WO2001007914A1

WO2001007914A1 - Fenretinide increases antibody cellular toxicity

Info

Publication number: WO2001007914A1
Application number: PCT/US2000/019838
Authority: WO
Inventors: Robert C. Seeger
Original assignee: Childrens Hospital Los Angeles; Childrens Hospital Los Angeles Research Institute
Current assignee: Childrens Hospital Los Angeles; Childrens Hospital Los Angeles Research Institute
Priority date: 1999-07-26
Filing date: 2000-07-21
Publication date: 2001-02-01
Anticipated expiration: 2002-01-26
Also published as: AU6227400A

Abstract

A method of treating a hyperproliferative disorder in a subject in need of such treatment is disclosed. The method comprises administering to the subject, in combination, a treatment effective amount of: (a) an immunocytokine that specifically binds to hyperproliferative cells (e.g., cancer cells) in said patient; and (b) a retinoid or a pharmaceutically acceptable salt thereof. The retinoid and the immunocytokine are preferably given in amounts that achieve a cytotoxic or cytostatic effect on the cells to which the immunocytokine specifically binds that is at least as great, and preferably greater than, the sum of the cytotoxic or cytostatic effect achieved by the same amounts of the compounds when given separately. Preferably, a synergistic amount of the two compounds is administered. Methods of treating infectious agents are also disclosed.

Description

FENRETINIDE INCREASES ANTIBODY CELLULAR TOXICITY

Related Applications

This application claims priority from United States Provisional Application Serial No. 60/145,580, filed July 26, 1999, the disclosure of which is incorporated by reference herein in its entirety.

Field of the Invention

The present invention concerns combination chemotherapy treatments for disorders such as cancer.

Background of the Invention

Fenretinide [HPR; all-trøns-N-(4-hydroxyphenyl)retinamide; CAS Registry number 65646-68-6] is known to have certain antitumor properties. See, e.g., D. Delia et al., Carcinogenesis 18, 943-948 (1997); N. Oridate et al., J. Natl. Cancer Inst. 89, 1191-1198 (1997). Fenretinide has been effective both in chemoprevention and chemotherapy of mammary gland tumors in experimental animals, does not accumulate in the liver, and exhibits a mild toxicity.

U.S. Patent No. 4,665,098 to Gibbs describes pharmaceutical compositions of fenretinide as useful for the treatment of breast and bladder cancer.

U.S. Patent No. 5,650,150 to Gillies describes recombinant antibody cytokine fusion proteins, where the fusion proteins are comprised of an immunoglobulin specific for a target cell such as a cancer or virus infected cell, and a cytokine such as lymphotoxin, TNF-α, IL-2, or GM-CSF.

Summary of the Invention A method of treating a hyperproliferative disorder in a subject in need of such treatment is disclosed. The method comprises administering to the subject, in combination, a treatment effective amount of: (a) an immunocytokine that specifically binds to hyperproliferative cells (e.g., cancer cells) in said patient; and (b) a retinoid or a pharmaceutically acceptable salt thereof. The retinoid and the immunocytokine are preferably given in amounts that achieve a cytotoxic or cytostatic effect on the cells to which the immunocytokine specifically binds that is at least as great, and preferably greater than, the sum of the cytotoxic or cytostatic effect achieved by the same amounts of the compounds when given separately. Preferably, a synergistic amount of the two compounds is administered.

Pharmaceutical formulations useful for carrying out the present invention are also disclosed.

Brief Description of the Drawings Figure 1 shows 18 hour antibody dependent cellular cytotoxicity (ADCC) as percent neuroblastoma survival for a first patient. Circles represent neutrophils + neuroblastoma; triangles represent neutrophils + neuroblastoma + hul4.18/GM-CSF.

Plot A represents pre-fenretinide ADCC survival; plot B represents post fenretinide

ADCC survival. Figure 2 shows 18 hour antibody dependent cellular cytotoxicity (ADCC) as percent neuroblastoma survival for a second patient. Circles represent neutrophils + neuroblastoma; triangles represent neutrophils + neuroblastoma + hul4.18/GM-CSF.

ADCC survival. Figure 3 shows 18 hour antibody dependent cellular cytotoxicity (ADCC) as percent neuroblastoma survival for a third patient. Circles represent neutrophils + neuroblastoma; triangles represent neutrophils + neuroblastoma + hul4.18/GM-CSF.

