Treatment of cancers

A new treatment schedule for administration of N-2-(dimethylamino)ethyl!acridine-4-carboxamide and other related carboxamide anticancer drugs in which the drug is administered in a divided-dose schedule comprising two or more administrations at frequent intervals, for example every hour. Schedules to produce cyclic peaks/troughs in plasma levels are mentioned. The compounds can be used for circumventing multidrug resistance in cancers and may, for example, be used in combination with other cytotoxic drugs, especially non-topo II inhibitors. Treatment of melanoma and advanced colon cancer is included.

The present invention relates to the treatment of tumours, especially to 
the treatment of melanoma and cancer of the colon, and to the 
circumvention of multidrug resistance in cancer treatments. 
BACKGROUND OF THE INVENTION 
For certain types of cancer, chemotherapy has been capable of rendering 
patients with responsive tumours free of disease. However, this responsive 
category does not include the most frequently encountered forms of 
malignant tumours. 
The most common types of cancer in western populations are colon, lung and 
breast cancer. Each of these can be treated to some extent with existing 
chemotherapy, with different drugs being used preferentially for each type 
of malignancy (for instance, doxorubicin, cyclophosphamide and 
methotrexate for breast cancer, 5-fluorouracil for colon cancer), but 
response rates are not good. In addition, melanoma is a disease which is 
increasing in incidence at an alarming rate among fair-skinned 
populations. In melanoma, only 25-30% of patients with disseminated 
disease respond to treatment, and only 5-10% sustain durable remission 
(Evans B. D., et al., Proc. Am. Soc. Clin. Oncol. 1990, 9, 276). 
There is therefore a great need for new types of cancer therapy, and a 
desperate need for such treatments for the above cancers in particular. 
The basis for the development of the majority of anticancer drugs used 
today has been a panel of mouse tumours including transplantable 
leukaemias, the Lewis lung carcinoma and the colon 38 adenocarcinoma. A 
number of human tumour xenografts in mice have also been used. In general, 
the leukaemias are the most sensitive to experimental agents, the 
xenografts are the most resistant and the Lewis lung and colon 38 are of 
intermediate sensitivity (Goldin A., et al., Eur. J. Cancer 1981, 17, 
129-142). 
The murine Lewis lung adenocarcinoma is a tumour which initially arose 
spontaneously in C.sub.57 Bl mice and which has a number of features which 
make it a good model for clinical carcinomas. It grows easily both in 
vitro and in vivo, and is aneuploid, heterogeneous, metastatic and 
resistant to many but not all clinical antitumour agents. Zacharski 
(Zacharski L. R., Haemostasis 1986, 16, 300-320) has concluded that 
although Lewis lung has the cytological appearance of a large-cell cancer, 
its rapid rate of growth, propensity to cause lethal metastases, as well 
as its susceptibility to combination chemotherapy, radiation and 
anticoagulant treatment, make it a good model for human small-cell lung 
cancer (SCLC). The colon 38 tumour arose in carcinogen-treated mice, and 
because it is sensitive to 5-fluorouracil it can be considered as a useful 
model for human colon cancer (Corbett T. H., et al., Cancer Chemother. 
Rep. 1975, 5, 169-186, and Cancer 1977, 40, 2660-2680). Human melanoma 
xenografts have been considered for some time as an appropriate model for 
the development of new anticancer drugs for melanoma (Taetle R., et al., 
Cancer 1987, 60, 1836-1841). 
The essence of treating cancer with cytotoxic anticancer drugs is to 
combine a mechanism of cytotoxicity with a mechanism of selectivity for 
tumour cells over host cells. The selectivity of a drug for a particular 
cancer will depend on the expression by that cancer of properties which 
promote drug action, and which differ from tumour to tumour. 
Currently available cytotoxic drugs can be broadly divided into four 
groups: those which react chemically with DNA (such as the alkylating 
agents and cisplatinum), those which disrupt DNA synthesis (such as the 
anti-metabolites), those which disrupt the mitotic apparatus (such as the 
Vinca alkaloids) and those which are directed against the cellular enzyme 
topoisomerase II ("topo II") in order to effect changes in the topological 
form of the DNA. 
DNA topoisomerases were named after the first method used to detect their 
activity. When incubated with closed circles of double-stranded DNA 
prepared from viruses or bacteria, topoisomerases enzymatically change the 
number of coils contained in each circle (circular forms of DNA with 
different degrees of coiling are called topo-isomers). The topoisomerases 
are perhaps better understood as enzymes which temporarily break one 
strand of the DNA double helix (topoisomerase I or "topo I") or which 
simultaneously break two strands of the DNA double helix ("topo II") in 
order to effect changes in the topological form of the DNA. 
Topoisomerases have two main functions in the cell. The first is to act as 
swivel points on the DNA in association with DNA and RNA polymerases 
during the biosynthesis of nucleic acids required for cell replication and 
gene expression. The second is to untangle the DNA strands of the daughter 
chromosomes following DNA replication prior to cell division. The DNA of 
chromosomes is organised as a series of loops on a protein-aceous 
"scaffold". After duplication of chromosomes and of the "scaffolds", the 
DNA loops must be separated. Since there are hundreds of thousands of DNA 
loops attached to each chromosome scaffold it is not hard to imagine the 
necessity for an enzyme which effectively removes tangles by passing one 
double DNA strand through another. This process absolutely requires topo 
II. 
Topo I acts by transiently breaking a DNA strand and attaching itself to 
one of the free ends of the broken DNA via the amino acid tyrosine. Topo 
II contains two identical protein subunits, each of which is capable of 
breaking a DNA strand and attaching itself to one of the free ends. With 
both DNA strands broken, a second DNA double helix can be allowed to pass 
between the two enzyme protein subunits, thus allowing not only swivelling 
but also untangling of DNA. The process is normally spontaneously 
reversible by cleavage of the enzyme-DNA links and re-sealing of DNA 
breaks to restore the DNA to its original form. 
Topo II-directed agents include a number of important clinical anti-cancer 
drugs such as anthracycline antibiotics (e.g. doxorubicin), 
epipodophyllotoxin derivatives (e.g. etoposide) and synthetic DNA 
intercalating drugs (e.g. amsacrine). These act by jamming the enzyme in 
its DNA-associated form (Liu L. F., Annu. Rev. Biochem. 1989, 58, 
351-375). Such molecular lesions might be expected to be innocuous, since 
the drug eventually dissociates itself from the complex and the DNA strand 
breaks are then repaired perfectly. However, in a small proportion of 
cases, the presence of drug causes the complex to be dissociated 
abnormally, generating some kind of DNA lesion which eventually leads to 
cell death. 
Although the antitumour activity of many of these agents has been known for 
many years, it is only since 1984 that the molecular target of action has 
been identified (Nelson E. M., Tewey K. M., Liu L. F., Proc. Natl. Acad. 
