Doxorubicin (adriamycin) is effective against a broad spectrum. of tumors, including acute leukemias and iraligiant'lyn homas. ^* It is also present as one of the ingredients in the multiple drug therapy of non-Hodgkin's lymphomas, carcinomas of the breast and small cell carcinomas and is particularly beneficial in such therapy against a wide variety of sarcomas including osteogenic, Ewing's and soft tissue. It is one of the most active onoolytic agents for treatment of metastatic aό enocarcinomas of the breast, carcinoma of the bladder, bronchogenic carcinoma and neuroblastoma. The activity against solid tumors coupled with good activity against leukemias and lymphomas makes doxorubicin a unique drug. It would probably be the most widely used onoolytic agent today were it not for its toxicity, which tcocicity is unrelated to its onoolytic activity. The chief side effect which limits the utility of doxorubicin is serious, irreversible myocardial toxicity with delayed congestive heart failure often unresponsive to any cardiac supportive therapy. The cardiotcocicity is apparently cumulative.

A great deal of research has been devoted to finding ways to decrease the cardiotoxicity of doxorubicin. The chief strategy so far has been to modify the doxorubicin structure chemically. The . result of such experimental work, however, has not yielded any compound with improved therapeutic ratio sufficient to replace doxorubicin

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in the clinic. As a consequence, doxorubicin is chiefly employed as one of several drugs used in a shot gun or multiple drug therapy where it can be administered at drug levels well below the cardio- toxic dose. „-

The synthesis and structure of a group of di- and trihdroxy- benzohydroxamic acids are disclosed in among other publications [Elford et al., r-τt. Rgs. _f 21, 844 (1979), yan't Riet et al., J. Hed. Qiem., 22,- _. 589 (1979) and Elford et al., Aovanπes in Enzyme Regulation,. 1Q _r 151 (1981)]. _. The compounds show excellent activity against 11210 leukemia. The most active compounds are in general those lacking orthohydroxyl and of these 3,4-dihydroxybenzohydroxamic acid appeared to be the most active. The possibility of combining one of these di- or tri-bydroxybenzohydroxamic acids with another onoolytic agent is not mentioned in the references. In particular, there is nothing in the refernces that indicates thata ribonucleotide reductase inhibitor would be suitable candidate for combination with doxorubicin to provide an antineoplastic therapeutic combination of enhanced activity and lowered toxicity.

It is an object of this invention to provide such therapeutic combinations and a method of treatment of neoplastic disease featuring the administration of doxorubicin at a reduced dose, thereby lessening the cardiotoxic effects with no loss in onoolytic activity.

A second aspect of this invention is the use of the di- and trihydroxybenzohydroxamic acids in combination with cyclophos- phamide to provide an antineoplastic therapeutic combination of enhanced antitum r activity and reduced toxicity.

Cyclophosphamide is the most widely used alkylating agent and probably the most frequently used antitumor drug of any type. The clinical ⁷ spectrum of activity for cyclophosphamide is very broad

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and similar to that of nitrogen mustard. Beneficial results have been observed in acute and chronic lymphoblastic leukemia, Bodgkin's disease, multiple myeloma, bronchogenic carcinoma, carcinoma of the breast and cervix, ovarian malignancies, as well as in neuroblastoma, retinoblastoma and other neoplasms of childhood. Cyclophosphamide is an essential component of many effective drug combinations. It is commonly used in combination with doxorubicin, vincristine, and predniεone. One frequently used combination is with methotrexate and flurouracil as adjuvant therapy after surgery for carcinoma of the breast. The most commonly encountered toxicity is that of depression of the blood leucocyte count.

A unique and potentially serious toxicity that occurs in 10-20% of patients is sterile cystitis. The hematuria may be very severe and, in some instances, fatal. The bladder damage appears to be due to excretion of toxic metabolities into the bladder. At high doses of cyclophosphamide, cardiac damage may be seen. Also cyclophosphamide is carcinogenic in animals, and the increasing number of reports of new tumors in patients treated with the drug make it likely that this complication occurs in man.

Pibonucleotide reductase inhibitors, particularly hydroxyurea, have been incorporated into combination chemotherapy of £1210 mice leukemia with platinum compounds and cyclophosphamide and are disclosed in other publications [Gale et. al., J. Natl. Cancer Tnst..57. 1363 (1976), Gale et. al., Cancer Treat. Rep..61 , 445 (1977), Gale et. al.. Cancer, 41, 1230 (1978) , and Gale et. al. Cancer Treat. Rep.. £3_, 449 (1979)]. The three-drug regimens were very effective and resulted in a high percentage of cures. However, there was only a very modest synergism and a rare cure (a 60 day survivor), when only cyclophosphamide and hydroxyurea were used in combination. The highest cure rate was

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20% and only occured in 4 of 13 experiments [Gale et. al., (1977 and 1978)]. When other ribonudeotide reductase inhibitors, quanazole and 4-methyl-5-amino-l-formyliεoquinoline thiosemicarbazone as well as hydroxyurea, were tested in a two-drug regimen involving cyclophos¬ phamide, all three reductase combinations again resulted in a modest synergistic effect; however no cures were reported in any regimens.