ADCC survival. Figure 4a shows that fenretinide (4-HPR) increases neutrophil-mediated

ADCC against neuroblastoma cells targeted with hul4.18/GM-CSF. SMS-K.CNR neuroblastoma cells were pre-incubated with indicated concentrations of fenretinide for 6 hours.

Figure 4b shows that fenretinide (4-HPR) increases neutrophil-mediated ADCC against neuroblastoma cells targeted with hul4.18/GM-CSF. LA-N-1 and

CHLA-90 neuroblastoma cells were preincubated with fenretinide (10 μM or with its vehicle (0.1% ethanol: V) for 6 hours.

Figures 5a-b shows that fenretinide increases neutrophil mediated ADCC. Figure 5a shows neutrophils from a patient receiving fenretinide 450 mg/m²/day were tested in the morning on day zero before fenretinide. E:T ratios are as graphed; 2000 LA-N-1 target cells pre-labeled with calcein. DIMSCAN analysis to quantify fluorescence remaining in target cells after 18 hours of ADCC. Figure 5b shows neutrophils from the patient of Figure 5 a tested six hours after taking fenretinide. E:T ratios are as graphed; 2000 LA-N-1 target cells pre- labeled with calcein. DIMSCAN analysis to quantify fluorescence remaining in target cells after 18 hours of ADCC.

Detailed Description of the Preferred Embodiments

The treatments employed herein may be used to inhibit growth and/or to induce cytotoxicity (by necrotic or apoptotic mechanisms, or both) in the target cells, which are generally hyperproliferative cells (including tumors, cancers, and neoplastic tissue, along with pre-malignant and non-neoplastic or non-malignant hyperproliferative cells).

Examples of tumors, cancers, and neoplastic tissue that can be treated by the present invention include but are not limited to malignant disorders such as breast cancers; osteosarcomas; angiosarcomas; fϊbrosarcomas and other sarcomas; leukemias; lymphomas; sinus tumors; ovarian, uretal, bladder, prostate and other genitourinary cancers; colon esophageal and stomach cancers and other gastrointestinal cancers; lung cancers; myelomas; pancreatic cancers; liver cancers; kidney cancers; endocrine cancers; skin cancers; and brain or central and peripheral nervous (CNS) system tumors, malignant or benign, including gliomas and neuroblastomas. Examples of premalignant and non-neoplastic or non-malignant hyperproliferative disorders include but are not limited to myelodysplastic disorders; cervical carcinoma-in-situ; familial intestinal polyposes such as Gardner syndrome; oral leukoplakias; histiocytoses; keloids; hemangiomas; hyperproliferative arterial stenosis, inflammatory arthritis; hyperkeratoses and papulosquamous eruptions including arthritis. Also included are viral induced hyperproliferative diseases such as warts and EBV induced disease (i.e., infectious mononucleosis), scar formation, and the like. The methods of treatment disclosed herein may be employed with any subject known or suspected of carrying or at risk of developing a hyperproliferative disorder as defined herein.

As used herein, "treatment" of a hyperproliferative disorder refers to methods of killing, inhibiting or slowing the growth or increase in size of a body or population of hyperproliferative cells or tumor or cancerous growth, reducing hyperproliferative cell numbers, or preventing spread to other anatomic sites, as well as reducing the size of a hyperproliferative growth or numbers of hyperproliferative cells. As used herein,

"treatment" is not necessarily meant to imply cure or complete abolition of hyperproliferative growths. As used herein, a treatment effective amount is an amount effective to result in the killing, the slowing of the rate of growth of hyperproliferative cells, the decrease in size of a body of hyperproliferative cells, and/or the reduction in number of hyperproliferative cells. The potentiating agent (or agents) is included in an amount sufficient to enhance the activity of the first compound, such that the two (or more) compounds together have greater therapeutic efficacy than the individual compounds given alone (e.g., due to synergistic interaction; reduced combined toxicity, etc.).

As used herein, the administration of two or more compounds "in combination" means that the two compounds are administered closely enough in time that the presence of one alters the biological effects of the other. The two compounds may be administered simultaneously (concurrently) or sequentially. Simultaneous administration may be carried out by mixing the compounds prior to administration, or by administering the compounds at the same point in time but at different anatomic sites or using different routes of administration.

The phrases "concurrent administration," "administration in combination," "simultaneous administration" or "administered simultaneously" as used herein, interchangeably mean that the compounds are administered at the same point in time or immediately following one another. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time. Subjects to be treated by the methods of the present invention include both human subjects and animal subjects for veterinary purposes. Animal subjects are preferably mammalian subjects including horses, cows, dogs, cats, rabbits, sheep, and the like. 1. Retinoids.

