Cancer

Treatment

Reviews

(1990)

17, 261-277

New tumor-inhibiting B. K. Keppler,

M. R. Berger*

metal

complexes

and M. E. Heimt

Anorganisch-Chemisches Institut der Universitiit Heidelberg, Im Neuenheimer Feld 270, 6900 Heidelberg; *Institut fCr Toxikologie und Chemotherapie am Deutschen Krebsforschungszentrum, Im Neuenheimer Feld 280, 6900 Heidelberg and f Onkologisches Zen&urn des Klinikums Mannheim der Universitiit Heidelberg, Theodor-Kutzer-Ufer, 6800 Mannheim 1, F.R.G.

The only inorganic drug to be in routine clinical cancer therapy is cisplatin (international non-proprietary name), cis-diamminedichloroplatinum(I1) (Figure 1). This drug was first synthesized by Michele Peyrone in 1844 (31). In 1969, Rosenberg discovered the tumorinhibiting activity of cisplatin. Rosenberg showed that cis-Pt(NH,),Cl,, cis-Pt(NHs)&l,, PtenCl, and PtenCl, (Figure 2)-all cis-configurated compounds-had an outstanding tumor-inhibiting effect in animals. In contrast, the corresponding trans- compounds were found to be inactive (29, 32-36). Cisplatin is frequently used in combination with other antitumor agents and is effective mainly against testicular and ovarian carcinomas, tumors of the bladder, and the head

H3N\p+/c’ H,N’ Figure

1. Cisplatin

(INN),

‘Cl

cis-diamminedichloroplatinum(II~,

widely

used

as anticancer

agent

in the clinic

today.

H*N\p+/c’ C

H3N\p+2’ H3N’

‘Cl

Cis-Diomminedichloroplatinum INN: Cisplotin

CiS-Dlamminetetrachloroplatinum

H 2 N’

(II)

‘Cl

1,2-Diominoethanedichloroplotinum

(III)

1,2-Diaminaethanetetrachioroplatinum

(II)

(IX)

Figure 2. Rosenberg was the first to examine these platinum complexes with regard to tumor-inhibiting Cir-Diamminetetrachloroplatinum(IV) was the substance that caused filament growth of&cscherichia in his experiments.

0305~7372/90/2&3261+

17 $03.00/O

0 261

1990 Academic

properties. coli bacteria

Press Limited

262

B. K.

KEPPLER

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and neck. Although cisplatin is highly active against some relatively rare tumors, it does not show any effect on common malignant tumours such as carcinomas of the lung, breast and colon or rectum. The activity of cisplatin against testicular carcinomas showed that it is possible to find anticancer drugs by inorganic chemistry. This has, of course, been a considerable impetus for inorganic chemists to search for new metal complexes which have similar good activity, but against those types of tumor that are responsible for the major share of cancer mortality.

The development

of new

tumor-inhibiting

metal

complexes

The development of new tumor-inhibiting metal complexes is basically characterized by the three following procedures (19): (1) synthesis and activity screen of ‘direct’ cisplatin derivatives, (2) trials with new metal complexes that do not have platinum as their central metal, and (3) linking of cancerotoxic platinum compounds or of other tumor-inhibiting metal complexes with carrier molecules or carrier systems in order to achieve an accumulation in certain tissues. The first of these three strategies is not very promising for finding substances with a different spectrum of activity than cisplatin. Experience has shown that the development of ‘direct’ derivatives will lead to substances that are not very different in therapeutic efficacy from the parent compound since their mechanism of action may be similar. The procedure is rather more justified in attempts to decrease toxicity or increase selectivity in comparison with cisplatin. The second strategy, tumor-inhibiting non-platinum compounds, on the other hand, should be more likely to act on tumors other than those affected by cisplatin, owing to the change in chemical properties in comparison with platinum. It will, however, be much more difficult in this field to find a compound that has any tumor-inhibiting effects at all, because it is not possible to proceed from an established structure-activity relationship as can be done with platinum compounds. Examples of the third possibility, i.e. the linking of cancerotoxic platinum compounds to carrier molecules, include platinum derivatives with hormone receptor affinity and osteotropic platinum compounds. Part of our research in the field of non-platinum complexes will be outlined in the following sections. In order to assess the future clinical potential of non-platinum complexes, it is best to subdivide them into drugs that are at a preclinical stage of development and drugs which have already qualified for clinical studies. Drugs in preclinical studies include tin compounds, metallocenes, gold compounds, and ruthenium complexes, among others. The ruthenium compounds are already relatively advanced and will be described in the following paragraphs. Some classic ruthenium complexes such as ‘ruthenium red’, cis-Ru(DMSO),Cl,, or cis-[Ru(NHs)4C12]Cl (Figure 3), are well known for their antitumor activity, but so far none of them and none of their derivatives have been able to qualify for clinical trials (1, 2, 5-7, 10, 11, 37-39, 41). One of the reasons for this may be that in biological experiments the compounds were hardly ever investigated in realistic and predictive tumor models. One of the best-studied ruthenium derivatives is cis-dichlorotetrakis(dimethylsulfoxide)ruthenium(II), cis-Ru(DMS0)4C1, (F’g1 ure 3). This complex is fairly soluble in water. It shows only marginal activity against the P388 leukemia, but it has good metastases-inhibiting effects on the Lewis lung tumor. Ruthenium red is even less active. Cis-Tetramminedichlororuthenium(III)chloride (Fig. 3), cis-[Ru(NHs),Cl,]Cl, is active

