WO2001003718A2 - Combination chemotherapy - Google Patents

Abstract

Synergistic antineoplastic combinations comprise diethylnorspermine and a protein selected from interferon-α and interleukin.

Description

COMBΓNATION CHEMOTHERAPY

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.: 60/142,594, filed July 7, 1999, the contents of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to a combination of a polyamine anticancer agent and either interferon or interleukin, and to the use of such combinations in the treatment of cancer.

BACKGROUND OF THE INVENTION

Cancer remains one of the most dreaded diseases afflicting mankind, and is one of the main causes of death for both men and women. Significant progress has been made in treating most forms of cancer, particularly with the large number of chemotherapeutic agents now available. Many such agents are used in combinations, as a way to increase efficacy and reduce concomitant toxicity.

Because not all forms of cancer are subject to treatment with chemotherapeutic agents and because patients often become resistant to certain agents, the need for new and better treatments continues. Lnited States Patent No 5,866,613, which is incorporated herein by reference, discloses a seπes of polyamine compounds which are said to be useful as neoplastic agents One such polyamine is N,N'-bιs-(3-ethylammopropyl)- propane-l,3-dιamme, also known as diethylnorspermine (DENSPM) and CI-1006 DENSPM has been shown to be clinically useful to treat a variety of neoplasms, including large and small cell lung carcinoma, human colon carcinoma, vaπous human brain tumors, human melanocytes, several human melanoma lines, and renal carcinoma

Interferon (IFN-α. or mterferon-α 2a or 2b, recombinant) is a protein containing 165 ammo acids, and is manufactured commercially by recombinant

DNA technology that employs a genetically engineered Escherichia coh bacterium containing DNA that codes for the human protein The product is used to treat neoplastic diseases such as leukemias. especially hairy cell leukemia and AIDS-related Kaposi's sarcoma, as well as myelogenous leukemia, melanomas, fol cular lymphoma. and Hepatitis C The product is available commercially

(Roferon®-A., Roche Laboratoπes. and Intron® A. mterferon-a 2b, Scheπng Corporation) and is supplied as steπle solutions for injection m several concentrations (3mιllιon IU (11 1 mcg/mL) per vial. 6 million IU (22 2 mcg'mL), 9 million IU (33 3 mcg/0 9 mL), 18 million IU (66 7 meg' 3 mL), and 36 million IU (133 3 mcg'ml) per vial) The product typically is administered as a subcutaneous, intralesional, intravenous or intramuscular injection, at a dose of about 3 to about 36 MIU (million international units) given daily for up to about 16 to 24 weeks

Interleukm is a lymphokme protein made by recombinant DNA technology Commercial forms, such as Proleukm® (Chiron, aldesleukm), differ slightly m structure from native human mterleukm-2, but exhibit the same anticancer activity Proleukin is supplied as a steπle, white to off-white, lyophihzed cake in single-use vials The protein generally is administered as monotherapy by intravenous injection for treatment of humans with neoplastic diseases such as metastatic renal cell carcinoma and metastatic melanoma. The product typically is administered by IV injection over about 15 minutes every 6 to 10 hours. Each course of treatment consists of two 5-day treatment cycles separated by a rest peπod A routine dosing, for example, involves IV injection of 600,000 IU/kg (0 037 mg/kg) every 8 hours for about 12 to 14 doses Following a rest peπod of about 9 days, the dosmg schedule is repeated. The total dosmg continues for about 28 days. Aldesleukin is the only approved treatment for renal cell carcinoma. Both aldesleukin and INF-α 2b are approved for treatment of melanoma

SUMMARY OF THE INV ENTION

This invention provides a combination composing DENSPM together with either IFN-α or interleukin. and a method for treating cancer composing admmisteπng a combination of DENSPM and either IFN-α or interleukin.

A preferred embodiment is a combination compπsing DENSPM together with interferon, and a method for treating patients suffeπng from neoplastic diseases responsive to treatment with these agents

Another preferred embodiment is a combination compπsing DENSPM together with interleukin. and a method for treating patients suffeπng from neoplastic diseases responsive to treatment with these agents. In another embodiment, this invention provides a kit compπsed of

DENSPM in one compartment, and either IFN-α or interleukin m a second compartment The kit facilitates usage of the combination by having both agents readily available for convenient dosmg

BRIEF DESCRIPTION OF FIGURES The Figure shows the tumor growth of transplanted renal cell carcinoma in rats following treatment with DENSPM and IFN-α alone and m combination DETAILED DESCRIPTION OF THE INVENTION

As used herein, "IFN" and "IFN-α" mean interferon-α, interferon-α 2a, and interferon-α 2b. IFN made by recombinant technology is commercially available. "Interleukin" means the natural human lymphokine protein or commercial variants thereof. Human recombinant interleukin-2, also known as aldesleukin (IL-2), is commercially available.

DENSPM means N,N'-bis-(3-ethylaminopropyl)-propane-l,3-diamine, and pharmaceutically acceptable salts thereof, such as the hydrochloride salt form. DENSPM is also known as diethylnorspermine, CI-1006, and PD 150497.

The combination of this invention utilizes generally the same amounts of active antineoplastic agents which are commonly employed individually. For example, DENSPM will be administered at about 5 to about 150 mg/kg of animal body weight. Typical dose regimens will be, for example, intravenous administration once per day for about 5 days at doses of about 50 to about

100 mg/m-/day. These doses will be repeated every 3 to 4 weeks. DENSPM can also be administered orally.

The IFN component of the present combination will be administered at its normal clinical dosages, for instance, from about 3 million international units (MIU) to about 60 MIUs. The IFN will be administered parenterallv at the rate of about 1 to 4 times per week, generally for about 10 to 20 weeks.

The interleukin will be administered by IV injections at doses of about

600,000 IU/kg about every 8 hours (18 million [18 x 10⁶] IU of commercial aldesleukin equals 1.1 mg of active protein). The combination provided by this invention has shown dramatic and synergistic antineoplastic activity when tested in standard assays commonly used to measure anticancer activity.