Retinoids or retinoic acid derivatives that can be used to carry out the present invention include those described in U.S. Patent No. 4,190,594 to Gander (the disclosures of all patent references cited herein are incorporated herein by reference). Such retinoids include retinoic acid or all trans-retinoic acid (ATRA), and retinoic acid derivatives, including but not limited to:

(A) esters of all-trans-retinoic acid having the following formula:

wherein X is a member selected from the group consisting of:

2-cyclohexylethyl; 10-carbomethoxydecyl; 4-hydroxybutyl; cholesteryl; mixed m- and p-vinylbenzyl; and 4-bromobenzyl;

(B) esters of all-trans-retinoic acid having the following formula:

wherein Y is a member selected from the group consisting of: cholesteryloxy; phenyl; 4-bromophenyl; 4-methoxyphenyl; 4-nitrophenyl; 4-hydroxyphenyl; 4-methylphenyl; 4-cyanophenyl; 4-ethoxyphenyl; 4-acetoxyphenyl; 2-naphthyl; 4-biphenyl; 2,5- dimethoxyphenyl; 2,4-dichlorophenyl; 2,4-dimethylphenyl; 3,4-diacetoxyphenyl; 3,4,5-trirnethoxyphenyl; and 2,4,6-trimethylphenyl; and

(C) amides of all-trans-retinoic acid having the following formula:

wherein Z is a member selected from the group consisting of: n-propylamino; tert- butylamino;l,l,3,3-tetramethylbutylamino; l-morpholino; 4-hydroxyphenylamino; 4- carbomethoxy-2-hydroxyphenylamino; beta-(3 ,4-dimethoxyphenyl)-ethylamino; 2- benzothiazolylamino; 1-imidazolyl; l-(2-nicotinoylhydrazolyl); 1-benzotriazolyl; 1- (1,2,4-triazolyl);

Particularly preferred is all-trα«s-N-(4-hydroxyphenyl)retinamide, also called fenretinide, which has CAS registry number 65646-68-6, and has the structure:

The foregoing compounds can be prepared in accordance with known techniques.

See, e.g., U.S. Patent No. 4,190,594 to Gander et al; U.S. Patent No. 4,665,098 to

Gibbs.

Additional retinoids that can be used to carry out the present invention include

C-Glycoside analogs of N-(4-hydroxyphenyl)retinamide-O-glucuronide. Such compounds and their preparation are known and described in U.S. Patents Nos.

5,663,377 and 5,599,953, both to Curley et al, the disclosures of which are incorporated by reference herein in their entirety. Such compounds may have the general formula:

where R is COOH, CH₂OH, or H, and n is 0 or 1.

Specific examples of such compounds include: 4-(retinamido)phenyl-C- glucuronide; 4-(retinamido)phenyl-C-glucoside; 4-(retinamido)phenyl-C-xyloside; 4- (retinamido)benzyl-C-glucuronide; 4-(retinamido)benzyl-C-glucoside; 4-

(retinamido)benzyl-C-xyloside; l-(β-D-glucopyranosyl) retinamide; and 1-(D- glucopyranosyluronosyl) retinamide.

2. Immunocvtokines. In general, an immunocytokine comprises a conjugate of an immunoglobulin and a cytokine. The immunoglobulin is selected to specifically bind (i.e., preferentially bind through the immunoglobulin hypervariable region) to the hyperproliferative cells (e.g., cancer cells) in the subject being treated. While the conjugate may be of any type, covalent conjugates are preferred. Irnmonocytokines are known and described in, for example, U.S. Patent No. 5,650,150 to Gillies, the disclosure of which is incorporated herein by reference in its entirety.

The immunocytokine may comprise any cytokine, including but not limited to interleukins such as interleukin-2 (IL-2), hematopoietic factors such as granulocyte- macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor alpha, lymphokines such as lymphotoxins, etc. IL-2 and GM-CSF are particularly preferred. The cytokine may be of any species of origin, but is preferably of mammalian origin and, in the practice of the present invention for humans, is preferably a human cytokine.