NEW

TUMOR-INHIBITING

METAL

COMPLEXES

263

L

L

L

L =

NH3

-I

Cl

DMSO cis-Ru(DMSO),Cl,

cis-Dichlorotetrakis(dimethylsulfoxide)ruthenium(II) Figure

3. Three

ruthenium

cis-[Ru(NH,),Cl,]Cl

fat-Ru(NH,),Cl,

cis-‘I’etramminedichlororuthenium(III)chloride

fac-‘l’risamminetrichlororuthenium(II1)

complexes

with

known

tumor-inhibiting

activity.

against the P388 leukemia and reaches T/C values of about 160% in this model. It can be dissolved in water fairly well (5). A third derivative, fac-trisamminetrichlororuthenium(III), fat-Ru(NH,)sCl, (F i g ure 3), is highly active in the P388 leukemia, but in contrast to the latter, it is insoluble relative to the tetrammine complex, in water (5). On, the basis of the ruthenium compounds described, some promising new representatives have been synthesized in Heidelberg during the last few years. To assess the antitumor activity of these ruthenium complexes relative to those known from the literature, we have tested some of the typical representatives in the P388 model (3, 8, 9, 17, 20, 2 l-23, 26). The results of this prescreening are summarized in Table 1. They convey some idea of the possible variations in the activity spectrum. The first three complexes in Table 1 are rutheniumhexachlorides in the oxidation stages III and IV and oxygen-bridged ruthenium chlorides with different, protonated heterocycles as cations. They are inactive, with T/C values < 125y0 (26). The synthesis

Table

1.

Comparison of the antitumor activity of different classes of ruthenium compounds in the P388 leukemia; B = nitrogen heterocycle; R = organic ligand; n = 14

Water-solubility (HB),Ru”‘CI, (HB),(Rd”CI,) (HB),(Ru”‘,OCI Ru”(DMSO),CI, Ru”(DMSO),.,B,CI, Ru”‘(RSR),CI, Ru”‘B Cl Ru”‘“‘&R),Cl cis-[Ru”‘(NH,),Cl,]Cl fat-Ru(NH,),CI, HB-lrans-IRu”‘B,Cl,] (HB),[Ru”‘BCl,] * In accordance

10.)

+ + + + with

reference

5.

T/C (“A) 100-120 100~120 100-120 125 loo-125 130-135 100-130 125-135 160* 190* 14OG200 140-200