Sci. USA 1984, 81, 1361-1364 and Tewey K. M., et al., Science 1984, 226, 
466-468). 
A number of mechanisms of resistance to topo II poisons have now been 
identified, and in many cases the development of resistance to one drug is 
accompanied by the simultaneous acquisition of resistance to a variety of 
other drugs. Since the mechanism of resistance determines the pattern of 
cross-resistance to other drugs, an understanding of these processes is of 
great importance to the strategy for the use of these agents. Several 
resistance mechanisms important to the use of these agents have now been 
characterised in experimental systems, including those involved in drug 
transport (Endicott J. A., Ling V., Annu. Rev. Biochem 1989, 58, 351-375), 
drug-target interaction (Beck W. T., Biochem, Pharmacol. 1987, 36, 
2879-2888) and drug detoxification (Deffie A. M., et al., Cancer Res. 
1988, 48, 3595-3602). 
Attempts to overcome multidrug resistance (mdr) clinically have been 
concerned mainly with the first of these mechanisms, a drug transport 
mechanism that pumps drug out of cells. Various inhibitors of this 
process, such as verapamil, are known and some have been used in 
combination with drugs such as doxorubicin and etoposide to treat cancer 
(Stewart D. J., Evans W. K., Cancer Treat. Rev. 1989, 16, 1-10, Judson I. 
R., Eur. J. Cancer 1992, 28, 285-289). 
Another approach is to design drugs which can overcome mdr. We have now 
discovered that the investigational drug acridine carboxamide ("DACA") 
appears to be one such drug. 
The compound tested was the dihydrochloride of 
N-2-(dimethylamino)ethyl!acridine-4-carboxamide of formula 
##STR1## 
and is described and claimed in EP 98098. That patent also describes and 
claims other acridine carboxamide compounds and their use for the 
treatment of tumours; more particularly, the treatment of Lewis lung 
tumours and leukaemia is described. 
Various other derivatives of DACA have been tested for their antitumour 
activity and the results are reported in the literature. Active, compounds 
are carboxamides having an unsubstituted or substituted aromatic ring 
system comprising two or more fused rings and having an oxygen or an 
aromatic nitrogen peri to the carboxamide side chain. As well as the other 
acridine carboxamides (EP 98098 and Denny W. A., et al., J. Med. Chem. 
1987; 30: 658-663), examples are phenyl quinoline and pyrido quinoline 
carboxamides (EP 206802 A, Atwell G. J., et al., J. Med. Chem. 1988; 31: 
1048-1052 and Atwell G. J., et al., J. Med. Chem. 1989; 32: 396-401), 
phenazine carboxamides (EP 172744 A and Rewcastle G. W., et al., J. Med. 
Chem. 1987; 30: 843-851), carboxamides having angular tricyclic 
chromophores: phenanthridine carboxamides (NZ Patent 215286, 1986 and 
Atwell G. J., et al., J. Med. Chem. 1988; 31: 774-779) and carboxamides 
having various linear tricyclic chromophores (Palmer B. D., et al., J. 
Med. Chem. 1988; 31: 707-712). These other compounds are structurally very 
similar to DACA and are able to act in the same way. 
DACA is a DNA-binding drug which acts at the same target, topoisomerase II, 
as do drugs such as amsacrine and etoposide. We have now found that it has 
a different in vitro cytotoxicity profile to these compounds and a number 
of advantages over existing clinical drugs in the class of 
topoisomerase-directed agents. 
Firstly, it is active against cell lines displaying both 
P-glycoprotein-mediated or "transport" resistance and "atypical" or 
"altered" multidrug resistance; in this respect it is unique among topo II 
inhibitors. 
DACA and related compounds may therefore be used to circumvent mdr. For 
this it may be used in combination with other cytotoxic drugs, more 
especially non-topo II inhibitors, and/or as a second-line treatment if 
first-line treatment fails because of the development of multidrug 
resistance.

DESCRIPTION OF THE INVENTION 
Accordingly, the present invention provides the use of DACA or other 
aromatic fused-ring carboxamide having an aromatic nitrogen atom or an 
oxygen atom peri to the carboxamide side chain or a physiologically 
tolerable acid addition salt thereof, for the manufacture of a medicament 
for overcoming mdr. 
The present invention further provides a method of overcoming mdr, wherein 
there is administered DACA or other aromatic fused-ring carboxamide having 
an aromatic nitrogen atom or an oxygen atom peri to the carboxamide side 
chain or a physiologically tolerable acid addition salt thereof. 
The present invention further provides a pharmaceutical preparation 
comprising 
(i) DACA or other aromatic fused-ring carboxamide having an aromatic 
nitrogen atom or an oxygen atom peri to the carboxamide side chain or a 
physiologically tolerable acid addition salt thereof, 
and 
(ii) a DNA-reactive agent, a DNA-synthesis inhibitor or an agent which 
disrupts the mitotic apparatus, in admixture or conjunction with a 
pharmaceutically suitable carrier. 
The present invention also provides a combined preparation for use in the 
treatment of cancer, comprising separate components (i) and (ii) above for 
simultaneous or sequential administration. 
Secondly, we have found that, unexpectedly, DACA is effective against 
advanced colon 38 tumours and an advanced melanoma xenograft in mice colon 
when administered in a divided dose schedule over a period of two hours. 
(In this context "advanced tumour" means that the tumour was more than 5 
mm in diameter at the time of measurement.) In contrast, a single 
administration of DACA at the maximum tolerated dose (150 mg/kg), which is 
curative against Lewis lung tumours growing as lung nodules in mice, was 
only marginally effective. 
Thirdly, DACA has the ability to cross the blood brain barrier, suggesting 
that rapidly growing brain tumours may also be treatable, more especially 
when administered in a divided dose schedule. 
Accordingly, the present invention provides the use of DACA or other 
aromatic fused-ring carboxamide having an aromatic nitrogen atom or an 
oxygen atom peri to the carboxamide side chain or a physiologically 
tolerable acid addition salt thereof, for the manufacture of a medicament 
for the treatment of detectable colon cancer, melanoma or brain tumours, 
more especially by administration of a divided dose, the constituent doses 
being administered at frequent intervals. 
The present invention further provides a method for the treatment of 
detectable colon cancer, melanoma or brain tumours, wherein there is 
administered DACA or other aromatic fused-ring carboxamide having an 
aromatic nitrogen atom or an oxygen atom peri to the carboxamide side 
chain or a physiologically tolerable acid addition salt thereof, more 
especially by administration of a divided dose, the oenstituent doses 
being administered at frequent intervals. 
There may, for example, be at least 2 administrations in total in the 
divided dose, administrations being at least every 2 hours, for example 
every hour or every 1/2 hour, for up to 4 hours. 