The immunoglobulin in the cytokine may be of any type, but is preferably an Ig heavy chain. The immunoglobulin may be of any species of origin, but is preferably mammalian. The immunoglobulin may be chimeric or humanized (that is, may comprise a recombinant molecule containing human portions and non human portions). The immunoglobulin and cytokine may be conjugated to one another in any suitable manner. For example, an Ig heavy chain and a cytokine which elicits a cytokine-specific biological response by a cell bearing a receptor for said cytokine may be used, said Ig heavy chain comprising an N-terminal variable region specific for a cell bearing a cell surface antigen (and preferably includes the CHI, CH2, and optionally the CH3 domains), said Ig heavy chain being joined at its carboxy-terminus by a peptide bond to the ammo terminal amino acid of the cytokine, as described in U.S. Patent No. 5,650,150 to Gillies.

3. Formulations and administration.

The active compounds described above may be formulated for administration in a single pharmaceutical carrier or in separate pharmaceutical carriers for the treatment of a variety of conditions. In the manufacture of a pharmaceutical formulation according to the invention, the active compounds including the physiologically acceptable salts thereof, or the acid derivatives of either thereof are typically admixed with, inter alia, an acceptable carrier. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the patient. The carrier may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose formulation, for example, a tablet, which may contain from 0.5% to 95% by weight of the active compound. One or more active compounds may be incorporated in the formulations of the invention, which may be prepared by any of the well known techniques of pharmacy consisting essentially of admixing the components, optionally including one or more accessory ingredients. The formulations of the invention include those suitable for oral, rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), topical (i.e., both skin and mucosal surfaces, including airway surfaces) and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular active compound which is being used.

Formulations suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association the active compound and a suitable carrier (which may contain one or more accessory ingredients as noted above). In general, the formulations of the invention are prepared by uniformly and intimately admixing the active compound with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture. For example, a tablet may be prepared by compressing or molding a powder or granules containing the active compound, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the compound in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Molded tablets may be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder.

Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the active compound in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin or sucrose and acacia. Formulations of the present invention suitable for parenteral or vaginal administration conveniently comprise sterile aqueous preparations of the active compound, which preparations are preferably isotonic with the blood of the intended recipient. These preparations may be administered by means of subcutaneous, intravenous, intramuscular, or intradermal injection. Such preparations may conveniently be prepared by admixing the compound with water or a glycine buffer and rendering the resulting solution sterile and isotonic with the blood.

Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include Vaseline, lanolin, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.

Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3 (6):318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound. Suitable formulations comprise citrate or bisVtris buffer (pH 6) or ethano I/water and contain from 0.1 to 0.2M active ingredient.

As noted above, the present invention provides pharmaceutical formulations comprising the active compounds (including the pharmaceutically acceptable salts thereof), in pharmaceutically acceptable carriers for oral, rectal, topical, buccal, parenteral, intramuscular, intradermal, or intravenous, and transdermal administration. The therapeutically effective dosage of any one active agent, the use of which is in the scope of present invention, will vary somewhat from compound to compound, patient to patient, and will depend upon factors such as the condition of the patient and the route of delivery. Such dosages can be determined in accordance with routine pharmacological procedures known to those skilled in the art, particularly in light of the disclosure provided herein.

For fenretinide, for systemic treatment, a dose to achieve a plasma level of about 1, 2, or 3 μM to 10 or 20 μM will be employed; typically (for oral dosing) 50 or 100 to 500 or 1000, 2000 or even 3000 mg/m² body surface area per day.

For the immunocytokine, the agent may be given at a dosage of .1 or .2 to 2, 4, or 8, 12 or 16 mg/m /day, for two, four, six or eight days. More particularly, the agent may be given to adults as a four hour intravenous infusion for four days at a dosage of 2 mg/m /day. For children, the dosage may be 250 μg/m /day for four days. The length of infusion may be increased up to 20 hours if desired.

4. Infectious agents.

While the present invention has been described primarily with reference to the treatment of hyperproliferative disorders, the invention may also be employed for the treatment of subjects infected with infectious agents, such as bacterial, fungal and viral agents with immunocytokines that specifically bind to target infectious agents. Infectious agents that can be treated by the present invention include both gram positive and gram negative bacteria. Examples of bacteria and other infectious agents that can be treated by the present invention include, but are not limited to, Salmonella, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Candida species, etc. Dosages, formulations and routes of administration as described above may be employed for the treatment of such infections.

The present invention is explained in greater detail in the following non- limiting examples.

EXAMPLE 1 Neutrophils from Pediatric Neuroblastoma Patients on Fenretinide Therapy Effectively Eradicate Tumor Cells Neuroblastoma, one of the most common solid childhood tumors, originates from neural crest cells. We investigated the effect of fenretinide during neutrophil mediated antibody dependent cellular cytotoxicity (ADCC) against human neuroblastoma cells. Neutrophils isolated from blood samples of neuroblastoma patients on Fenretinide were isolated and their ability to kill neuroblastoma cells before Fenretinide was administered and 6 hours after Fenretinide was administered were investigated.