264

B. K.

KEPPLER

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of derivatives of the complex Ru(DMSO),CI, (Table 1) with the aim of obtaining compounds of the type RLI(DMSO).+.,B,CI~ (B = heterocycle) was successful only in the case of pyridine and pyridazine derivatives. The resulting compounds exhibited little tumorinhibiting activity, with T/C values below 125%. Hence, they do not have any advantages over Ru(DMSO)~CI, itself (26). The following two complexes in Table 1 are derivatives of fat-Ru(NH,),Cls. Like the latter, they cannot be dissolved in water. We also obtained two complexes of the general formula Ru(RSR)sCI, (RSR = phenylmethylthioether) and the corresponding derivative with ethyl instead of phenyl. Both these complexes are active in the P388 model, but the T/C values of 130% are not very promising. The activity loss of the thioether complexes relative to the trisammine complex may be explained by the difference in molecular structure. Ru(NH,)&Il, is facially configurated whereas the thioether complexes are meridionally configurated. This was confirmed by X-ray analyses (26). The antitumor activity of the compounds of the general formula RUB&I, (B = heterocycle) (Table 1) is not very promising. Only the derivatives with 4-dimethylaminopyridine, pyridine-4-aldehyde, and pyridazine reached T/C values of 12576, which indicate biological activity. All T/C values obtained fall clearly below that of the trisammine complex, which served as standard. Evidence suggests that this may be due to the fact that these derivatives, just as the thioether complexes, are not facially but meridionally configurated (26). We then examined water-soluble ruthenium complexes for antitumor activity. These include binuclear carboxylates of ruthenium. They were selected because of the wellknown tumor-inhibiting activity of the binuclear carboxylates of rhodium and rhenium. The schematic structures of two ruthenium complexes and the T/C values they reached in the P388 system are shown in Figure 4. Acetate and proprionate served as ligands. Both substances are water-soluble and faintly but significantly active, with T/C values of 125 and 133O/, (26).

R=CH30f

C,H,

Dose mmol/kg Ru,(OOCCH,),Cl* Ru,(OOCC,H,),Cl*

*Synthesis Figure

4. Antitumor (median

activity survival

0.21 0.16 0.06 according

to reference

Wk 100 85 32

Therapy on days 1 1 1,5,9

T/C (%) 125 125 133

40.

of two dirutheniumtetracarboxylates time of treated animals/median

survival

against the P388 leukemia; T/C time of control animals) x 100.

(O/o) =

NEW

TUMOR-INHIBITING

METAL

i “\R”/c’ Cl’ ‘Cl

HfP

COMPLEXES

265

2 HIi@

e

Figwe 5. General

structures

of the tumor-inhibiting ruthenium compounds and (HB),(RuBCl,); B = heterocycle.

of the general

formulas

HB(RuB,Cl,)

The water-soluble ruthenium species of the general formulas HB(RuB&I,) and (HB)2(RuBCIj) (Fig. 5), which have been developed by us, show much better activity. Some of the representatives of these classes even reach T/C values of 140-200% in the P388 leukemia. The heterocycles in the anions are mostly trans-orientated. Derivatives with only one heterocycle in the anion are usually a little less active than their corresponding counterparts with two heterocycles. The compounds are synthesized with purified ruthenium(III)chloride and the corresponding heterocycles in a solution ofhydrochloric acid or of ethanol/hydrogen chloride (see Method 1, Table 2). The reaction is fairly difficult to produce and depends on variables such as pH, temperature, and concentration (26). Complexes of the type HB(RuB,Cl,) can in principle be cis- or tramconfigurated. Characterization of the complexes was carried out by means of various methods of spectroscopy such as NMR, IR, and Mossbauer spectroscopy. The trans- configuration was confirmed by X-ray analysis (21, 22, 26). Table 3 compares the results of trans-imidazolium-bisimidazoletetrachlororuthenate(III), HIm(RuIm,C14), ICR in different transplantable tumor models. The column in the middle represents the evaluation parameter-survival time or tumor weight. Apart from showing good activity in the P388 leukemia, ICR reaches T/C values of about 2500/, in the Walker 256 carcinosarcoma, and of about 150% in the Stockholm ascitic tumor. Survival time increases of > 300% could be achieved in the MAC 15A tumor, a transplantable colon adenocarcinoma (3, 26). The subcutaneously transplanted melanoma B16 and the intramuscularly transplanted sarcoma 180 were treated intravenously in order to provide proof of systemic activity. Therapy resulted in a significant reduction of tumor volume to 15% and 450/,, respectively, compared to the control animals (10076). The next important step in preclinical trials includes considerably more sophisticated models for the delimitation of the clinical spectrum of new substances. It was in the course

Table

2.