The combinations provided by this invention have been evaluated in several assay systems, and the data can be analyzed utilizing a standard program for quantifying synergism, additivism, and antagonism among anticancer agents The program preferably utilized is that descπbed by Chou and Talalay, m "New Avenues in Developmental Cancer Chemotherapy," Academic Press, 1987, Chapter 2

The method is based on the median-effect pπnciple of the mass-action law using an enzyme kinetic system as a model. The equation is simple and descπbes the relationships between dose and effect regardless of the shape of the dose-effect curve Two basic equations constitute the pillars of this methodology To relate dose and effect for a single drug in the simplest way possible, the median-effect equation deπved by Chou is given by

where the πght side represents the dose and the left side represents the effect, in which f_a and f_u are the fractions affected and unaffected, respectively, D is the dose, D_m is the median-effect dose signifying the potency, and m is a coefficient signifying the shape of the dose-effect curve From this equation Chou and Talalay deπved the general equation for two or more drugs

(D), α (D), (D),

+ (D)_:

(D , (D , (D , (D ,

where m = 1 is for first-order Michaelis-Menten-type kinetics and m >1 (or m <1) is for higher order (or lower order) Hill-type kinetics When alpha = 0, the third term on the πght side disappears and when alpha = 1 , the third term is conserved

Alpha = 0 is used for mutually exclusive drugs and alpha = 1 is used for mutually nonexclusive druss For drugs that have the same or similar modes of action, the effects of both drugs are mutually exclusive For drugs that have different modes of action or act independently, the effects of both drugs are mutually nonexclusive

A plot of fraction affected (F^ versus combination index (CI) is called the F_a-CI plot This plot indicates synergism, additivrty, or antagonism of two drugs at vaπous effect levels in a mixture that is seπally diluted If several mixtures are made, it is possible to estimate the optimal combination ratio for maximal synergy Different effect levels usually give different degress of synergism, additivism, or antagomsm CI values <1 indicate synergism, CI values >1 indicate antagonism, and CI values that are one or hover around one indicate additivity

For anticancer agents, synergism at high effect levels (F^) is clinically more relevant than synergism at low F_a levels

The synergistic combinations provided by this invention have been evaluated in standard chemotherapy studies in animals The assay was conducted as descπbed in Examples 1 and 2 Clinical evaluations are descπbed in

Example 3

EXAMPLE 1

MATERIALS AND METHODS

Antitumor agents CI-1006 (PD 150497, N,N'-bιs[3-(ethylammo)propyl]-l,3-propanedιamιne

HC1, DENSPM) was dissolved in steπle 0 9% saline and administered mtrapeπtoneally in 0 5-mL doses CI-1006 was given 3 times a day for 5 days at vaπous doses (equivalent to 64, 84, 1 13, and 150 mg/kg/day of CI-1006 as free base) Interferon-α (Roferon-A. IFN-α) was obtained from Hoffmann-LaRoche Inc (Nutley, NJ) Interleukin- 2 (Proleukin, IL-2) was obtained from Chiron

Therapeutics (Emeryville, CA) The IFN-α and IL-2 were diluted for injection in steπle 0 9% saline. Starting with the first CI-1006 treatment, both IFN-α and IL-2 were given subcutaneously (SC) once a day for 14 days IFN-α was administered at doses of 5 x 10° and 10 x 10" IU/kg/day The following treatment sequences were evaluated (1) treatment with CI-1006 and IFN-α started at the same time, (2) CI-1006 administered before IFN-α treatment started, and

(3) IFN-α administered before CI-1006 treatment started.

Mice

A498 Human renal cell carcinoma was grown as a SC xenograft m female, athymic (NCr nu/nu) mice These mice were obtained from the NCI's animal program at Fredenck Cancer Research and Development Center, Maryland The

SK-MEL-5 human melanoma was grown as a xenograft m CD-I nu/nu mice These mice were obtained from Charles River UK Ltd. and Charles Ri er France Ltd The mice were housed in steπle, filter capped, polycarbonate cages containing steπle bedding. Steπle feed (Teklad Sterhzable 8656 Mouse Diet) and water were provided ad libitum All animal handling procedures were done m laminar flow biosafety hoods located in stenle bamer rooms. Lighting in the banner rooms was controlled as 12-hour light and dark cycles, and temperature was maintained at 72 ± 4°F

Tumor Models The A498 renal cell carcinoma (deπved from an undifferentiated renal cell carcinoma in a 52-year-old woman) was used to evaluate the antitumor activity of the vaπous CI-1006 and IFN-α combinations The A498 tumor was maintained by seπal SC implantation of tumor fragments (30-40 mg) over the πb cage in the axillary region of NCr nu nu mice When re-established from frozen fragments, the tumor was not used for antitumor studies until its doubling time had stabilized

(usually by passages 3 or 4 times) The A498 human renal carcinoma xenograft model has been shown to be refractory to many standard anticancer drugs The SK-MEL-5 human melanoma was used to evaluate the antitumor activity ot the combinations of CI-1006 and IL-2 As descnbed below, the SK-MEL-5 tumor was grown in cell culture to obtain enough cells to establish it as a xenograft m athymic CD-I mice

Evaluation of Antitumor activity

For the A498 tumor, mice were pooled, implanted SC with 30 to 40 mg tumor fragments as descπbed above, and then randomized into the vaπous treatment and control groups Chemotherapy was started when the median tumor burden per mouse was 200 mg (range 150-250 mg) Tumor sizes were measured with a vernier ca per twice weekly

To establish the SK-MEL-5 xenograft tumor in mice, the tumor was harvested from subconfluent cultures and resuspended in phosphate-buffered saline at a concentration of 7 x 10' cells per mL Athymic CD-I mice were implanted subcutaneously in the πght flank with 7 x 10°^" cells m a volume of 0 1 mL and assigned to treatment groups composed of 10 mice Chemotherapy was started when the tumors were in the range of 150 to 250 mg

Assuming a density of 1 rag/mm-³, tumor weight was calculated from cahper measurements by the following equation for a prolate ellipsoid (Geran RI, Greenberg NH, Macdonald MM, Schumacher AM, Abbott BJ Protocols for screening chemical agents and natural products against animal tumors and other biological systems Cancer Chemotherapy Rep, 1972,3(Part 3) 51)

Tumor weight (mg) = (a χ O-)l2,

where a and b are the tumor length and width, respectively, in mm

The following parameters were used to evaluate antitumor activity (1) partial tumor response, (2) complete tumor response, and (3) tumor free survival A paπial response (PR) is defined as the fraction of mice that had at least a 50° o reduction in measurable tumor mass A complete response (CR) is defined as the^' fraction that have a 100% decrease in measurable tumor mass. Clinically, the complete response rate is the most important indicator of the effectiveness of chemotherapy (DeVita VT Jr. Principles of cancer management: Chemotherapy. In: Cancer-Principles and Practice of Oncology. V.T. DeVita, S. Hellman. and S.A. Rosenberg eds, Lippincott-Raven Publishers, Page 334, 5^th edition, 1997). A measure of the durability of a complete remission is the tumor free survival rate. This is the fraction of mice that do not have a measurable tumor mass at the end to a study. This criterion is the preclinical counterpart of the relapse-free survival parameter that is used clinically to evaluate chemotherapy (DeVita VT Jr. Principles of cancer management: Chemotherapy. In: Cancer-Principles and

Practice of Oncology. V.T. DeVita, S. Hellman, and S.A. Rosenberg eds, Lippincott-Raven Publishers, Page 334, 5^th edition, 1997).