Afurther objective of this invention is toprovide therapeatic combinations of polyφdroxybenzc-hyόxoxamic acids and cyclophosphos— phamide and a method of treatment of neoplastic disease featuring the administration of cyclophosphamide at a reduced dose, thereby reducing the toxic side effects (including cystitis, cardiac damage and the potential for carcinogenic transformation) with no loss of onoolytic activity.

DESCRIPTION OF THE INVENTION In fulfillment of the above and other objects, this invention provides a combination of do ^" xoτub1 cin and a hydroxybenzohydroxamic acid of the formula:

wherein one of R and Rr is OH and the other is H or OH.

Hydroxybenzohydroxamic acids represented by the above formula include 3,4-dihydroxybenzohydroxamic acid, 3,5-dihydroxybenzohydroxamic acid and 3,4,5-trihydroxybenzbhydroxamic acid.

Doxorubicin and a hydroxybenzohydroxamic acid of formula I are administered intravenously to the patient suffering from neoplastic disease. Vials containing a lyophilized mixture of doxorubicin. and a hydroxybenzohydroxamic acid are reconstituted with preferably isotonic saline solution USP containing 0.9« sodium chloride. Doxorubicin is usually present as the hydrochloride salt. Sterile water may also be used to reconstitute the lyophilized mixture. The reconstituted solut ns are then administered to the patient. The usual dosace level for doxorubicin hydrochloride is 60-75 mg./m or 100-130 g. per 75 kg. (average weight for humans). These dosage levels are equivalent to dosages of 1.33-1.73 g./kg of mammalian body weight. The hydroxybenzohydroxamic acid according to formula I above are administered at a rate of 12-26 mg./kg. of mammalian

2 body weight or 900-1950 mg. per patient: 530-1150 mg./m .

Ordinarily, doxorubicin hydrochloride is administered

2 at the higher dose levels 60-75 mg./m only once every 21 days although an alternate regimen is available in which half the dosage is given on three successive days, followed by a 3 week waiting period. The benzohydroxarr.ic acids of formula I are relatively nontoxic and can be administered daily, if desired. In our novel therapeutic method, dose levels of doxorubicin

20-60% of the usual therapeutic.dose can be employed ^' (12-15 to 36-45 mg./m ; 20-75 mg. per 75 kg. person) coupled with 12-26 mg./kg. or 530-1150 mg./m of the beπzohydroxaπic acid of formula I. It is an advantage of our novel combination of oncolytϊc drugs that, since the doxorubicin hydrochloride can be administered in our combination in far lower doses (as low as 20S of the usual dose) with equal therapeutic effect, it can also be administered more often without any increase in cumulative toxicity. As will be demonstrated below, it is possible in a therapeutic combination to administer as little as .27 mg./kg. or

12.mg./m 2 up to 1.0 mg./kg. of doxorubicin hydrochloride accompanied by 12-26 mg./kg. of a hydroxybenzohydroxamic acid of formula I above with comparable therapeutic results.

For intravenous administration a lyophilized vial containing 50 mg. of doxorubicin hydrochloride can be reconstituted v/ith 25 ml. of isotonic saline and from 500-1500 mg. of a benzhydroxamic acid dissolved therein. Naturally, if . more isotonic saline is required ^' o give a clear solution at the higher levels of benzohydroxamic acid, more saline can be added without affecting the efficacy of the treatment. Alternatively, the doxorubicin hydrochloride and hydroxybenzohydroxamic acid of formula I can be dissolved in v/ater and lyophilized. The vials

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containing the combined lyophilized solids are then reconstituted with normal saline or sterile water to give the desired solution ready for intravenous injection.

After administering the combination of doxorubicin and a hydroxybenzohydroxamic acid of formula I once every three weeks or at lower doses on successive.days, it is usual to follow this combined dosace by administration of the benzohydroxamic acid alone at the same dose level daily or on alternate days for a brief period of time. Because of its lower toxicity, it is possible to administer far larger quantities of the benzohydroxamic acid by itself v/hile doxorubicin hydrochloride is administered far less frequently. The entire regimen can be repeated every three weeks provided toxic levels of doxorubicin are not found.