Method

I:

Preparation

of the Ruthenium HOEt, HCl* HT F

Commercial ‘RuCl, x 3H,O’ RuCl, x 3H,O + 3B + HCI R&l, x 3H,O + 3B + 2HCl Method

2:

‘RuCl, Ru”‘!”

x 3H,O’ solution

*la

= 1N HCl;

HCl;

HB(RuB,Cl,)

HCI

cont. HCl sGFr&Gx

+ Hg

lb = cow.

species

lc = HCl/abs.

and

(HB),(RuBCl,).

solution of Ru”’ HB(RuB,CI,) + 3H,O (HB),(RuBCl,) + 3H,O green solution HB(RuB,Cl,)

, ethanole

of Ru”“”

+ Hg,Cl,J

B. K.

266 Table

3.

Survey of the experimental, tumor systems

Tumor

model

KEPPLER

ET

antitumor activity both transplantable

Evaluation parameter

P388 leukemia Walker 256 carcinosarcoma Stockholm ascitic tumor B16 melanoma, S.C. growing Sarcoma 180, i.m. growing MAC 15A colon tumor AMMN-induced colorectal tumors of the rat ST, median survival toxymethylmethylnitrosamine.

AI,.

time;

of ICR in different and autochthonous,

T/C

ST ST ST TW TW ST TW

TW,

median

Optimum value

(y/o)

200 250 150 15 45 > 300 10

tumor

weight;

AMMN,

ace-

of these investigations that the good activity of ICR in the AMMN-induced colorectal tumors of the rat was found. This is a particularly valuable model, because it is well suited for predicting the clinical activity of new compounds in this type of tumor. The tumors are induced by the carcinogen acetoxymethylmethylnitrosamine (AMMN), and they are macroscopically and microscopically very similar to the corresponding human tumors. Figure 6 shows these tumors in the colon of a rat, together with the structure of the carcinogen that induces these tumors. The sensitivity of these tumors to chemotherapeutic agents is almost the same as that of the corresponding human tumors. The tumors are not sensitive to cisplatin therapy and do not respond to treatment with alkylating agents such as cyclophosphamide. At present 5-fluorouracil is the only drug in clinical use to produce a certain reduction of tumor volume. This effect can also be reproduced in the experimental model (3, 26). Apart from ICR, some other ruthenium derivatives have been tested in this model. Beside ICR, trans-indazolium-bisindazoletetrachlororuthenate(III), HInd(RuInd,Cl,), shows the best activity. The results obtained with these two compounds in comparison to cisplatin and 5-fluorouracil are given in Figure 7. Three different experiments are summarized there. The tumor volume of the control animals was always standardized to lOOq/,, with the result that the therapeutic efficacy of the different substances can be directly compared. Cisplatin is completely inactive in this model, just as it is in the clinical tumor type. The effect of 5-fluorouracil on comparable clinical tumors was also reproduced in this model. Tumor volume decreased to 40%, compared to that of controls. ICR, given at a dose of 7 mg/kg twice a week over ten weeks, reduced tumor volume to 20”/, in one case and to 10% in the other. The indazole derivative-HInd(RuInd&l,)-had already turned out to be less toxic in chronic use, and hence it could be administered at a higher dose. The result was a decrease in tumor volume to only 5% or tumor growth inhibition of 95%. Final evaluation showed that in this group about one third of the animals were tumor-free. This is evidence of successful and non-toxic therapy of colorectal tumors with this drug. These findings are remarkable since cisplatin is completely inactive in this type of tumor. The results promise well for clinical activity (3, 9, 26). As far as drug toxicity is concerned, ultrastructural investigations revealed that the liver and the kidney are the main target organs of TCR at a dose of 110 mg/kg and 2 ml of

NEW

TUMOR-INHIBITING

METAL

COMPLEXES

267

(a) H3C,

0=-N

,N--CH,-o-c-w,

ii

(b)

Figure 6. (a) Structure of acetoxymethylmethylnitrosamine, a carcinogen that was used to induce the colon tumors as shown in (b). These tumors developed in the colon of a rat 20 weeks after the carcinogen was applied for the first time. The multiple adenotumors resemble human tumors already from their outward appearance. (Photo: M. R. Berger, German Cancer Research Center, Heidelberg, FRG).