Thus, the present invention also provides the use of DACA or other aromatic 
fused-ring carboxamide having an aromatic nitrogen atom or an oxygen atom 
peri to the carboxamide side chain or a physiologically tolerable acid 
addition salt thereof, for the manufacture of a divided-dose medicament 
for the treatment of tumours, including melanoma and colon and brain 
tumours, by a treatment regime comprising 2 to 4 administrations of drug 
over a period of up to 4 hours, for example 2 to 4 hours. 
The present invention also provides a method for the treatment of tumours, 
including melanoma and colon tumours, which comprises the administration 
of a divided dose of DACA or other aromatic fused-ring carboxamide having 
an aromatic nitrogen atom or an oxygen atom peri to the carboxamide side 
chain or a physiologically tolerable acid addition salt thereof, 2 to 4 
constituent doses being administered over a period of up to 4 hours, for 
example 2 to 4 hours. 
Fourthly, we believe that suitable DNA-binding compounds will reduce the 
toxicity of DACA when administered in conjunction with a divided high-dose 
schedule of DACA. DNA-binding compounds include, for example 
9-aminoacridine; such compounds have the ability to inhibit the antitumour 
activity of DACA. 
Accordingly, the present invention provides the use of a DNA-binding agent 
in combination with DACA or other aromatic fused-ring carboxamide having 
an aromatic nitrogen atom or an oxygen atom peri to the carboxamide side 
chain or a physiologically tolerable acid addition salt thereof, to reduce 
the host toxicity of DACA or specified other compound. 
Doses in mice of 100 to 300 mg/kg, especially 150 to 250 mg/kg, more 
especially substantially 200 mg/kg, administered as a divided dose over a 
period of 2 to 4 hours, have proved suitable. Administration to humans of, 
for example, substantially 800 mg/m.sup.2 of DACA or equivalent amount of 
other carboxamide, should be mentioned, but lower or higher amounts may 
also be possible. As explained above, advantageous results are obtained 
when the dose is administered as a divided dose, producing a high plasma 
level followed by a drop in level and then a high level again. Thus, the 
use of substantially 800 mg/m.sup.2 for the total of the constituent doses 
of a divided dose should be mentioned. 
Compounds suitable for use according to the present invention are those of 
the general formula 
EQU ArCONH(CH.sub.2).sub.n Y (I) 
in which 
Ar represents an unsubstituted or substituted ring system comprising two or 
more fused aromatic rings and having an aromatic nitrogen atom or an 
oxygen atom peri to the carboxamide side chain, 
Y represents C(NH)NH.sub.2, NHC(NH)NH.sub.2 or NR.sub.4 R.sub.5, where each 
of R.sub.4 and R.sub.5 separately is H or lower alkyl optionally 
substituted by one or more of the same or different substituents selected 
from hydroxy, lower alkoxy and amino functions, or R.sub.4 and R.sub.5 
together with the nitrogen atom to which they are attached form a 5- or 
6-membered heterocyclic ring optionally containing a further hetero atom; 
and 
n represents an integer from 2 to 6, and their physiologically tolerable 
acid addition salts and N-oxides thereof. 
The ring system may comprise, for example, three fused aromatic rings, 
preferably linear, or two fused aromatic rings with a carbocyclic or 
heterocyclic aromatic ring as substituent. Of the fused aromatic rings, 
one or more may be heterocyclic. 
In a preferred embodiment of the present invention there is used a compound 
of the general formula 
##STR2## 
in which R.sub.1 represents H, CH.sub.3 or NHR.sub.o, where R.sub.o is H, 
COCH.sub.3, SO.sub.2 CH.sub.3, COPh, SO.sub.2 Ph or lower alkyl optionally 
substituted with hydroxy, lower alkoxy and/or amino functions; 
R.sub.2 represents H or lower alkyl, halogen, CF.sub.3, CN, SO.sub.2 
CH.sub.3, NO.sub.2, OH, NH.sub.2, NHSO.sub.2 R.sub.3, NHCOR.sub.3, 
NHCOOR.sub.3, OR.sub.3, SR.sub.3, NHR.sub.3 or NR.sub.3 R.sub.3 (where 
R.sub.3 is lower alkyl optionally substituted with hydroxy, lower alkoxy 
and/or amino functions), and/or may represent the substitution of an aza 
(--N.dbd.) group for one of the methine (--CH.dbd.) groups in the 
carbocyclic ring, 
Y represents C(NH)NH.sub.2, NHC(NH)NH.sub.2 or NR.sub.4 R.sub.5, where each 
of R.sub.4 and R.sub.5 separately is H or lower alkyl optionally 
substituted with hydroxy, lower alkoxy and/or amino functions, or R.sub.4 
and R.sub.5 together with the nitrogen atom to which they are attached 
form a 5- or 6-membered heterocyclic ring optionally containing a further 
hetero atom; 
n represents an integer from 2 to 6; 
X.sub.1 represents H, and 
X.sub.2 represents a phenyl or pyridyl ring unsubstituted or substituted by 
a substituent R.sub.6, or 
X.sub.1 and X.sub.2, together with the carbon atoms to which they are 
attached, form a fused benzene ring unsubstituted or substituted by a 
substituent R.sub.6, and 
R.sub.6 represents lower alkyl, halogen, CF.sub.3, CN, SO.sub.2 CH.sub.3, 
NO.sub.2, OH, NH.sub.2, NHSO.sub.2 R.sub.3, NHCOR.sub.3, NHCOOR.sub.3, 
OR.sub.3, SR.sub.3, NHR.sub.3 or NR.sub.3 R.sub.3 (where R.sub.3 is lower 
alkyl optionally substituted with hydroxy, lower alkoxy and/or amino 
functions); or a phenyl ring optionally further substituted by lower 
alkyl, halogen, CF.sub.3, CN, SO.sub.2 CH.sub.3, NO.sub.2, OH, NH.sub.2, 
NHCOR.sub.3, NHCOOR.sub.3, OR.sub.3, SR.sub.3, NHR.sub.3 or NR.sub.3 
R.sub.3 (where R.sub.3 is lower alkyl optionally substituted with hydroxy, 
lower alkoxy and/or amino functions); and/or may represent the 
substitution of an aza (--N.dbd.) group for one of the methine (--CH.dbd.) 
groups in the ring; 
or a physiologically tolerable acid addition salt, or, especially when 
X.sub.1 .dbd.H and X.sub.2 is unsubstituted or substituted phenyl or 
pyridyl, a 1-N-oxide thereof. 
A compound of the general formula Ia in which X.sub.1 and X.sub.2 complete 
a fused ring and R.sub.1 represents an unsubstituted or substituted phenyl 
group should also be mentioned. 