As evidence of the efficacy of our novel therapeutic method, the combination of drugs were administered to mice bearing a transplanted tumor, L-1210 leukemia. Synergistic results were obtained by the combined therapy of doxorubicin and hydroxybenzohydroxamic acid, specifically 3,4-dihydroxy¬ benzohydroxamic acid, in this experiment, as set forth below in Tables I and II. In this procedure, L-1210 leukemia was

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maintained by weekly passage of 10 L-1210 cells intraperitoneally into g

DBA/2 mice. Diluted ascitic fluid, 0.1 ml. (10 cells), was adminis¬ tered p to female B6D2F. mice weighing about 20 g. Drugs were administered intraperitoneally to the animals. Injections containing doxorubicin were given only on day 2 and the 3,4-dihydroxybenzo- hydroxamic acid was given on days 2, 3 and 4.

In the Tables, column 1 gives the medication, either a control group receiving nothing, doxorubicin hydrochloride by itself, 3,4- hydroxybenzohydroxamic acid by itself or various combinations of the two drugs, column 2 gives the dosage in mg./kg., column 3 the mean life span, column 4 the percent increase in life span over controls and column 5 the percent cures.

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. TABLE 1

Percent

Mean . Increase

Dosage Life In Life

Medication Mg./Kg. Percent

Span Span Cures*

Control — 10.9 — 0

doxorubicin hydrochloride plus

3,4-dihydroxy- fcnnzohydroxaπric acid 6 + 430 28.25 159 25

17 + 430 3S.0 258 25

29 ÷ 430 8.5 -22 O

6 + 645 31.25 187 25

17 + 645 24.0 120 0

2S + 645 11.5 6 O

* Survivors calculated at day 60 **Four mice per group

5 U STITUTE H

TA3LE II

• Percent

Mean ^• Increase

• Dosage . . Life . In Life Percent

Kεdication Hq./ g. - ^■ .Span Span Cures*

Control** — 9.1 — 0

Doxorubicin HC1 6 13.3 46

* 0

17 16 76 0

29 26.3 ^' 189 33

3,4-dihydroxy- 430 12 32 0 benzoπydroxE ic 645 13.6 49 0 acid -

Doxorubicin hydro¬ 6 + 430 13.7 51 0 chloride plus 3,4- 17 + 430 32.7 259 67 dihydrαxybeπ∑o- 29 ÷ ^' 430 33.7 270 . 67 hydroxEmic acid ^• 6 + 645 32 252 67-

17 + 645 ^' 27.7 204 33

29 ÷ 645 9.7 7 G

* Survivors calculated on day 35 ** Three mice per group

As can be seen from the above Tables, 3, -dihydroxy¬ benzohydroxamic acid at any dose level gives very little increase in life span and in one experiment (Table I - 645 mg./kg.) life span v/as no better than control. Doxorubicin by itself gave substantial increase in life span with one s ^vivor at the highest dose level tested. However, combinat =.£ of lower levels of doxorubicin with 275 or 430 mg./kg. of 3,4-dihydroxybenzo¬ hydroxamic acid gave substantial increases in life span plus survivors when compared with the dose level of doxorubicin by itself or of 3,4-dihydroxybeπzohydroxamic acid by itself. 'T . increaε- life spans tended to fall off at the higher dose levels for do*.-"ubicin indicating toxicity. The data clearly indicates that doxorubicin hydrochloride can be administered at dose levels as low as 202 of the usual dose level (6 mg. vs. 29 mg.) with varying amounts of 3,4-dihydroxybenzohydroxamic acids with no loss in efficacy vs. L-1210 leukemia.

Doxorubicin derivatives with comparable antitumor effect and toxicity can be employed in place of doxorubicin in our novel methods and formulations — see chapter by Arcarnone in Anticancer Agents Based on Natural Product Models (Ed. Cassady and Douros, Academia Press 1980 - New York) for lists of such derivatives.

SUBSTITUTE SHEET

Thls invention also provides a combination of cyclophosphamide and a hydoxybenzohydroxamic acid of the formula:

CONHOH

wherein one of R and R ¹ is OH and the other is H or OH.

.Hydroxybenzohydroxamic.acids represented by the above formula include3,4-dihydroxybenzohydroxamic acid, 3,5-dihydroxybenzohydroxamic acid and 3,4,5-trihydroxybenzohydroxamic acid.

Cyclophosphamide and a polyhydroxybenzohydroxamic acid of formula I are administered intravenously to the patient Buffering from neoplastic disease. Vials containing a lyophilized mixture of cyclophosphamide and a hydroxybenzohydroxamic acid are reconstituted with preferably isotonic saline solution OSP containing 0.9% sodium chloride. Sterile water may also be used to reconstitute the lyophilized mixture. The reconstituted solutions are then administered to the patient. The usual dosage level for cyclophosphamide is 600-1200 mg./m ² or 1215-2430 mg. per 75 kg. (average weight for humans). These dosage levels are equivalent to dosages of 16.2-32.4 mg kg. of mammalian body weight. The hydroxybenzohydroxamic acids according to formula I above are administered at a rate of 12-26 mg/kg of mammalian body weight or 900-1950 mg. per patient; 530-1150 mg/πr%