solubilizer per 100 g of mouse. Other findings include erythropenia and an increase in creatinine and liver enzymes (28). To study chronic toxicity, both the imidazole and the indazole compound were given twice a week at equimolar doses. ICR was administered at 10 mg/kg over 7 weeks, and the indazole compound at 13 mg/kg over 8 weeks. There was no mortality in either experiments, but the animals treated with ICR had significant weight loss in the final 2 weeks, which led to treatment being interrupted after week 7. In contrast, no toxicity was observed with the indazole compound up to week 8. These results were somewhat unexpected. Long-term treatment with the indazole compound has also proven useful in the treatment of colorectal tumors (3, 26). Stability of the pharmaceutical formulation of ICR and its derivatives is an important aspect particularly in connection with clinical use. HPLC investigations showed that the half-life of the compound is roughly 400 min. This means that during the first 30 min more than 95% of the complex remains undecomposed. This stability is quite sufficient for infusion therapy in the clinic. One of the two compounds-trans-imidazolium-bisimidazoletetrachlororuthenate(III),

268

B. K.

Control

Cisplatin

I.5 (0.0049

KEPPLER

ET

5-FU

1 40 (0.066)

AL.

ICR

Hlnd(Rulnd,CI,)

7 (0.015)

I3 (0.022)

1

J Dose-mg/kg

(mmol/kg)

Figure 7. Test results of the two ruthenium compounds ICR = HIm(RuIm,Cl,) and HInd(RuInd,CI,) in autochthonous colorectal tumors of the rat, compared to cisplatin and 5-fluorouracil. Doses were given twice a week over ten weeks. The reduction of tumor volume represented by the shaded columns is statistically significant compared to the control group.

HIm(RuIm,Cl,), HInd(RuInd,Cl,)-will

Non-platinum

ICR,

and trans-indazolium-bisindazoletetrachlororuthenate(III), be slated for clinical studies.

complexes

in clinical studies budotitane

and the development

of

The high standards required of a new drug to be accepted for clinical studies can be seen from the fact that in the last few decades only four non-platinum complexes have been tested clinically on an international level. These include germanium-132, carboxyN-(3-dimethylaminopropyl)-2-aza-8,8ethylgermaniumsesquioxide; spirogermanium, diethyl-%germaspiro-4,5-decanedihydrochloride; gallium salts, and budotitane (INN) (Figure 8). The two germanium compounds and the gallium salts have passed clinical phase I studies and are now about to enter phase II studies, but so far no really promising

NEW

TUMOR-INHIBITING

METAL

COMPLEXES

269

[(G~CH~CH~COOH)~O,]~ Germanium132 Carbaxyethylgermaniumsesquioxide

Gallium salts e.g., Ga(N0313 Galliumnitrate

.2HCI

Spirogermanium N-~3-Dimethylaminopropyl~-2-aza-8,8-diethlyl-8germaspiro-4,5-decanedihydrochloride

I CH, Budotitane (INN) Dlethoxybis(l-phenylbutone-l.3-dionato)titanium(IP)

Figure 8. Four

non-platinum

complexrs

currently

under

clinical

trials

field of indication has been found. Budotitane clinical phase I studies are now almost complete, and the development of this titanium compound can therefore be described in more detail. Budotitane belongs to the class ofbis(fl-diketonato) metal complexes. Antitumor activity of some representatives of these complexes was reported as early as 1982. Here we dealt with the cis-dihalogenobis(l-phenyl-l,3-butanedionato)titanium(IV) complexes with fluoride, chloride, and bromide as further ligands. These complexes effected a doubling or trebling of survival time of animals with the Walker 256 carcinosarcoma or murine leukemias (13, 14). In later studies budotitane, diethoxybis( 1-phenylbutane-1,3dionato)titanium(IV), Ti(bzac),(OEt)*, was selected from this class of compounds for further development (4, 8, 12, 15, 16, 18, 24-26, 30). The bis(P-diketonato) metal complexes can be synthesized from the corresponding metal tetrahalogenides or tetraalkoxides and the diketonates in an anhydrous organic solvent (Figure 9). An exception must be made for the corresponding molybdenum compounds, where the basis is molybdenumpentachloride. The compounds synthesized are six-coordinated, quasi-octahedrally configurated compounds, which may occur either in the cis- or the tram- form (Figure 10). The cZS- configuration is usually favoured, although the trans-isomer should actually be preferred for steric reasons. In the case of the benzoylacetonato complexes, which have been the centre of our investigations and which produce a maximum of antitumor activity, trans-isomers can be obtained only with extremely bulky

B. K.

KEPPLER

0

0

270

ET

AI,.

anhydrous,

MX4+2 RI‘KI

organic

solvent

R3

H

Rz

M[ RJLRJLR3]2x.+ 2HX Figure

9. General

synthesis

of M(p-diketonato)2X2

complexes. X = Hal. R = organic group.