RESULTS AND CONCLUSION

The antitumor effect that is produced when CI-1006 is used in combination with IFN-α is shown in Tables 1 through 4. For the combination of

CI-1006 with IFN-α, the following treatment sequences were evaluated: (1) the sequence where treatment with CI-1006 and EFN-α were started at the same time, (2) the sequence where CI-1006 was given before IFN-α, and (3) the sequence where IFN-α was given before CI-1006. Treatments were started when the median tumor mass of the A498 renal cell carcinoma was approximately 200 mg.

Delaying treatment until tumors are this size provides a more stringent evaluation of antitumor activity, and allows for the use of the clinically relevant endpoints of partial and complete tumor responses.

The antitumor effects seen when treatments with CI-1006 and IFN-α were started at the same time are shown in Table 1. CI-1006 was given at doses that ranged from 64 to 150 mg kg/day. There were no drug associated deaths or significant weight loss associated with any CI-1006 dose. However, it should be noted that in some studies the high dose of CI-1006 is very close to a LD J Q dose (see Tables 2 and 4). The tumor growth delays seen with CI-1006 alone ranged frorn 1 1 5 to 21 2 days These represent net tumor cell kills of 0 4 to 0 8 logjo, respectively Similar antitumor activities were seen in other studies with these doses (see Tables 2-4) There were no partial tumor responses seen with any dose of CI- 1006 However, complete tumor regressions were seen in 20% of the mice treated with 64 mg/kg' day of CI-1006 No drug associated deaths or significant weight losses were seen with IFN-α alone at doses of 5 or 10 x 10° umts/kg/day IFN-α at these doses produced tumor growth delays of 1 1 9 and 20 8 days, respectively These delavs represent net tumor cell kills of -0 4 and 0 1 \og\_Q No complete or partial tumor responses were produced by treatment with either dose of IFN-α alone There was one possible drug associated death and a 9% weight loss w hen CI-1006 was given at 150 m kg'day with IFN-α at 5 x 10°^" units kg/day No deaths or significant weight losses were seen when the other doses of CI-1006 were given in combinations of CI-1006 and the low IFN-α dose The tumor growth delays produced with the dose combinations of CI-1006 and the low IFN-α dose ranged from 27 4 to 34 3 days The growth delays represent net tumor cell kills of 0 7 to 1 1 logjo Based on tumor growth delays, the antitumor effect was approximately additive when treatments with CI-1006 and the low IFN-α dose were started at the same time No partial tumor regressions were seen when CI- 1006 was given with the low IFN-α dose However, 40% to 100% complete response rates were seen when CI-1006, at doses of 64 to 150 ma'kg/day, was given with the low IFN-α dose Ten to 20% of the mice were still tumor free when the study ended 98 days after the last IFN-α treatment A 13% weight loss was seen when CI-1006 was given simultaneously at 150 mg-kg/day with IFN-α at the high dose of 10 x 10°^" units/kg/ day However, no deaths were seen with any dose of CI-1006 given in combination with the high

IFN-α dose The dose combinations of CI-1006 with the high IFN-α dose produced tumor growth delays that ranged from 42 to 71 3 days These growth delays represent net tumor cell kills of 1 5 to 2 9 log i Q Based on the tumor growth delay values, the antitumor effect was greater than additive when treatments with CI-1006 and the high IFN-α dose were started at the same time. There were 100% complete tumor regressions with all doses of CI-1006 given with the high IFN-α dose. Forty to 50% of the mice were still tumor free when the study ended 98 days after the last IFN-α treatment.

The increased antitumor activity described above was confirmed in a second study where treatments with CI-1006 and IFN-α were started on the same day. The results of this study are shown in Table 2. However, in the second study the antitumor activity was not pronounced. For example, 100% complete tumor regressions were not seen with all doses of CI-1006 in combination with the high

IFN-α dose. Instead, partial tumor responses were seen with most CI-1006 doses in combination with the low and high JJFN-a doses.

Table 3 shows the antitumor effect that is produced when a full course of treatment with CI-1006 was given before treatment with IFN-α started. No drug associated deaths or significant weight losses were seen when CI-1006 was given at doses of 64 to 150 mg/kg/day. These doses produced tumor growth delays of 10.5 to 16.5 days, which represent net tumor cell kills of 0.3 to 0.5 logi r No partial tumor regressions were produced by CI-1006 alone. CI-1006 at doses of 113 and 150 mg/kg/day produced a 10% complete response rate. Mice with the complete responding tumors were still tumor free 66 days after the last IFN-α treatment. Like the other studies, no deaths or significant weight losses were seen after treatment with IFN-α alone at the low or high doses. The tumor growth delays produced by these doses were 10.2 and 11.9 day, which represent net tumor cell kills of -0.1 and 0.0 logi Q- Treatment with IFN-α alone at either dose produced no partial or complete tumor responses. When CI-1006 was given at doses of 64 to 150 mg/kg/day before treatment with the low IFN-α dose, no deaths or significant weight losses were seen. The rumor growth delays ranged from 33.1 to 44.8 days when CI-1006 was given before IFN-α. These growth delays represent net tumor cell kills of 0.6 to 1.1 logi Q. Tumor growth delay values indicate the antitumor effect was greater than additive when CI-1006 was given before IFN-α. These growth delays represent net rumor cell kills of 0.6 to 1.1 log ιo- The tumor growth delay values indicate the antitumor effect was greater than additive when CI-1006 was given before treatment with the low