Inour novel therapeuticmethod, doselevelsofcyclophosphamide at 20-50% of the usual therapeutic dose can be employed (120-240 to 300-600 mg./m ² ; 243-486 to 608-1215 mg. per 75 kg. person) coupled

SHEET

with 12-26 g. g _* or 530-1150 mg/m ² of the benzohydroxamic acid of formula I. It is an advantage of our novel combination of onoolytic drugs, that since the cyclophosphamide can be administered in our combination in far lower doses (as low as 20% of the usual dose) with equal therapeutic effect, it can also be administered more often without any increase in cumulative toxicity. As will be demonstrated later, it is possible in a therapeutic combination to a&niniεter as little as 20% of the usual therapeutic dose of cyclophosphamide (120-240 mg./m ² ) accompanied by 26 mg./kg. of a hydroxybenzohydroxamic acid of formula I above and obtain comparable therapeutic results.

For intravenous administration, a lyophilized vial containing 100 mg. of cyclophosphamide can be reconstituted with 25 ml. of isotonic saline and from 500-1500 mg. of a benzohydroxamic acid dissolved therein. Naturally, if more isotonic saline is required to give a clear solution at the higher levels of benzohydroxamic acid, more saline can be added without affecting the efficacy of the treatment. Alternatively, the cyclophosphamide and hydroxybenzohydroxamic acid of formula I can be dissolved in water and lyophilized. The vials containing the combined lyophilized solids are then reconstituted with normal saline or sterile water to give the desired solution ready for intravenous injection.

As evidence of the efficacy of our novel therapeutic method, the combination of drugs were a&ninistered to mice bearing a transplanted tumor, L1210 leukemia. Synergistic results were obtained by the combined therapy of cyclophosphamide and hydroxybenzohydroxamicacid, specifically 3,4-dihydroxybenzohydroxamic acid, in this experiment, as set forth below in Tables III and IV. In this procedure, LI210 leukemia was maintained by weekly passage of 10 ⁵ 1-1210 cells intraperitoneally

o INSTITUTE SHEET

into DBA/2 mice. Diluted aεcitic fluid, 0.1 ml. (10 ⁵ cells), was administered i.p. to female BδDSLF^ mice weighing about 20 g. Drugs were administered intraperitoneally to the animals. Injections of drugs were given only on day 2.

In the Tables, column 1 gives the medication, either a control group receiving nothing, cyclophosphamide by itself, 3,4-dihydroxybenzo- hydroxamic acid by itself or various combinations of the two drugs, column 2 gives the dosage in mg./kg-r column 3 the mean life span, column "*the percent increase in life span over controls and column 5 the percent cures.

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TABLE III

Percent

,. Mean Increase

Dosage Life In Life Percent

Medication }5- _2_ 2r_- Span Cures* ,

Control — 7.75 — 0

Cyclophos¬ phamide 71.5 10.5 35 0

3,4-dihydroxy benzohydroxamic acid 493 9.25 19 0

Cyclophos¬ phamide plus 3 ,4-dihydroxy- 71.5 . acid 493 60* 675* 100

2.5 x 10 ⁵ L1210 cells i.p. Three mice per group Cyclophosphamide given i.p. on day 2 3,4-dihydroxybenzohydroxamic acid given i.p. only on day 2

^♦ Survivors are calculated as day 60

SUBSTITUTE SHEET

TftBT.fi TV

Percent

Mean Increase Dosage Life In Life Percent

Medication JS- ∑-M. . .Span Span Cu r s *

Control 8.25

Cyclophos¬ 67.5 10.5 27 0 phamide 305 48.0 482 100

3, -dihydroxy benzohydroxa ic acid 494 9.2 12 0

Cyclophos¬ phamide plus 3,4-dihydroxy- 67.5 benzohydroxamic + acid 494 48.0 482 100

2.5 x 10 ⁵ L1210 cells i.p. Four mice per group Cyclophosphamide given i.p. on day 2 3,4-dihydroxybenzohydroxamic acid given i.p. only on day 2

^♦ Survivors are calculated as day 48

As can be seen from the above Tables, 3,4-dihydroxybenzo¬ hydroxamic acid gave very little increase in life span. Cyclophosphamide by itself at 67.7 or 71.5 mg.Ag« also resulted in only a very modest increase in life span of 27 and 35%, respectively. However, when both agents were administered, substantial increases in life span resulted. In these two experiements, the combination treatmentproduced cures in all the animals. This very εynergeεtic result was much greater than expected on past results with other ribonucleotide reductase inhibitors. Toachieveacomparableeffectiveresultwithcyclophosphamide alone required a dose of cyclophosphamide almost 5 times greater (Table

//

JV.).

ΓOREΛ ^* I

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