or OR;

M = Ti,

Zr,

Hf,

Ge,

Sn;

substituents such as iodide or p-dimethylaminophenoxy, as proved by means of NMR spectroscopy. Within the cis- and the tram- form different isomers are possible. Their number depends on whether the bound diketone carries the same substituents in I- and 3-position or different ones. At room temperature, however, the isomers within the cis- and the transform can convert into one another in solution, with the result that it cannot be determined which isomer is responsible for biological activity (24, 25). The M@diketonato),X, complexes are relatively difficult to dissolve in water, and they are very susceptible to hydrolysis. Thus a specific formulation had to be found for these compounds. We developed what is known as a coprecipitate, consisting of cremophorEL, propylenglycol, and the drug in the ratio of 9 : 1 : 1. It is easy to produce a micellar solution of the drug in water from this coprecipitate, which will then remain undecomposed over several hours and which is suitable for infusion therapy. This galenic formulation was used for most of the preclinical experiments and the clinical studies carried out so far (18).

Structure-activity

relation

of tumor-inhibiting complexes

his (B-diketonato)

metal

We have synthesized and examined about 200 bis (/I-diketonato) complexes. Table 4 shows a few of these, which have been selected to give an impression of the structure-activity relation. The complexes are listed in the order of increasing activity in the sarcoma 180 ascitic tumor model.

X

Figure

10. Structures

ofthe

Bis(fi-diketonato)

metal complexes

M(b-diketonato)2X,,

cis- and trans-configuration.

NEW Table

TUMOR-INHIBITING

METAL

COMPLEXES

tumor-inhibiting assessed in the

271

4.

Structure activity relation of complexes, M(P-diketonato),X,, system at a dose of 0.2 mmol/kg

fi-diketonato metal sarcoma 180 ascitic

b-diketonate

M

X

Ti

OEt

go-100

Ti

OEt

130-170

Ti

OEt

130-170

Ti

OEt

ZOO-250

Ti

OEt

300

T/C

(“lo)*

0 &

/

1

*T/C animals)

(“/“) = (median x 100.

survival

time of treated

animals

vs. median

survival

budotitane

time of control

It is evident that the complex with unsubstituted acetylacetone is entirely inactive. Activity increases when space-filling groups and, above all, planar aromatic ring systems, e.g. phenyl groups, are substituted at the diketonato ligand. The complex with benzoylacetone as ligand has the best activity of all. This complex, the ultimate example in Table 4, is budotitane. Other aromatic systems at the diketonato ligand, even those which contain heteroatoms such as sulfur, nitrogen and oxygen, show similar activity, but do not have any substantial advantages over budotitane (25). The fact that the planar aromatic system in the periphery of the molecule is important for antitumor activity can also be realized when considering that substitution of this system often leads to a decrease in activity. This, for example, is the case with methoxy groups, nitro groups, and halogen groups, among others (16). We also investigated the dependence of antitumor activity on the central metal and found that activity remains virtually unchanged when titanium is replaced by zirconium, whereas the compound with hafnium as central metal is already

272

B. K.

KEPPLER

ET

AI.

less active, and a marked decrease in antitumor activity can be observed in the molybdenum and tin compounds up to a virtual inactivity in the germanium compound (25). The nature of the leaving group X does not seem to contribute much to the antitumor activity of the substance class. Ethoxide, chloride, bromide, and fluoride with benzoylacetone as ligand show excellent tumor-inhibiting activity. The same derivatives do not show any activity when they have acetylacetone as diketonate ligand. The four derivatives of the type Ti(acac),X, with X = F, Cl, Br, or OEt are inactive with T/C values between 90 and 100~~. In contrast to this, the four Ti(bzac),X, derivatives reach T/C values up to 300%. They are thus highly active, irrespective of the hydrolizable group X. However, pharmaceutical behaviour is considerably influenced by this leaving group, because stability in water clearly increases in the order iodine < bromine < chlorine < fluorine < OR. Thus budotitane, rather than other, analogous compounds, was chosen for further development. The iodine compound is too unstable to be considered for further development, and the bromine and fluorine compounds, apart from pharmaceutical disadvantages, have disadvantages over ethoxide as to the way in which the hydrolizable group is physiologically tolerated (25).