IFN-α dose. A 10% partial response rate was produced when CI-1006 was given at 113 and 150 mg/kg/day before IFN-α. More importantly, treatment with these CI-1006 doses before IFN-α produced complete response rates of 70% to 100%. Ten to 40%o of the mice were tumor free when the study ended 66 days after the last IFN-α treatment. There was one unexplained death in the group of mice that were treated with CI-1006 at 113 mg/kg/day before treatment with the high IFN-α dose. There were no significant weight losses for the other mice in this group. In groups treated with the other doses of CI-1006, there were no deaths or significant weight losses. Treatment with CI-1006 at doses of 64 to 150 mg/kg day before treatment with the high IFN-α dose produced tumor growth delays that ranged from 33.4 to 51.8 days. These delays represent net tumor cell kills of 0.6 to 1.3 log \ Q. Like the combination with the low IFN-α dose, pretreatment with

CI-1006 before treatment with the high IFN-α dose produced a synergistic antitumor effect. A 10% partial response rate was seen only in the group that was pretreated with 113 mg/kg/day of CI-1006 before the high IFN-α dose. The complete response rates were either 80% or 100% when CI-1006 was given at the other doses before the high IFN-α dose. The percentage of tumor free mice at the end of the study ranged from 0% to 80%, with the highest percentage in the group treated with the highest doses of CI-1006 and IFN-α. The antitumor effects that are produced when IFN-α is given before

CI-1006 are shown in Table 4. Similar to the repeat study for the simultaneous treatment combination (Table 2), one possible drug related death was seen in the group treated with 150 mg kg/day of CI-1006 alone. In the other CI-1006 dose groups, no deaths or significant weight losses were seen. Treatment with CI-1006 produced tumor growth delays that ranged from 2.2 to 15.9 days. The net tumor cell kill values for these delays are -0.1 to 0.4 logiQ. A 10%> partial response rate was seen in the group of mice treated with CI-1006 at 84 mg kg/day. Ten percent complete response rates were seen in the groups treated with CI-1006 at 64, 84, and 150 mg/kg/day. In the groups treated with 64 and 84 mg/kg/day, 10% of the mice were tumor free when the study ended 54 days after the last CI-1006 treatment. IFN-α alone at the low and high doses did not cause any deaths or significant weight losses. These doses produced tumor growth delays of 11.1 and 7.1 days, which represent net tumor cell kill values of -0.1 and -0.1 logi n. A 10% complete response rate was seen in the group treated with the high IFN-α dose. The mouse with the responding tumor was still tumor free when the study ended 44 days after the last IFN-α treatment. In all the groups treated with the low IFN-α dose before treatment with the CI-1006 doses, no deaths or significant weight losses were seen. For these treatment groups, the tumor growth delay values ranged from 15.1 to 22.1 days. These delays represent net tumor cell kills of -0.1 to 0.2 log jo- Only one complete rumor response was seen in the group pretreated with the low IFN-α dose followed by treatment with 64 mg/kg/day of CI-1006. Like the low IFN-α dose, no deaths or significant weight losses were seen when the mice were treated with the high IFN-α dose before CI-1006. The tumor growth delays produced by the various dose combinations ranged from 14.2 to 25.1 days. These represent net tumor cell kill values of -0.1 to 0.3 days. At best, these tumor growth delays represent a positive antitumor effect. One partial tumor response was seen in the group treated first with the high IFN-α dose and then 64 mg/kg/day of CI-1006. A 20% complete response rate was seen with the dose combination of high dose IFN-α followed by CI-1006 at 84 mg/kg/day; the complete response rate was 10% for the other dose combinations. . These results indicate the antitumor action of the combination of CI-1006 and IFN-α is greater than additive when they are given simultaneously. They also indicate that IFN-α and CI-1006 have non-overlapping dose limiting toxicities under these studv conditions.

TABLE 1

Antitumor Effect of the Combination of CI- 1006 and IFN- α Against Advanced Stage A498 Human Renal Tumor Xenograft Model.

Effect o f S: imultaneous Treatment With Cl- 1006 and IFN-α

CI- 1006 IFN - No of % Weight Antitumor Effect

Dose' ¹ Schedule Dose^a Schedule Deaths Change^b % CR % PR^d T-C^e(+) Net Log ₁₀ Kιll^f % Tumor Fιee^B

64 Days 15- 19 - - 0/10 -4 5 20 0 19 9 0 8 20

84 - - 0/10 -4 3 0 0 21 2 0 9 0

1 13 - - 0/10 -8 7 0 0 18 3 0 7 0

150 - - 0/10 -4 3 0 0 1 1 5 0 4 0

- 5 χ 10⁶ Days 15 28 0/10 -4 5 0 0 1 1 9 -0 4 0

- 10 x 10⁶ 0/10 14 5 0 0 20 8 0 1 0

64 Days 15- 19 5 χ 10⁶ Days 15-28 0/10 -4 5 40 0 27 4 (31 8) 0 7 20

84 0/10 -4 5 50 0 27 5 (33 1) 0 7 0

1 13 0/10 -4 5 60 0 28 5 (30 2) 0 8 10

150 1/10 -8 7 100 0 34 3 (23 4) 1 1 10

64 Days 15 19 10 x 10⁶ Days 15-28 0/10 -14 5 100 0 62 4 (40 7) 2 5 50

84 0/10 - 10 0 100 0 69 0 (42 0) 2 8 50

1 13 0/ 10 -4 5 100 0 71 3 (39 1) 2 9 40

150 0/10 - 13 0 100 0 42 0 (32 3) 1 5 40

T he dosLs of CI- 1006 (diethylnoispermine) are m nig/kg/day of the free base The daily CI-1006 doses weie given as 3 equally divided doses every 8 hours I he doses of IFN α are in units/kg/day II N u was given once a day Treatments were not started until Day 15, when ihe tumors weighted approximately 200 mg

A weight loss is the maximum seen during treatment, a weight gain is for the weight seen at the end of lieatment

A complete response is a lumor that had a 100% decrease in tumoi mass during the study

A paitial response is a lumor thai had at least a 50% decrease in tumor mass during the study

I -C is Ihe difference in days lor the treated and control tumors to reach 750 mg I he values in parenlhesis repiesent Ihe T-C values for an additive antitumor effect

Net log _jo tumor cell kill was calculated from the I C value as described in Materials and Methods

The percent lumor free represents Ihe mice that had an undetectable lumor when Ihe study ended on Day 126

TABLE 2 Antitumor Effect of the Combination of CI- 1006 and IFN-α Against Advanced Stage A498 Human Renal Tumor Xenograft Model:

Effect of Simultaneous Treatment With CI-1006 and IFN-α

CI- 1006 IFN -α No of % Weight Antitumor Effec t

Dose³ Schedule Dose^a Schedule Deaths Change¹" % CR^C % R^d T -C^e(+) Net I .og₁₀ ill^r % Tumor Free⁶