Another class of compounds is, for example, represented by the general 
formula Ib 
##STR3## 
in which 
R.sub.7 represents H or up to three substituents, at positions selected 
from 2 to 4 and 6 to 9, wherein any two or all of the substituents may be 
the same or different and the substituents are selected from lower alkyl 
radicals; lower alkyl radicals substituted by one or more of the same or 
different substituents selected from hydroxy, lower alkoxy and/or amino 
functions; OH; SH; OCH.sub.2 Ph; OPh; NO.sub.2 ; halogen; CF.sub.3 ; 
amino; NHSO.sub.2 R.sub.3, NHCOR.sub.3, NHCOOR.sub.3, OR.sub.3 and 
SR.sub.3 (where R.sub.3 has the meaning given above; and 
CONH(CH.sub.2).sub.n' Y' (where n' and Y' are as defined below), there 
being a maximum of one CONH(CH.sub.2).sub.n' Y' group; or any two of 
R.sub.7 at adjacent positions represent --CH.dbd.CH--CH.dbd.CH-- as part 
of an extra benzene ring or --O--CH.sub.2 --O-- (methylenedioxy) and the 
third of R.sub.7 has any one of the meanings given above with the 
exception of an OH at position 2; 
Y and Y', which may be the same or different, each has the meaning given 
above for Y; and n and n', which may be the same or different, each has 
the meaning given above for n; 
and physiologically tolerable acid addition salts, 5- and 10- mono-N-oxides 
and 5,10-di-N-oxides thereof. 
These compounds may be prepared by methods known per se, for example by 
methods described in EP 98098 A, in EP 206802 A and in EP 172744 A or by 
analogous methods. 
When used herein, the term "lower alkyl" denotes an alkyl group having from 
1 to 5, preferably 1 to 4, carbon atoms. 
An amino function as substituent of a lower alkyl radical represented by 
any of R.sub.3, R.sub.4, R.sub.5, R.sub.o and R.sub.7 may be unsubstituted 
or, for example, substituted by one or two lower alkyl groups (where lower 
alkyl has the meaning given above), especially by one or two methyl 
groups. Thus, for example, an amino substituent of a lower alkyl radical 
represented by R.sub.3, R.sub.4, R.sub.5, R.sub.o and/or R.sub.7 may be 
NH.sub.2, NHCH.sub.3 or N(CH.sub.3).sub.2. 
A lower alkoxy group as substituent of a lower alkyl radical represented by 
R.sub.3, R.sub.4, R.sub.5, R.sub.o and/or R.sub.7 is especially a methoxy 
group. 
A heterocyclic radical represented by R.sub.4 and R.sub.5 and the nitrogen 
atoms to which they are attached may, if desired, contain an additional 
hetero atom, and is 5- or 6-membered. An example is a morpholino group. 
Examples of optionally substituted lower alkyl groups include those 
substituted by hydroxy, lower alkoxy or an amino function, for example 
lower alkyl optionally substituted with hydroxy, amino, methylamino, 
dimethylamino or methoxy. when X.sub.1 +X.sub.2 complete a fused benzene 
ring such lower alkyl groups are preferably unsubstituted or substituted 
with hydroxy and/or amino groups. 
In a NR.sub.3 R.sub.3 group the two R.sub.3 substituents may be the same or 
different, but are preferably the same. 
A preferred class of compound of the above formula I where X.sub.1 
represents H and X.sub.2 represents a phenyl or pyridyl ring is where 
R.sub.1 represents H, and, more especially, 
R.sub.2 represents H, 
Y represents N(CH.sub.3).sub.2, 
n represents 2 and 
if X.sub.2 represents a pyridyl ring, that ring is unsubstituted, and 
if X.sub.2 represents a phenyl ring that ring is unsubstituted or 
substituted by halogen, NO.sub.2 or OCH.sub.3. 
A pyridyl ring represented by X.sub.2 is preferably a 4-pyridyl ring. 
The use of an acridine carboxamide of the general formula 
##STR4## 
where R.sub.1 and n have the meanings given above, R.sub.8 represents H or 
up to two of the groups CH.sub.3, OCH.sub.3, halogen, CF.sub.3, NO.sub.2, 
NH.sub.2, NHCOCH.sub.3, and NHCOOCH.sub.3 placed at positions 1-3 and 5-8, 
and/or may represent the substitution of an aza (--N.dbd.) group for one 
of the methine (--CH.dbd.) groups in the carbocyclic ring; 
and 
Y represents C(NH)NH.sub.2, NHC(NH)NH.sub.2, or NR.sub.4 R.sub.5, where 
each of R.sub.4 and R.sub.5 is H or lower alkyl optionally substituted 
with hydroxyl and/or amino groups; 
and where any lower alkyl radical has up to 4 carbon atoms, 
and the physiologically tolerable acid addition salts thereof, should 
especially be mentioned. 
A preferred subclass of these compounds of formula 
I' are those where 
R.sub.1 represents H or NH.sub.2, 
R.sub.8 represents up to two of 1-, 5-, 6-, 7- and 8-NO.sub.2, 5- and 
6-CH.sub.3, and 5-Cl, 
Y represents NHC(NH)NH.sub.2, N(CH.sub.3).sub.2, or NHCH.sub.2 CH.sub.2 OH, 
and 
n represents 2. 
Compounds specifically identified in EP 98098 A, EP 206802 A and EP 172744 
A and in the literature references given above should also be mentioned. 
When any of R.sub.2, R.sub.6 and R.sub.8 represents the substitution in the 
ring of an aza group for one of the methine groups, that ring may be 
unsubstituted or substituted as specified above. 
Compounds of the general formula Ia or I' in which R.sub.6 or R.sub.8 
represents the substitution of an aza group for one of the methine groups 
and which optionally contains a further R.sub.6 or R.sub.8 substituent(s) 
are novel, and as such form part of the present invention. 
The compounds used according to the invention, including compounds of 
formulae Ia and Ib, form pharmaceutically acceptable addition salts with 
both organic and inorganic acids. Examples of suitable acids for salt 
formation are hydrochloric, sulphuric, phosphoric, acetic, citric, oxalic, 
malonic, salicylic, malic, fumaric, succinic, ascorbic, maleic and 
methanesulphonic acids. 
When used as a means of circumventing mdr, in combination with another 
cytotoxic drug, for example a DNA-reactive agent, a DNA-synthesis 
inhibitor or an agent which disrupts the mitotic apparatus, the compound 
of the general formula I may be administered together with, before or 
after the other cytotoxic drug. DACA could be given, for example, up to 
.+-.2 days of a second drug, or alternatively could be given as a separate 
or alternating course with another cytotoxic drug and separated by a 
period of bone marrow or other host tissue recovery, generally 3 to 4 
weeks. DACA may, for example, be administered by intravenous infusion 
using the divided dose regime mentioned above, for example as a series of 
2 to 4 administrations over a period of 2 to 4 hours. 
