer in a warm-blooded animal such as a human, which comprises administering to said animal an effective amount of a compound of Formula (I) as defined in claim 1 or a pharmaceutically acceptable salt or derivative thereof, before, after or simultaneously with an effective amount of Gemcitabine.

5. The method of claim 4 in which both the compound of Formula (I) or salt or derivative thereof and Gemcitabine are administered together with a pharmaceutically acceptable excipient or carrier.

6. The method of claim 4 in which the compound of Formula (I) is 2[(2-bromoethyl)- 2,4-dinitjro-6-[[[2-(phosphonooxy)ethyl]amino]-carbonyl]anilino]ethyl methanesulfonate.

7. A therapeutic combination treatment comprising the administration of an effective amount of a compound of Formula (I) as defined in claim 1 or a pharmaceutically acceptable salt or derivative thereof, optionally together with a pharmaceutically acceptable excipient or carrier, and the simultaneous, sequential or separate administration of an effective amount of Gemcitabine, optionally together with a pharmaceutically acceptable excipient or carrier, to a warm-blooded animal such as a human in need of such therapeutic treatment.

8. The treatment of claim 7 in which the compound of Formula (I) is 2[(2- bromoethyl)-2,4-dinitro-6- [[[2- (phosphonooxy) ethyl] amino] -carbonyl] anilino] ethyl methanesulfonate.

9. A combination product comprising a compound of Formula (I) as defined in claim 1 or a pharmaceutically acceptable salt or derivative thereof, and Gemcitabine, for use in a method of treatment of a human or animal body by therapy.

10. The product of claim 9 in which the compound of Formula (I) is 2[(2-bromoethyl)- 2,4-dinitro-6-[[[2-(phosphonooxy)ethyl]amino]-carbonyl]anilino]ethyl methanesulfonate.

11. A pharmaceutical composition which comprises a compound of Formula (I) as defined in claim 1 or a pharmaceutically acceptable salt or derivative thereof, and Gemcitabine, in association with a pharmaceutically acceptable excipient or carrier.

12. The composition of claim 11 in which the compound of Formula (I) is 2[(2- bromoethyl) -2,4-dinitro-6- [[[2- (phosphonooxy) ethyl] amino] -carbonyl] anilino] ethyl methanesulfonate.

13. A kit comprising a compound of Formula (I) as defined in claim 1 or a pharmaceutically acceptable salt or derivative thereof, and Gemcitabine.

14. The kit of claim 13 in which the compound of Formula (I) is 2[(2-bromoethyl)-2,4- dinitJro-6-[[[2-(phosphonooxy)ethyl]arnino]-carbonyl]anilino]ethyl methanesulfonate.

15. A kit comprising: a) a compound of Formula (I) as defined in claim 1 or a pharmaceutically acceptable salt or derivative thereof in a first unit dosage form; b) Gemcitabine in a second unit dosage form; and c) container means for containing said first and second dosage forms.

16. The kit of claim 15 in which the compound of Formula (I) is 2[(2-bromoethyl)-2,4- dimtto-6-[[[2-φhosphonooxy)etiiyl]amino]-carbonyl]anilino]ethyl methanesulfonate.

17. The use of a compound of Formula (I) as defined in claim 1 or a pharmaceutically acceptable salt or derivative thereof and Gemcitabine in the preparation of a medicament for producing an anti-cancer effect in a warm-blooded animal such as a human.

18. The use of claim 17 in which the compound of Formula (I) is 2[(2-bromoethyl)- 2,4-dinitro-6-[[[2-(phosphonooxy)ethyl]amino]-carbonyl]anilino]ethyl rnethanesulfonate.

20. The use of a compound of Formula (I) as defined in claim 1 or a pharmaceutically acceptable salt or derivative thereof and Gemcitabine in the preparation of a medicament for the treatment of cancer in a warm-blooded animal such as a human.

21. The use of claim 20 in which the compound of Formula (I) is 2[(2-bromoethyl)- 2,4-dinitro-6-[[[2-(phosphonooxy)emyl]amino]-carbonyl]anilino]ethyl methanesulfonate.