64 Days 19-23 - - 0/10 +4.3 10 0 15 5 0 3 0

84 - - 0/10 0 0 10 0 15.2 0.3 10

1 13 - - 0/10 0.0 0 0 13.5 0.3 0

150 - - 1/10 0 0 0 0 13.1 0 3 0

- 5 x 10⁶ Days 19-32 0/10 -8.0 0 0 13.4 0 0 0

- 10 x 10⁶ 0/10 -4.3 10 0 14.0 0.0 0

64 Days 19-23 5 x 10⁶ Days 19-32 0/10 -4.3 100 0 34.9 (28.9) 0.6 40

84 0/10 +4.2 80 20 37.2 (28.6) 0.7 20

1 13 0/10 -4.3 50 30 29.3 (26.9) 0.5 10

150 0/10 +4.2 90 10 31.0 (26.5) 0.5 20

64 Days 19-23 10 x 10⁶ Days 19-32 0/10 0 0 50 20 25.2 (29.5) 0.4 20

84 0/10 -4.2 90 10 39.1 (29.2) 0.8 30

1 13 0/10 -4 3 100 0 42.9 (27.5) 0.9 50

150 0/10 -4.2 90 10 36.6 (27.1) 0.7 0

The doses of CI- 1006 (diethylnorspermine) are in mg/kg/day of the fiee base. The daily CI- 1006 doses were given as 3 equally divided doses every 8 hours The doses of lFN-cc are in unils/kg/day IFN-u was given once a day Treatment with CI- 1006 was not started until Day 19, when Ihe tumors weighted approximately 200 g

A weight loss is ihe maximum seen during tieatment, a weight gain is tor the weight seen al the end of treatment

A complete response is a lumor that had a 1 0% decrease in lumor mass during the study

A partial response is a lumor that had at least a 50% decrease in tumor mass during the study

1 -C is the dillcrence in days for Ihe treated and control tumors to reach 750 mg The values in parenlhesis repiesent the T-C values for an additive antitumor effect

Net log _j lumor cell kill was calculated from the T-C value as dcsci ibed in Materials and Methods

The percent lumor free represents Ihe mice that had an undetectable tumor when Ihe study ended on Day 1 14

TABLE 3

Antitumor Effect of the Combination of CI- 1006 and IFN- α Against Ad- vanced Stag e A498 Human Renal Tumor Xenograft Model:

Effect of Treatment With CI-1006 Before Treatment With IFN-α

CI- 1006 IFN - No of % Weight Antitumor F.ffec t

Dose^{: 1} Schedule Dose^a Schedule Deaths Change^b % CR^C % PR^d τ-c ) Net I -°B 10 Kιll^f % Tumoι Fιee^e

64 Days 15- 19 - - 0/10 -4 2 0 0 10.5 0 3 0

84 - - 0/10 -8.7 0 0 13.5 0 4 0

1 13 - - 0/10 -8.7 10 0 1 1.6 0 3 10

150 - - 0/10 -8.7 10 0 16.5 0 5 10

- 5 10⁶ Days 15-28 0/1 -4 2 0 0 10 2 -0.1 0

- 10 x 10⁶ 0/10 -4.5 0 0 1 1.9 0 0 0

64 Days 15- 19 5 x 10⁶ Days 20-33 0/10 -8.7 70 0 32.6 (20.7) 0.6 10

84 0/10 -8.7 100 0 33.1 (23.7) 0 6 40

1 13 0/10 -8.7 80 10 33.1 (21.8) 0 6 10

150 0/10 -8.7 90 10 44.8 (26.7) 1.1 30

64 Days 15- 19 10 x 10⁶ Days 20-33 0/10 -8.7 80 0 40.0 (22.4) 0.9 0

84 0/10 -4 3 100 0 47 0 (25.4) 1 1 30

1 13 1/ 10 -8.3 80 10 33.4 (23.5) 0.6 30

150 0/10 -8.7 100 0 51.8 (28.4) 1.3 80

The doses of CI- 1006 (diethylnorspermine) are in mg/kg/day of the fiee base. The daily CI- 1006 doses were given as 3 equally divided doses every 8 hours The doses of IFN-α are in umls/kg/day IFN-α was given once a day Treatment with CI- 1006 was not started until Day 15, when the lumors weighled approximately 200 g

A weight loss is the maximum seen during treatment, a weight gain is loi the weight seen at the end of tieatmenl

A complete response is a lumor that had a 100% decrease in lumor mass during the study

A partial response is a tumor that had at least a 50% decrease in tumor mass din ing Ihe study

T-C is Ihe dillcience in days for the treated and control lumors to leach 750 mg The values in parenthesis repiesent Ihe T-C values for an additive antitumor effect

Net log _j o luinoi cell kill was calculated from the I -C value as described in Materials and Methods

1 he pei cent lumor free represents Ihe mice thai had an undelcctable lumoi when the study ended on Day 99

TABLE 4 Antitumor Effect of the Combination of CI-1006 and IFN-α Against Advanced Stage A498 Human Renal Tumor Xenograft Model:

Effect of Treatment With IFN-α Before Treatment With CI-1006

CI- 1006 IFN -α No. of % Weight Antitumor Effect

Dose^a Schedule Dose" Schedule Deaths Change^b % CR^C % PR^d T-C^e(ϊ ) Net Lo ₁₀ Kill^r % Tumor Free⁸

64 Days 20-24 - - 0/10 0 0 10 0 2 2 -0.1 10

84 — - 0/10 -4.3 10 10 15.9 0.4 10

1 13 - - 0/10 -4.3 0 0 14.9 0.4 0

150 - - 1/10 -4.5 10 0 9.5 0.2 0

- 5 x 10⁶ Days 20-33 0/10 0.0 10 0 1 1.1 -0.1 10

- 10 x 10⁶ 0/10 -4.5 0 0 7.1 -0.2 0

64 Days 34-38 5 x 10⁶ Days 20-33 0/10 +4.8 10 0 15.1 ( 13.3) -0.1 10

84 0/10 -4.5 0 0 22.1 (27 0) 0.2 0

C

1 13 0/10 -8 7 0 0 16.0 (26.0) -0.1 0

150 0/10 -9.1 0 0 21.6 (20.6) 0.1 0

64 Days 34-38 10 x 10⁶ Days 20-33 0/10 -4.3 10 10 14.2 (9.3) -0.1 10

84 0/10 0.0 20 0 21.3 (23.0) 0.1 10

1 13 0/10 -4.5 10 0 23.6 (22.0) 0.2 0

150 0/10 0.0 10 0 25.1 ( 16.6) 0.3 10

T he doses of CI- 1006 (dielhylnorspeimine) are in mg/kg/day of the free base The daily CI-1006 doses were given as 3 equally divided doses every 8 hours The doses of IFN-α are in units/kg/day IFN-u was given once a day Treatment with IFN-α was not started until Day 20, when ihe lumors weighted approximately 200 mg