Suitable DNA-reactive agents are, for example, cisplatin, cyclophosphamide, 
bleomycin and carboplatin. Suitable DNA synthesis inhibitors are, for 
example, 5-fluorouracil, 5-fluorodeoxyuridine and methotrexate; and 
suitable agents that disrupt the mitotic apparatus are, for example, taxol 
and suitable Vinca alkaloids, for example, vincristine, vinblastine and 
vindesine. These agents should be used in a treatment schedule which has 
been found optimal for antitumour effect; for example, cyclophosphamide 
may be used at monthly intervals, and vincristine at monthly or weekly 
intervals. 
There are four main types of multidrug resistance related to topo II 
inhibitor: 
(a) No change in the topoisomerase enzyme but increased transport of the 
drug out of the cell. DACA is not susceptible to this, while etoposide and 
doxorubicin are. 
(b) No change in the enzyme, but an increase in drug detoxifying enzymes. 
This applies to doxorubicin but not to etoposide or DACA. 
(c) A quantitative change (decrease) in the amount of topoisomerase II 
enzyme. DACA, etoposide and doxorubicin are equally susceptible, since DNA 
damage depends on the amount of enzyme present. Since the amount of topo 
II is regulated during the cell division cycle, cytokinetic resistance, 
whereby non-cycling cells resist the effects of topo II agents, may 
involve this type of resistance. 
(d) A qualitative change in the topoisomerase enzyme, a result either of a 
switch in gene expression (there are two genes for topoisomerase II and 
one is normally dominant) or of a mutation in the gene, or of a change in 
modification of the enzyme after it has been synthesised. This results in 
a change in drug-target interaction. The qualitative change is accompanied 
by a differential change in sensitivity: in general, cells become highly 
resistant to amsacrine, moderately resistant to etoposide and doxorubicin, 
and, we have ascertained, minimally resistant to DACA. 
Investigation of the cytotoxicity of DACA under conditions of continuous 
drug exposure in a variety of human and mouse cell lines and in a panel of 
60 human cell lines revealed IC.sub.50 values (defined as drug 
concentration required over approx. 5 cell doubling times for the 
reduction of the final cell culture density by 50%) ranging from 0.09 
.mu.M to 3.4 .mu.M, and a mean IC.sub.50 value for human cell lines of 1.3 
.mu.M. The latter value compared with 2 .mu.M for the 4-pyridyl quinoline 
analogue, 0.76 .mu.M for amsacrine, 0.1 .mu.M for the amsacrine analogue 
4'-(9-4-N-methylcarboxamido!-5-methyl!-acridinylamino)methanesulphon-m-a 
nisidide ("CI-921") and 81 .mu.M for etoposide. Whereas the patterns of 
cytotoxicity of amsacrine, CI-921 and etoposide in the human cell line 
panel were very similar, those of DACA and its pyridoquinoline analogue 
were quite different, suggesting differences in mode of action. 
A multidrug resistant subline of P388 murine leukaemia (P/ACTD) was tested 
for sensitivity to DACA. This line was cross-resistant to actinomycin D, 
doxorubicin, mitoxantrone, etoposide and vincristine. Its resistance to 
vincristine was overcome by the presence of verapamil (10 .mu.M). It 
stained for the presence of P-glycoprotein, consistent with the presence 
of transport-mediated multidrug resistance. This line was sensitive to 
DACA in vitro and in vivo, suggesting that DACA may be useful in at least 
some types of multidrug resistance. 
DACA was also able to overcome, to a large extent, other mechanisms of 
multidrug resistance, as demonstrated in a series of sublines of Jurkat 
leukaemia cells which were highly resistant to amsacrine, etoposide and 
doxorubicin. Two of these lines had been selected for resistance to 
amsacrine, and were more than 100-fold cross-resistant to amsacrine but 
only 2- to 4-fold cross-resistant to DACA. These lines exhibited 
resistance mechanisms which were distinguishable from transport-mediated 
multidrug resistance. We believe that this ability to overcome resistance 
mechanisms accounts for the different IC.sub.50 patterns observed with the 
human cell line panels. 
It is apparent that in many tumours, regrowth during therapy is associated 
with resistance. The type of resistance is not yet properly characterised, 
but if it involves the mechanisms discussed above, DACA may be useful for 
second time treatment, especially in the divided dose regime mentioned 
above. 
The use of the above compounds and combinations in the treatment of sarcoma 
and of lung, breast, ovarian and testicular cancer should especially be 
mentioned. 
The use of compounds of the general formula I to treat colon tumours has 
been suggested previously, but there has been no evidence of their 
suitability for this treatment and there has been no indication that they 
are effective in test systems even with delay of initiation of treatment 
beyond day 2 or 3 after tumour implantation, as is usual in tests. There 
has also been no disclosure of high activity against such tumours. Such 
high activity would not have been expected since the most closely 
structurally related topo II inhibitor, amsacrine, is inactive. 
An initial experiment against advanced colon 38 tumour (on day 11 after 
implantation), using the same schedule of administration as used for Lewis 
lung (3 injections at 4-day intervals) gave only a modest growth. delay (4 
days). 
However, by adjustment of the administration schedule of DACA, growth delay 
of the advanced colon 38 tumour (5-8 mm in diameter) was increased to more 
than 21 days. Thus, while intermittent schedules (270 mg/kg q.sup.4 
days.times.3; 400 mg/kg q.sup.7 days.times.3) provided only modest growth 
delays (3 days and 7 days, respectively), repeated injection schedules (4 
injections at 30 minute intervals; 180+120+120+120 .mu.mol/kg, q.sup.7 
days.times.3) provided a 21 day growth delay. Such results were completely 
unexpected. 
We have found that a low drug concentration for a long time (for example 6 
hours) is much more toxic than a high concentration for a correspondingly 
shorter time. We believe these unusual "self-inhibitory" properties of 
DACA may be of help in the new divided dose administration strategy. 
Because DACA diffuses more slowly in solid tumours such as colon tumours, 
than in normal tissues, peak drug concentrations in tumours are lower than 
in normal tissues: an obvious disadvantage. However, because, as we have 
found, higher concentrations of DACA are less inhibitory than lower ones., 
the adjustment of the dosing strategy may provide partial protection of 
normal tissues. 
DACA or other compound of the general formula I may be administered, for 
example, in a divided dose over a period of up to 4 hours, for example 2 
to 4 hours, followed by a rest, for example for 3 to 4 weeks. The dose may 
be divided into two to four administrations over the 2 to 4 hour or other 
administration period, and the first dose may be larger than the others, 
that is, as a loading dose; administrations may be given intravenously. 
For example, a short-term intravenous infusion of 10 to 30 minutes (for 
example 15 minutes) may be used, followed by a further such infusion 
after, for example, 1 hour. This schedule differs from that normally used 
for other cytotoxic agents, which involves periods of intravenous infusion 
administered daily, for example for 3 to 7 days, or long-term intravenous 
infusion over a number of days, for example for a week. 
We have found that DACA also has activity against melanoma cell lines and 
human tumour xenografts of these lines, and it is believed that this 
activity is improved by the same strategy. 