Antitumor

activity

of budotitane

in other

transplantable

tumor

models

Antitumor activity in other transplantable tumor models is summarized in Table 5. T/C values of > 300% were observed in the Stockholm ascitic tumor, in the Ehrlich ascitic tumor, and in the MAC 15A colon tumor, which is a transplantable colon adenocarcinoma. T/C values of 200% were reached in the Walker 256 carcinosarcoma. The subcutaneously transplanted sarcoma 180 can be cured with intravenous budotitane therapy. Tumor weight of the intramuscularly growing sarcoma 180 is reduced to 30%, compared to the control experiment ( lOOoh), given intravenous therapy. It is interesting to note that budotitane would not have been taken into account in a primary screening in the leukemias P388 or L 12 10, because it is only marginally active in these models [T/C = roughly 1300/,). Th ese quick-growing leukemias are certainly not

Table

5.

The most transplantable

important tumors

Tumor Sarcoma 180 ascitic tumor Sarcoma 180 tumor, subcutaneously growing Sarcoma 180 tumor, intramuscularly growing Walker 256 carcinosarcoma P388 leukemia Stockholm ascitic tumor Ehrlich ascitic tumor MAC 15A colon tumor

results

of budotitane

Evaluation parameter

therapy

Optimum value

ST TW

> 300 0

TW

30

ST ST ST ST ST

200 130 > 300 > 300 > 300

in

T/C

T/C values > 3000/ mean that a high percentage of animals is cured; T/C (7;) = (median tumor weight or survival time of treated animals us. median tumor weight or survival time of control animals) x 100; ST = median survival time; TW = median tumor weight.

NEW

COMPLEXES

273

the right model for finding substances that are active in slow-growing tumors. However, the slow-growing tumors, of all cancers, present cancer therapy today ( 18,25).

tumors such as colon the biggest problem in

Budotitane

therapy

TUMOR-INHIBITING

results

METAL

on autochthonous, tumors

AMMN-induced,

colorectal

The high predictivity of autochthonous tumor models for the clinical situation and, more specifically, the high predictivity of AMMN-induced colorectal tumors has already been described. Figure 11 compares the activity of 5-Ruorouracil, cisplatin, and budotitane in this model. Budotitane is markedly more active than 5-fluorouracil. It reduces tumor volume to about 20% of the initial value. 5-Fluorouracil effects a tumor remission up to 40”;, of tumor volume, whereas cisplatin, with a value of about 120%, seems to stimulate tumor growth. Stimulating effects are not infrequent with inactive compounds. These results could be the basis of an interesting clinical indication for budotitane. This is especially important because colon tumors are among the most frequent causes of death from cancer (4, 25).

Toxicity

of budotitane

When budotitane is given to female SD rats in a single intravenous administration, the LDS, is about 80 mg/kg. In mice the LDjo is about twice as high. Given i.p., rats and mice will tolerate somewhat more than twice the maximum tolerated i.v. dose. A dose-limiting factor for budotitane is hepatotoxicity, with multiple focal necroses of the liver at about the level of the LD50. There were also signs of lung toxicity owing to hemorrhagic pleural effusions and hemorrhagic oedematous areas in the lung, which, however, were only found at the level of the highest lethal doses given. Chronic doses at 10 and 20 mg/kg, given twice per week over 10 weeks, were tolerated without problems, with only low and reversible liver toxicity. Laboratory parameters such as the liver enzymes GOT and GPT, as well as LDH, were raised. No myelosuppression was detected. Independent investigations in other laboratories confirmed the level of the LDso to be at about 6070 mg/kg in single intravenous administration. The other toxicological parameters were also confirmed. In addition, signs of extramedullar blood formation were found in the liver and in the spleen. There was mild nephrotoxicity, and, serum alkaline phosphatase was raised. In chronic dosing, up to 18 mg/kg twice a week over 12 weeks were tolerated without mortality, i.e. a cumulative dose of 432 mg/kg, which is seven times the LD,, of a single dose. In this experiment we also observed a mqrked increase in creatinine and urea, which points to nephrotoxicity. Budotitane did not cause emesis in experiments with pigeons. Mutagenicity of budotitane was examined by means of the salmonella typhimurium mammalian microsome assay of Ames. No evidence of a mutagenic potential was found (27). Toxicology of budotitane, particularly chronic dosage, shows that it is well tolerated at levels which could be considered for treatment. Mild, reversible liver toxicity is a prominent feature. Nephrotoxicity is evident only at high doses. Lack of myelosuppression is an advantage of budotitane 124, 25).