A weight loss is the maximum seen during treatment, a weight gain is for the weight seen at the end of tiealment

A complete response is a lumor that had a 100% decrease in tumor mass during the study

A paitial response is a tumor that had at least a 50% decrease in tumor mass din ing the study l -C is the difference in days for the treated and conliol tumois to leach 750 mg T he values in parenlhesis icpresent the T -C values for an additive antitumor effect

Net log _j o lumor cell kill was calculated fiom Ihe T-C value as described in Materials and Methods he peicent tumoi free represents the mice that had an undelectable tumoi when ihe study ended on Day 77

EXAMPLE 2

The general procedure described above in Example 1 is repeated with CI-1006 alone and interleukin alone, and the combination treatment of CI-1006 plus interleukin, except that the SK-MEL-5 human melanoma is used to infect athymic CD-I mice.

EXAMPLE 3

The combinations provided by this invention will be evaluated clinically according to the following general procedure. Patients with solid tumors (renal cell carcinoma and melanoma) will be given escalating doses of DENSPM in combination with fixed doses of IFN-α. In a similar, patients with solid tumors will be given escalating doses of DENSPM in combination with fixed doses of IL-2.

Preliminary data from studies in mice with human renal cell xenografts (A498) exploring the activity of DENSPM in combination with human IFN-α demonstrates as many as 10/10 complete responses in the combination arm and

0/10 in each of the single agent treatment arms. In addition, EFN-α (as well as IL-2) is approved for the treatment of melanoma and is used extensively in patients with renal cell carcinoma. Both agents have demonstrated antitumor activity and, except for nausea, do not have over-lapping toxicities. Based on the safety data from the QD x 5-day schedule in Phases 1 and 2, it is clear that doses of DENSPM above 100 mg/m^/day (free base) are not well-tolerated due to diarrhea, abdominal pain, nausea and vomiting, and facial swelling. In addition, antitumor activity was seen at this dose level. Therefore, the DENSPM dose in this study will not be escalated beyond 100 mg/m^. Three dose levels (60, 80, and 100 sj -) given QD for 5 days repeated every 3 weeks will be investigated.

Three patients will be entered at each dose level. Up to 10 additional patients will be entered at the proposed Phase 2 dose level for DENSPM. The dose of IFN-α 2b that will be used in this study, 10 MU SC 3 times a week, is based on the review of treatment results for published trials of IFN-α which concluded that the highest response rates for single agent IFN therapy have been obtained with uninterrupted schedules, and with doses in the range of 10 million International

Units/m^ daily on an alternate-day schedule. The SC dose of IFN, 10 MU, administered 3 times a week, will be held constant throughout the study unless a dosage decrease is required to manage toxicity.

The objectives of this study are to identify the maximum tolerated dose (MTD) and the dose-limiting toxicities (DLT) of DENSPM, administered IV once-a-day for 5 days at doses of 60, 80, and 100 mg m^/day (free base) over 15 minutes repeated every 3 weeks, in combination with 10 MU IFN-α 2b administered SC 3 times a week and to recommend a dose for Phase 2. Any evidence of efficacy will also be noted. Safety will be assessed by spontaneous adverse event reporting and clinical laboratory measurements. Plasma and tissue pharmacokinetics of DENSPM will also be investigated.

This is an open-label, multicenter, Phase 1 study of escalating doses of DENSPM in combination with a fixed SC dose of IFN in patients with advanced solid tumors. Treatment with DENSPM is given as a 15-minute IV infusion at doses of 60, 80, and 100 mg/m^"7day (free base) QD for 5 consecutive days repeated every 3 weeks. Interferon is administered SC 3 times a week at a dose of 10 MU. A typical dosing schedule is shown in Table 5 below. A minimum of 3 patients will be entered at each dose level of DENSPM. Escalation of the dose of DENSPM to higher dose levels requires 3 patients at the current dose level to have completed one full course (21 days) without a DLT. If one of the first 3 patients experiences a DLT, 3 more patients will be enrolled at that dose level (for a total of 6 patients) before escalation to the next dose level. Patients who do not complete a course of therapy for reasons other than a DLT will be replaced in order to achieve a full 3 or 6 patients per cohort. Once the MTD has been identified (DLT >2 patients at any dose level), additional patients will be entered at the previous (proposed Phase 2) dose level. Up to 10 additional patients may be entered at the dose proposed for Phase 2 studies. TABLE 5

Dosmg Schedule for DENSPM and IFN

DENSPM U UUU uuuuu

IFN (SC) u u u u u u u u u u u u u u u

Days on D1 D8 D15 D22 D29 D36 D43

Which doses are administered

STUDY POPULATION

Patient-Selection Criteria

Inclusion Cπteπa The inclusion criteria are as follows:

• Male or female at least 18 years of age;

• Women of childbearing potential are eligible, but must not be breast-feeding, must have a negative pregnancy test, and must be using a reliable method of contraception;

• Histologically confirmed and documented diagnosis of either renal cell carcinoma, melanoma, or any other solid tumor which is refractory to standard therapy or for which standard therapy does not exist; • Patients must have received <2 prior cytotoxic chemotherapy regimens (in addition to any adjuvant treatment);

• Kamofsky Performance of 60 (See Appendix B.l), determined within 1 week prior to first DENSPM treatment;

• Expected survival >9 weeks; and • Capable of giving written informed consent.

These cπteπa are mandatory and must be met to provide evaluable data. Exclusion Criteria

Presence of any of the following at baseline (within 1 week prior to initial dose) excludes a patient from entering the study until such condition is resolved:

Laboratory Abnormalities • Creatinine clearance <60 mL/min (by Cockcroft-Gault equation see

Appendix C)

- Total bilirubin >ULN,

- SGPT or SGOT >ULN.

- Absolute neutrophil count <1500/μL. and - Platelet count <100.000/μL.