The cytotoxicity of DACA was assessed in a panel of primary human melanoma 
cultures derived from fresh surgical melanoma specimens. IC.sub.50 values 
ranged from 0.2 o 1.5 .mu.M, and a feature of the data was the ability of 
DACA to kill much higher proportions of cells (&gt;99%) in some cultures, as 
compared to a maximum of 90% for etoposide. 
A further experiment was carried out using human melanoma line, implanted 
subcutaneously in nude (athymic) mice. Treatment was started when the 
tumours were 4-7 mm in diameter. DACA was administered ip as a divided 
dose (2.times.100 mg/kg body weight at 0 and 60 min) and a second similar 
administration (2.times.100 mg/kg) was given after 7 days. A growth delay 
of 30 days was obtained. 
The positive results achieved by DACA in these treatments is surprising 
since melanoma is more difficult to treat with chemotherapy than are other 
forms of tumour. 
Moreover, Berger et al. (Berger D. P., Winterhalter B. R., Flebig H. H., 
"Conventional chemotherapy" in "The Nude Mouse in Oncology Research", 1st 
ed. London: CRC Press, 1991, 165-84, ed. Boven and Winograd) states that 
melanoma xenografts are resistant to treatment by doxorubicin and 
etoposide, so activity by a drug in this class is completely unexpected. A 
summary of the activity of various other agents against subcutaneous 
melanoma xenografts growing in nude mice is given by Berger et al. as 
follows: 
______________________________________ 
Percentage of xenografts 
Drug responding 
______________________________________ 
Topo II inhibitors 
Doxorubicin 5% (total of 5 studies) 
Etoposide 0% (2 studies) 
Other drugs 
Bleomycin 0% 
Cisplatin 13% 
Cyclophosphamide 11% 
Dacarbazine 17% 
5-Fluorouracil 14% 
Methotrexate 7% 
Mitomycin C 32% 
Vinblastine 10% 
Vincristine 43% 
______________________________________ 
In pharmacokinetic studies using radioactive (tritium-labelled) DACA high 
levels of active ingredient have been found in all tissues, including 
brain, with a long elimination t.sub.1/2 of 37-176 h. As determined by 
HPLC, the tissue concentrations of DACA 1 h after intraperitoneal 
administration of drug (400 .mu.mol/kg) were 45, 185, 139, and 57 
.mu.mol/kg in brain, liver, kidney and heart, respectively. The 
corresponding AUC values (AUC=area under the plasma concentration-time 
curve) were 218, 547, 492 and 147 .mu.mol.h/l , respectively, as compared 
to the plasma AUC of 26.6 .mu.mol.h/l. DACA showed relatively high rates 
of passage across the blood brain barrier. We believe that administration 
of DACA, especially at the new divided dose-high constituent dose 
frequency regime mentioned above, will be helpful in combating brain 
tumours. With the exception of nitrosoureas, few of the antitumour agents 
currently in use possess the physicochemical properties required for 
adequate penetration of the blood-brain barrier (Greig M. H. (1987), 
Cancer Treat. Rep. 11: 157). 
We also propose the use of DACA and related compounds with a "rescue" 
treatment with a second drug which by itself is not an active agent but 
which displaces DACA or the other compound from the DNA. This DNA-binder, 
or chemoprotector, should have a lower intrinsic toxicity and less 
efficient tissue distribution properties than the cytotoxic agent, thus 
sparing rapidly growing and highly vascularised normal tissues such as 
bone marrow from cytotoxic effects. Use of the new schedule of 
administration of DACA or other compound of the general formula I or a 
physiologically tolerable acid addition salt or 1-N-oxide thereof, 
combined with "rescue" treatment with a chemoprotector, should especially 
be mentioned. Timing of administration of the chemoprotector will depend 
on the pharmacokinetics of DACA or other drug used. The chemoprotector 
may, for example, be administered at the same time as or up to 30 minutes 
after one or more of the constituent doses of a divided dose of that drug; 
by such means doses of, for example, 200 mg/kg or even 300 mg/kg of 
DACA--doses which are normally toxic--may be possible. 
The following Examples illustrate the invention. 
EXAMPLES 
EXAMPLE 1 
Activity of DACA against cultured multidrug resistant human leukaemia cells 
Materials and Methods 
Acridine carboxamide hydrochloride, synthesised in the Cancer Research 
Laboratory (Atwell G. J., et al., J. Med. Chem. 1987, 30, 664-669), and 
amsacrine isethionate, obtained from the Parke-Davis Division of the 
Warner-Lambert Company, Ann Arbor, USA, were dissolved in 50% v/v aqueous 
ethanol to make stock solutions of 2-5 mmol/l and stored at -20.degree. C. 
Other cytotoxic drugs were available either from the NCI repository (Monks 
A., et al., J. Natl. Cancer Inst. 1991, 83, 757-766) or were obtained as 
described in Marshall E. S., et al., J. Natl. Cancer Inst. 1992, 84, 
341-344 and Finlay, et al., Eur. J. Cancer Clin. Oncol. 1986, 22, 655-662. 
Cell lines were from the NCI repository except for MM-96 (Dr. R. 
Whitehead, Ludwig Institute, Melbourne, Australia), FME (Dr. K. M. Tveit, 
Norwegian Radium Hospital, Oslo, Norway) and Jurkat normal and 
multidrug-resistant lines (Dr. K. Snow and Dr. W. Judd, Department of 
Cellular and Molecular Biology, University of Auckland). Melanoma tissue 
was obtained from patients with pathologically confirmed metastatic and 
recurrent melanomas under Auckland Hospital Ethical Committee guidelines. 
Cells were released by digestion of tissue (at 50 mg.ml.sup.-1) with 
collagenase (1 mg.ml.sup.-1) and DNAase (50 .mu.g.ml.sup.-1) with 
continuous stirring at 37.degree. C. for 1 to 2 hours, and cultured as 
previously described (Marshall E. S., et al., J. Natl. Cancer Inst. 1992, 
84, 341-344). 
Tumour cell lines were cultured in 96-well plates. Growth of NCI cell lines 
was assessed using sulphorhodamine B staining (Skehan P., et al., J. Natl. 
Cancer Inst. 1990, 82, 1107-1112), that of the leukaemia lines with 
(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium (MTT) staining 
(Mosmann T., J. Immun. Methods 1983, 65, 55-63) and that of the primary 
human tumour material by .sup.3 H-thymidine incorporation (Marshall E. S., 
et al., J. Natl. Cancer Inst. 1992, 84, 341-344). Primary tumour material 
was cultured in 96-well plates in which the wells were coated with agarose 
to inhibit selectively the growth of normal cells. (Marshall E. S., et 
al., J. Natl. Cancer Inst. 1992, 84, 341-344). Primary cultures were 
incubated at 37.degree. C. in sealed perspex boxes containing a humidified 
atmosphere of 5% CO.sub.2 and 5% O.sub.2 in nitrogen for 7 days. 