B. K. KEPPLER

274

-

Control

Dose:

mg/kg

ET AL.

Cisplatin

0

5-FU

Budotitane

40

IO

1.5

Figure 11. Comparison of the antitumor activity of cisplatin, 5-fluorouracil, and budotitane in autochtonous, AMMN-induced colorectal tumors of the rat. The compounds listed were given twice per week over ten weeks after tumor appearance. The reduction of tumor volume, which is represented by the shaded columns, is statistically significant, in contrast to the control group (Kruskal Wallis Test).

Budotitane

clinical

phase

I study

In 1986 a phase I study with budotitane in cancer patients was begun. In the first part of the study only single doses were investigated, and in the second part budotitane was administered twice a week over four weeks. Seven different dosages were tried in single dose infusions, namely 1, 2, 4, 6, 9, 14, and 21 mg/kg of bodyweight. First signs of drug toxicity appeared at 9 mg/kg, when a patient complained about an impairment of the sense of taste shortly after the infusion. This side-effect continued to be present also at higher doses, and finally a complete loss of taste was observed. This impairment, however, was entirely reversible in all cases and was present only for a few hours after budotitane

NEW

TUMOR-INHIBITING

METAL

COMPLEXES

275

infusion. From a dose of 14 mg/kg onwards, a minor increase in liver enzymes and in lactate-dehydrogenase was observed. At 21 mg/kg, dose-limiting nephrotoxicity was seen with a rise in urea and creatinine (grade 2 WHO criteria). This side-effect was also entirely reversible after some weeks, and urea and creatinine levels returned to normal. No signs of myelotoxicity were found at any dose. The maximum tolerated budotitane dose thus ranges between 14 and 21 mg/kg. In pharmacokinetic investigations blood was taken from patients at 10 min, 1, 2, 8, and 24 h and 7 days after budotitane infusion. The titanium level in the serum and in the erythrocytes was determined by means of atomic absorption spectroscopy. With doses of 14 mg/kg of budotitane, the highest titanium levels were found at 2 h after the infusion (225 pg/g)-levels between 5 and 12 pg/g were found with doses of 21 mg/kg. Titanium was detected in the serum even after 7 days and on one occasion even after 4 weeks. In the erythrocytes, titanium was found at a concentration between 1 and 2 pg/g. After single dose studies, side-effects of chronic budotitane were screened. Proceeding from a maximum tolerated single dose that is slightly below 2 1 mg/kg, a total dose of 2 1 mg/kg (800 mg/m’) was fixed for repeated applications, which was divided into eight applications of 100 mg/m’ twice a week over 4 weeks. Three patients have been treated on the basis of this scheme without any dose-limiting toxicity occurring. In the meantime dosages have been increased to 120 and 150 mg/kg. At this highest dose, only ageusia occurred as sideeffect. It can be assumed that it is possible to considerably increase dosages, because in animal experiments, too, the maximum tolerated total dose in repeated applications was many times higher than in single applications (12, 25, 26). The clinical side-effects resemble those of the preclinical toxicology. The phase I study also confirmed the prediction based on the experiments with pidgeons that budotitane, unlike cisplatin, would not cause emesis. The absence of this side-effect is of the utmost importance for the compliance of the patient. Intensive vomiting, as usually happens with cisplatin therapy, frequently leads to patients interrupting therapy. As had also been predicted in preclinical studies, the main side-effects turned out to be hepato- and nephrotoxicity. Phase II studies will show the extent to which preclinical expectations of the activity of budotitane in adenocarcinomas of the gastrointestinal tract can be confirmed.

Outlook The possibility of being able to cure testicular carcinomas by cisplatin in combination with other drugs provided the impetus for inorganic chemists to develop more new substances. Promising trends can be seen amongst tumor-inhibiting titanium compounds; the development of budotitane; tumor-inhibiting ruthenium compounds and in drug targeting with platinum complexes showing osteotropic properties and affinity to hormone receptors. The future will show the place the new inorganic substances can attain in cancer therapy. It is to be hoped, however, that they can make a contribution to the improvement of cancer treatment. References 1. Alessio, E., Attia, G., Quadrifoglio,

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