• Treatment with any other investigational drugs within 4 weeks prior to first dose:

• Radiation therapy or cytotoxic chemotherapy within 3 weeks (6 weeks for nitrosoureas or mitomycin C) prior to first dose; • Hormone therapy, immunotherapy, or other biologic therapy within 2 weeks prior to first dose;

• Concurrent serious infection;

• Life-threatening illness (unrelated to tumor); and

• Symptomatic or suspected brain metastases.

NOTE: Patients with documented brain metastases are not excluded if:

1. They have had a surgical excision of the lesion(s) or have completed a course of cranial irradiation;

2. They have discontinued or have been on stable doses of corticosteroids for at least 1 month; and 3. They are currently asymptomatic (may have a residual but stable central nervous system [CNS] finding). ProhibiteαV Allowable Medications or Precautions

Once a patient begins DENSPM treatment, the addition of mvestigational agents or other cancer treatments will confound the assessment of safety and efficacy and is therefore not allowed. This restπction precludes the addition of cytotoxic, hormonal, lmmunologic, or biologic agents, or radiotherapy

(therapeutic or palliative) while the patient continues on this protocol.

In order to ameliorate the "flu-like" syndrome produced by IFN, acetaminophen 650 mg should be admimstered 4 hours pnor to and at the time of the initial IFN dose. MegaceD (megestrol) used as an appetite stimulant is allowed.

Antiemetics may be used at the investigator's discretion for prevention and/or treatment of nausea or vomiting. DENSPM is mildly to moderately emetogenic and premedication for nausea and vomiting is recommended

STUDY METHODOLOGY If a patient has measurable disease and if a computed axial tomography

(CAT) scan or a magnetic resonance imaging (MRI) scan is being used to document measurable disease at baseline, the baseline scan must be done withm 3 weeks pnor to first study treatment. Kidney, liver, and marrow function tests must be obtained withm 1 week pnor to first study treatment.

Safety Assessments

All patients receiving any amount of study drug will be evaluated for safety The safety parameters include all laboratory abnormalities, physical examination findings, and spontaneous reports of adverse events repotted to the investigator by patients.

Dose-Limitmg Toxicity

Any of the following conditions are considered to be dose-hmitmg toxicities (DLT)

• Treatment-related CNS adverse event of Grade 2 or higher: and • Any treatment-related non-hematologic adverse event of Grade 3 or higher (does not apply to alopecia, Grade 3 nausea and vomiting m the absence of antiemetics)

• Grade 3 anemia, Grade 4 thrombocytopenia or neutropema

Maximum-Tolerated Dose

The MTD is that dose level which produces a DLT in 2 patients at any dose level or the attainment of a DENSPM dose level of 100 mg/m^, whichever occurs first

Efficacy Parameter(s) Efficacy is the achievement of an objective response to treatment, defined as either a CR or PR

In patients who have measurable disease at baseline, investigators should identify one or more (up to 3) bidimensionally measurable lesιon(s) withm each patient Dunng the treatment phase, investigators must re-assess and measure these same disease sites, using the same methods, after the completion of every other course of DENSPM treatment.

Other Efficacy Parameter(s)

Record the following events related to the medical condition being studied as efficacy parameters

Time to Objective Response

Time to objective response among responders is the interval (number of days) between the first day of study treatment and the start of the confirmed PR or CR

Duration of Objective Response Duration of objective response among responders is the interval (number of days) between the start of the confirmed PR or CR and the first date that progression occurs Time to Progression

Time to progression is the interval (number of days) between the first day of study treatment and the first date that progression occurs

Clinical Benefit Clinical benefit will be determined by improvement m performance status, improvement in disease related symptoms, reduction m narcotic analgesic requirements, mixed objective tumor response (reduction in size of some lesions without progression at any other site), etc Such benefits must be considered to be related to the administration of study drugs Evidence of such benefit will be based upon clinical judgment of the attending physician

Continuation of Treatment

Based on the patient safety and disease assessment, the investigator must determine whether to continue study treatment according to the following categones

Patients With Objective Response

Patients who achieve a PR will continue to receive DENSPM and IFN treatment indefinitely until a reason for withdrawal occurs

Patients who achieve a CR will continue to receive DENSPM and IFN treatment until the patient completes 2 courses of treatment beyond the confirmation of the CR or another reason for withdrawal occurs Such patients remain on study and must be evaluated for disease status every 6 weeks

Patients With Stable Disease

Patients who do not meet the cntena for either progression or objective response are considered to have stable disease They may remain on therapy

Patients With Progressive Disease

Patients who develop rapidly progressing disease, new lesions, and/or clinical detenoration may be withdrawn from the study Patients who require palliative radiotherapy to new or progressive tumor lesions are considered to have progressive disease and should be withdrawn from the study Withdrawal of Patients From Study Treatment Phase Make every effort withm the bounds of safety and patient choice to have each patient complete the study Patients are considered to have completed the treatment phase of the study when any of the following occur

• Patient has completed 2 courses of treatment after a complete response;

• Disease progression,

• Unmanageable adverse event;

• Patient refuses further therapy; and

• Investigator concludes that it is in the patient's best interest to discontinue therapy (e g , poor patient tolerance, poor compliance with protocol momtonng, etc)

Clearly indicate on the appropnate CRF the reason for withdrawal from the study treatment phase

Study Completion The study is completed when the MTD is identified or safety is demonstrated at the 1 OO-mg/m^ dose level for DENSPM and all patients have completed smdy treatment and a minimum follow-up penod of 1 month When this occurs, a data cut-off date will be established by Parke-Davis; all CRFs are to be completed up to that date and retneved by Parke-Davis. In addition, Parke- Davis reserves the nght to terminate this study for any reason at any time. Clearly indicate on the appropnate CRF the completion of the study by a patient

Follow-Up Data Requirements

When a patient discontinues study medication, an end-of- treatment evaluation of disease and safety parameters must be conducted See Appendix A 1 for the tests and procedures that are required at this time.

The end-of-Treatment evaluation is followed by a 30-day follow-up penod to monitor the outcome of any abnormal laboratory test results or treatment- related adverse events that were present at the time study drug was terminated, and to observe for any delayed adverse events or patient death At the end of the 30-day follow-up period, record any new adverse events that occurred during this period. No further reporting of new adverse events is required after the initiation of any subsequent chemotherapy unless the study drug was considered to have contributed to the new adverse event.

STUDY MEDICATION

EFN Dosing

Interferon-α 2b (Intron-Schering) will be supplied by the site and is administered SC 3 times a week in a dose of 10 MU per day. See package insert for safety precautions and adverse event profile.