5-Methyl-.sup.3 H!-thymidine (20 Ci.mmol.sup.-1 ; 0.04 .mu.Ci per well), 
thymidine and 5-fluorodeoxyuridine (each at final concentrations of 0.5 
.mu.M) were added in medium to cultures (20 .mu.l per well) 24 h before 
terminating the cultures. Cells were aspirated on to glass fibre filters 
using a multiple automated sample harvester (LKB Wallac OY Beta 
Harvester). The filter discs were washed for 15 seconds with water, dried, 
and the amount of tritium retained quantified by liquid scintillation. 
IC.sub.50 values were defined as in Paull K. D., et al., J. Natl. Cancer 
Inst. 1989, 81:1088-1092, where growth, as indicated by staining or 
thymidine incorporation, corresponded to 50% of that of the control 
cultures. DELTA values were determined for groups of logarithmic IC.sub.50 
values as deviations from the mean, with positive DELTA values 
representing higher drug sensitivity relative to the mean. Variances of 
DELTA values were expressed as standard deviations in log.sub.10 units. 
Comparison of DELTA values was made using Pearson correlation 
coefficients. Resistance factors were defined as the ratios of IC.sub.50 
values between the resistant line and the parent line. Statistical 
evaluation was performed either with NCI programmes, with RS/1 software 
(BBN Research Systems, Cambridge, Mass., USA), or with Sigmaplot (Jandel 
Scientific, San Rafael, Calif., USA). 
Results 
The effect of DACA on the growth of cultured cells was assessed by 
continuous drug exposure. DACA inhibited the growth of two human Jurkat T 
cell leukaemia lines, one diploid (L) and the other tetraploid (B1), with 
IC.sub.50 values each of 380 nM. These values were similar to those of 
other human leukaemia cell lines, which ranged from 290 to 760 nM 
(CEM-CCRF, 410 nM; MOLT-4, 290 nM; Daudi, 400 nM; Raji, 370 nM, U937, 590 
nM; HL-60, 620 nM; K-562, 760 nM). Four multidrug-resistant cell lines, 
developed from JL and JB1 by in vitro exposure to increasing 
concentrations of doxorubicin (L.sub.D and B1.sub.D) or amsacrine (L.sub.A 
and B1.sub.A) (Finlay G. J., et al., J. Natl. Cancer Inst. 1990, 82, 
662-667, Snow K., et al., Br. J. Cancer 1991, 63, 17-28) were tested. 
DACA was compared with six other drugs including four topo II poisons, 
doxorubicin, mitozantrone, etoposide and amsacrine. Resistance factors for 
the topo II poisons were consistently higher than those for DACA (Table 
1). In contrast, the topoisomerase I poison camptothecin showed no cross 
resistance, and the mitotic inhibitor vincristine showed a different 
pattern of resistance with the B1.sub.D line having the highest resistance 
(Table 1). 
TABLE 1 
______________________________________ 
Drug-resistant Jurkat Leukaemia sublines. 
Resistance factors 
doxo- mitoxan- etopo- amsa- campto- 
vincris- 
rubicin trone side crine 
DACA thecin 
tine 
______________________________________ 
L.sub.A 
3.8 42 11 130 2.0 1.0 1.5 
L.sub.D 
16 160 93 110 2.5 0.97 3.6 
B1.sub.A 
11 59 22 240 3.9 0.48 2.0 
B1.sub.D 
15 8.4 83 8.8 1.9 0.86 10 
______________________________________ 
One method of providing a visual comparison of the patterns of resistance 
is to plot DELTA values (Paull K. D., et al., J. Natl. Cancer Inst. 1989, 
81, 1088-1092) where the differences in bar lengths are used as a measure 
of relative resistance. FIG. 1 shows a comparison of DELTA values in log 
mean graph format for DACA, amsacrine, etoposide, doxorubicin, 
vincristine, 5-fluorouracil, camptothecin and mitozantrone for the panel 
of multidrug resistant Jurkat leukaemia lines using MTT staining; JL/AMSA 
and JB/AMSA were selected for resistance to amsacrine and JL/DOX and 
JB/DOX were resistant to doxorubicin. DACA clearly shows a pattern 
distinct from doxorubicin. 
A second method of comparing agents is to plot resistance factors for one 
of the lines against another. Since the Jurkat lines exhibited 
predominantly "altered topoisomerase" resistance (Finlay G. J., et al., J. 
Natl. Cancer Inst. 1990, 82, 662-667, Sugimoto Y., et al., Cancer Res. 
1990, 50, 7962-7965), the resistance factors for one of these (L.sub.A) 
was plotted versus the resistance factors for a P-glycoprotein positive 
multidrug resistant P388 Leukaemia line (P/DACT) which exhibits transport 
resistance (Baguley B. C., J. Natl. Cancer Inst. 1990, 82, 398-402). The 
results (FIG. 2) indicate that DACA is unique when compared to other topo 
II agents in that it is able to overcome two different multidrug 
resistance mechanisms. Qualitatively similar graphs are obtained when the 
resistance factors of the other resistant Jurkat lines are plotted on the 
abscissa, or those from a P-glycoprotein positive, vinblastine resistant 
human leukaemia line (CEM/VLB.sub.100) (Qian X., Beck W. T., Cancer Res. 
1990, 50, 1132-1137) are plotted on the ordinate. 
DACA was also compared with three other topo II agents using a panel of 
cell lines (data provided by Dr. Ken Paull from the National Cancer 
Institute, USA) encompassing a number of tumour types, and using protein 
staining. The mean IC.sub.50 for DACA was 2,100 nM, as compared with 
amsacrine (520 nM), etoposide (21,000 nM) and doxorubicin (140 nM). The 
results, presented as DELTA plots, are compared with corresponding plots 
for three other topoisomerase II poisons in FIG. 3. The variance of DELTA 
values was considerably smaller for DACA (0.24 units) than it was for 
amsacrine (0.61 units) etoposide (0.55 units) or doxorubicin (0.44 units). 
The differences in DELTA values for amsacrine, etoposide and doxorubicin 
for primary human cultures imply that intrinsic resistance mechanisms 
exist and are partially overcome with DACA. 
DACA was also compared in a series of 12 primary melanoma cultures. Tissue 
was excised from human malignant melanomas and cultured using a modified 
96-well assay system in which the cells were cultured on agarose and 
assayed for proliferation using the .sup.3 H-thymidine incorporation assay 
as described in Marshall E. S., et al., J. Natl. Cancer Inst. 1992, 84, 
341-344. The mean IC.sub.50 for DACA was 590 nM, as compared with 
amsacrine (128 nM), etoposide (2,200 nM) and doxorubicin (56 nM). DELTA 
values for DACA, amsacrine, etoposide and doxorubicin are compared in FIG. 