Figures 1-3 illustrate the increased kill of tumor cells by neutrophils.

Neuroblastoma survival decreases significantly for all patients in the Post-Fenretinide period (B). To see a progressive increase in cytotoxicity, neutrophils to neuroblastoma ratios of 10:1, 25:1 and 50:1 were plotted. The greatest cytotoxicity is seen in plot (B), with neutrophikneuroblastoma = 50:1, where the neuroblastoma survival decreased to 9% of the control (100% survival). The hul4.18/GM-CSF, provided by Steve Gillies and described in S Gillies et al., Bioconjug. Chem. 4, 230-

235 (1993), is an antibody fusion protein (immunocytokine) which (1) allows the neutrophils to target neuroblastoma cells, and (2) localizes a cytokine, GM-CSF, to the tumor site to trigger neutrophil activity against neuroblastoma. Our experiments demonstrate for the first time that neutrophils are activated by Fenretinide in vivo to kill neuroblastoma cells more effectively in vitro. These date indicate that fenretinide is useful in combination with immunocytokine therapy in the treatment of cancer. Furthermore, we have shown that fenretinide treatment of tumor cells renders them more susceptible to neutrophil killing with the immunocytokine (see Figure 4).

EXAMPLE 2 Neutrophils from patients receiving fenretinide according to a phase 1 protocol who are treated at Children's Hospital Los Angeles have been evaluated with the calcein-DIMSCAN ADCC assay. Data is available for 6 patients and 15 time points (pre and 6 hours post fenretinide); patients received 450, 600 or 800 mg/m²/day of fenretinide. A representative case is shown in Figure 5. Comparing pre and 6 hours post fenretinide ADCC with a nested analysis of variance demonstrated no effect upon cytotoxicity without immunocytokine. For hul4.18 alone, there was a trend for fewer viable cells at 25 and 50:1 effector to target (E:T) ratios. However, for hul4.18/GM-CSF, there were significantly fewer viable cells after fenretinide at an E:T ratio of 50:l (p=0.035). The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

That which is claimed is:

1. A method of treating a hyperproliferative disorder in a subject in need of such treatment, comprising administering to said subject, in combination, a treatment effective amount of: (a) an immunocytokine that specifically binds to hyperproliferative cells in said patient; and

(b) a retinoid or a pharmaceutically acceptable salt thereof.

2. A method according to claim 1, wherein said hyperproliferative cells are cancer cells.

3. A method according to claim 1, wherein said retinoid is selected from the group consisting of fenretinide, retinoic acid and the pharmaceutically acceptable salt thereof.

4. A method according to claim 1 , wherein said immunocytokine comprises a conjugate of an immunoglobulin and a cytokine, wherein said immunoglobulin specifically binds to said hyperproliferative cells.

5. A method according to claim 1, wherein said cytokine is selected from the group consisting of granulocyte-macrophage colony stimulating factor and interleukin-2.

6. A pharmaceutical formulation useful for treating a hyperproliferative disorder in a subject in need of such treatment, said formulation comprising, in combination, a treatment effective amount of:

(a) an immunocytokine that specifically binds to hyperproliferative cells; and

(b) a retinoid or a pharmaceutically acceptable salt thereof.

7. A pharmaceutical formulation according to claim 6, further comprising a pharmaceutically acceptable carrier.

8. A pharmaceutical formulation according to according to claim 6, wherein said retinoid is selected from the group consisting of fenretinide, retinoic acid and the pharmaceutically acceptable salt thereof.

9. A pharmaceutical formulation according to claim 6, wherein said immunocytokine comprises a conjugate of an immunoglobulin and a cytokine, wherein said immunoglobulin specifically binds to said hyperproliferative cells.

10. A pharmaceutical formulation according to claim 6, wherein said cytokine is selected from the group consisting of granulocyte-macrophage colony stimulating factor and interleukin-2.

11. A method of treating an infectious agent in a subject in need of such treatment, comprising administering to said subject, in combination, a treatment effective amount of:

(a) an immunocytokine that specifically binds to said infectious agent in said patient; and

(b) a retinoid or a pharmaceutically acceptable salt thereof.

12. A method according to claim 11, wherein said infectious agent is a bacteria.