DENSPM Dosing

The dose of DENSPM in the initial treatment course is 60 mg m^/day. After 3 patients have been entered at this level, and provided that no dose-limiting toxicities are observed, 3 more patients each will be entered into the next dose levels of 80 and 100 mg m^. The appropriate dose should be withdrawn from the vial and further diluted in 50 mL of normal saline and infused IV over 15 minutes into a peripheral vein once a day for 5 consecutive days. Subsequent courses are to be administered every 3 weeks (21 days after the start of the prior course, e.g.. Days 1-5, 22-26, and 43-47, etc).

Treatment Courses A course of treatment is 21 days long. The administration of DENSPM is to be completed over 5 consecutive days. Every effort should be made to deliver a 5-day course of treatment. If treatment with DENSPM is interrupted, either because of an adverse event or for another reason, that treatment course should be resumed, if medically appropriate, as long as it can be completed within a total of 7 days. If it cannot be completed within a total of 7 days, that treatment course should be terminated. The patient should begin their new course of DENSPM no sooner than 21 days after having started the prior course, once the conditions for recover/ have been satisfied. Conditions for Recovery

Nonhematologic toxicities have recovered to either Grade 0 or 1 (or to baseline for the patient).

Treatment Delay If recovery has not occurred by the scheduled start of the next course of

DENSPM, the subsequent course of DENSPM should be delayed by weekly intervals until these conditions have been met. The IFN should be continued during this time.

Dosage Reductions The premise of this combination trial is to explore the safety of escalating doses of DENSPM in combination with a fixed dose of IFN. Therefore, every attempt should be made to maintain the dose of IFN at 10 MU.

DENSPM Dose Reduction

The daily DENSPM dose in the patient's next course must be reduced to the next lower dose level if the prior course was associated with dose-limiting toxicity. Patients who require a dose reduction for dose-limiting toxicity may not subsequently be re-treated at higher doses.

Interferon Dose Reduction

If necessary, the dose of IFN may be reduced to 5 MU in patients who experience dose-limiting toxicities at their current dose. Interferon dosing may also be interrupted completely until side-effects have abated. The dose of IFN should be escalated back to 10 MU as soon as clinically possible.

Preparation and Stability

Interferon-α 2b (Intron-Schering) will be supplied from commercial sources.

DENSPM is supplied as a lyophilized powder packaged in 10-mL clear glass vials containing 150 mg of free base. The dosage form is physically and chemically stable when stored at room temperature. Stability programs are ongoing. The "use before date" may be extended as additional long-term stability data becomes available.

When reconstituted with 5 mL of Water for Injection (USP), the resulting solution contains 30 mg/mL of DENSPM (free base) and is chemically and physically stable for 96 hours at room temperature.

When further diluted in 50 mL normal saline, the resulting admixture is chemically and physically stable for up to 24 hours when stored at room temperamre (data not available for refrigeration). After reconstitution, drug should be used within 8 hours since it contains no preservative. Compatibility studies were performed only with normal saline in polyvinyl chloride (PVC) containers, connected to a PVC administration set, over a concentration range of 0.1 to 10 mg'mL. Therefore, only this IV system may be used for infusions of DENSPM.

DATA ANALYSIS AND STATISTICAL CONSIDERATIONS

Efficacy Analysis

All efficacy data will be reviewed by the Sponsor to ensure that evaluability and response determinations meet protocol definitions.

To be considered evaluable for response, a patient must have completed one full course of DENSPM treatment with a response assessment made by the investigator.

The percentage of patients attaining a CR or PR will be noted. Time to response and duration of response will be determined for responding patients.

Time to progression will be determined for all patients. Patients not having the end point for these analyses (i.e., not progressing or lost to follow-up) will be right-censored based on the latest data available. If reasonable, product-limit estimates of time to progression will be calculated.

The Kaplan-Meier estimate and a 95% confidence interval for the median number of days to event will be provided for time to response, duration of response, and time to progression. The number and percentage of patients who demonstrate a clinical benefit will be provided. .Among those patients demonstrating a clinical benefit, the number and percentage for each reason given will be provided. The mean, standard deviation, median, minimum, and maximum number of days of clinical benefit will be provided.

Statistical Power and Sample Size Considerations

Since this is a Phase 1 study, there is no statistical rationale included for sample size determination or for efficacy analyses. It is expected that up to 19 patients will be entered. However, depending on the safety profile observed, it is possible that with expansion or elimination of dose levels, sample size could be larger or smaller than these initial estimates.

The foregoing data establish the synergistic antineoplastic activity of DENSPM together with IFN-α or interleukin. The agents are available individually, or can be packaged together in a kit, which is a further embodiment of the invention. The kit will have 2 compartments (or more for multiple doses), wherein DENSPM is presented in one compartment, and either IFN-α or interleukin is presented in the second compartment. The kits thus provided facility dosing at the site of patient treatment.

Claims

What is claimed is

1 An antineoplastic combination compnsmg diethylnorspermine and a protein selected from mterferon-α and interleukin, wherein said components are present m amounts which are effective to treat a neoplastic disease

2. A combination compnsmg diethylnorspermine and mterferon-a 2a.

3 A combination compnsmg diethylnorspermine and interferon-α 2b

4 A combination compnsmg diethylnorspermine and interleukin

5 A method for treating a patient suffenng from a neoplastic disease compnsmg administenng antineoplastic effective amounts of diethylnorspermine and a protein selected from mterferon-a and interleukin

6 A method according to Claim 5 compnsmg administenng diethylnorspermine and interferon-α 2a.

7 A method according to Claim 5 compnsmg admimstenng diethylnorspermine and mterferon-a 2b

8 A method according to Claim 5 compnsmg admimstenng diethylnorspermine and interleukin

9 A method according to Claim 5 wherein the neoplastic disease is renal cell carcinoma

10. A method according to Claim 5 wherein the neoplastic disease is melanoma.

11. A method according to Claim 5 wherein the diemymorspermine and the protein are administered sequentially.

12. A method according to Claim 5 wherein the diemymorsperrnine and the protein are administered simultaneously.

13. A kit comprising in one compartment an amount of diethylnorspermine, and in a second compartment an amount of a protein selected from interferon-α and interleukin.

14. A kit according to Claim 11 comprising m^thymorspermine and interferon-α.

15. A kit according to Claim 11 comprising diethymorspermine and interleukin.