4. The variance of DELTA values was smaller for DACA (0.39 units) than for 
amsacrine (0.54 units), etoposide (0.66 units) or doxorubicin (0.63 
units). The differences in DELTA values for amsacrine, etoposide and 
doxorubicin for primary human cultures again imply that intrinsic 
resistance mechanisms exist and are partially overcome with DACA. 
EXAMPLE 2 
Activity of DACA against advanced colon 38 and melanoma in mice 
Materials and Methods 
Colon 38 carcinoma was obtained from the Mason Research Institute 
(Worchester, Mass., USA) and was grown in BDF.sub.1 hosts. Tumour 
fragments (1 mm.sup.3) were implanted subcutaneously in anaesthetised 
mice. Tumours had grown to the appropriate size 9 days after implantation. 
A melanoma tumour line (WADH) was developed in the Cancer Research 
Laboratory. Tumour cells were grown in culture and 1.times.10.sup.6 cells 
were implanted intradermally into the flank of nude (C57BI/J genetic 
background) mice. Mice were grown under sterile surroundings until tumours 
were of appropriate size. 
Tumours were measured 3.times. (colon 38) or 2.times. (xeno-graft) weekly 
with callipers and tumour volumes calculated as 0.52a.sup.2 b, where a and 
b were the minor and major tumour axes. Tumour growth delays were measured 
at a time when tumour volumes of treated and control animals had increased 
by 4-fold. 
Results 
The effect of DACA on the growth of advanced colon 38 tumours in mice was 
investigated by implanting tumour fragments subcutaneously and allowing 
them to grow until they had reached a diameter of 5-8 mm. I.p. treatment 
of mice with a single maximum tolerated dose of DACA (150 mg/kg body 
weight), a treatment which was known to induce cures of intravenously 
implanted Lewis lung tumours (Finlay G. J., Baguley B. C., Eur. J. Cancer 
Clin. Oncol. 1989, 25, 271-277) caused only a slight growth delay (5 days; 
FIG. 5). However, when a divided dose (200 mg/kg) was administered over a 
period of 0.5-4 hours, greater delays were unexpectedly observed (Table 
2). Repetition of these divided doses provided a substantial growth delay 
(23 days; FIG. 5) which was longer than that obtained with the maximum 
tolerated dose of amsacrine (2 days), cyclophosphamide (6.5 days) or 
5-fluorouracil (13 days). 
TABLE 2 
______________________________________ 
Tumour growth delays (colon 38) treated with DACA 
Total dose 
Schedule Growth delay (days) 
______________________________________ 
100 ip single dose (SD) 4 
150 ip SD 5 
150 .times. 3 
ip SD every week .times. 3 
7 
150 ip 2 doses, 0, 60 min 5 
200 ip SD toxic 
200 ip 2 doses, 0, 30 min 7 
200 ip 2 doses, 0, 60 min 10, 12 (2 expts) 
200 ip 2 doses, 0, 24 hours 
6 
200 ip 4 doses, 0, 30, 60, 90 min 
6 
200 iv 1 hour infusion 7 
200 iv 3 hour infusion 6.5 
200 .times. 3 
ip (4 doses, 0, 30, 60, 90 min) .times. 3 
23 
______________________________________ 
Note: the 4 dose schedule was 65 + 45 + 45 + 45 mg/kg 
A further experiment was carried out using human melanoma line, implanted 
subcutaneously in nude (athymic) mice using an inoculation of one million 
cells of a human melanoma cell line designated WADH. Treatment was started 
when the tumours were 4-7 mm in diameter. DACA was administered ip as a 
divided dose (2.times.100 mg/kg body weight at 0 and 60 min) and a second 
similar administration (2.times.100 mg/kg) was given after 7 days. A 
growth delay of 30 days was obtained (FIG. 6). 
EXAMPLE 3 
Exploitation of the self-inhibitory properties of a drug in the therapy of 
solid tumours 
One of the characteristics of solid tumours is that because of the poor 
vascularisation, oxygen, nutrients and chemotherapeutic drugs must diffuse 
for longer distances than they do in normal tissue (Wilson W. R., Denny W. 
A., Radiation Research: a Twentieth Century Perspective, 1st ed. v. 2. New 
York: Academic Press, 1992:796-801). In the case of antitumour agents, a 
gradient of drug concentration is established with the lowest drug 
concentration at greatest distances from the capillary. Since in all cases 
examined so far with existing clinical agents, cytotoxicity is related in 
a positive fashion to drug concentration, it follows that those areas most 
remote from the tumour blood supply are protected from drug cytotoxicity, 
a so-called "pharmacological sanctuary". 
DACA is a DNA intercalating agent which acts on topo II and has the unusual 
property of inhibiting its own toxicity at concentrations above 5 .mu.m. 
It also inhibits the formation of DNA-protein cross-links above 5 .mu.M, 
consistent with the hypothesis that self-inhibition of DNA-protein 
cross-links is related to self-inhibition of toxicity. A simple model for 
this behaviour is that in order for topo II to form its complex with DNA 
(i.e. to form DNA-protein cross-links) it requires the presence of a 
DNA-drug complex (probability=p), surrounded on each side by drug-free DNA 
(probability=(1-p)). It follows that the probability of forming a 
productive complex is p(1-p).sup.2. When this function is plotted against 
experimental cytotoxicity data for DACA (Haldane A., et al., Cancer 
Chemother. Pharmacol. 1992, 29, 475-479), a good approximation is obtained 
(FIG. 7). 
FIG. 7 can also be plotted as toxicity versus cell-associated drug (using 
unpublished data from the Cancer Research Laboratory which relates 
external drug concentration to cell-associated drug). It can be seen from 
FIG. 7 that if a tumour concentration gradient is established whereby the 
area of the tumour closest to the capillary has, for example, a 
concentration of 1800 .mu.mol/kg, areas of the tumour which are more 
remote from the capillary, although having a lower drug concentration, 
will have higher cytotoxicity. Furthermore, host tissues, which have good 
blood supplies, will have high tissue drug concentrations and thus lower 
cytotoxicity. By this principle, DACA (and other compounds of this general 
class) could have a selectivity mechanism for solid tumours which is not 
possessed by other agents. 
The practical application of this hypothetical situation requires that free 
drug plasma concentrations (and corresponding tissue concentrations of 
drug) fall into the range which will provide selectivity (i.e. greater 
than 1000 .mu.mol/kg tissue). Preliminary results (Dr. James Paxton, 
personal communication) indicate that when DACA is administered at a 
maximally tolerated single drug dose (150 mg/kg body weight), drug 
concentrations in normal tissues (e.g. liver, spleen) slightly exceed 1000 
.mu.mol/kg. This principle may be exploited further by drug design or by 
combining DACA administration with that of a second chemoprotector agent 
which increases the self-inhibition of DACA (i.e. the descending part of 
the curve in FIG. 7) and thus lowers the average tissue drug concentration 
required for the application of this principle.