Europ. d. Canter Vol. 13, pp. 433--435. Pergamon Press 1977. Printed in Great Britain

Pharmacokinetics of Adriamycin and Adriamycin-DNA Complex in L1210 Mice and Men

M. STAQUET,*~ M. ROZENCWEIG,* M. DUARTE-KARIM* and Y. KENIS*

&bltraetmAdriamydn when linked to desoxyribonucleic acid gives higher plasma concentrations than adriamycin injected at the same dose.

In mice, under certain conditions, the therapeutic activity of A D M - D N A is higher than AD M.

In men, both forms of the drug induce the same toxicity but for a C × t bigger for A D M - D N A than for ADM.

INTRODUCTION

AD~A~rI'CIN is an anthracycline antibiotic which induces objective responses in many solid tumors and hematologic malignancies [1-3]. Its mechanism of action seems to be the binding of the drug to cellular DNA by inter- calation between base pairs, and inhibition of RNA synthesis [2, 3].

The therapeutic usefulness of adriamycin is however restricted by its toxicity on the bone marrow and heart muscle. Trouet et al. [4, 5], using the concept of lysosomotropic cancer chemotherapy, have been able to decrease toxicity and to increase activity in L1210 leukemia in DBA 2 mice by binding adriamycin and DNA. The concept of lysosomotropic chemotherapy implies that an antitumoral drug firmly linked to an endocytisable and digestible carrier will penetrate only cells with a high endocytic activity. In the cell, the carrier will be digested by the lysosome and the drug will become free to diffuse. Cells which do not possess both high endocytic and mitotic activities would then be protected from the action of the drug.

The present investigation was performed to compare the pharmacokinetic properties of adriamycin when infused alone or complexed with DNA.

*Service de MSdecine et d'Investigation Clinique, Institut Jules Border, Brussels, Belgium.

]'Service de Statistique M~dicale, Facult~ de M~decine, Universit~ Libre de Bruxelles, Belgium.

MATERIAL AND METHODS

The adriamycin-DNA complex (ADM- DNA) was prepared as described by Trouet et al. [4] by mixing adriamycin and a DNA solution (Herring sperm type VII ; Sigma, Saint Louis) with a Molar ratio of DNA mononucleotides to adriamycin equal to 20. The apparent binding constant of ADM to DNA is 0"41 • 106 M -I [5].

L1210 leukemia was obtained from Trouet [5] and maintained in DBA2 female mice (Ch. Rivers Laboratory). Ascites tumor cells were counted in a hemocytometer and diluted in Hank's balanced solution. 0.1 ml (10 s cells) was injected i.v. into DBA2 mice, 8-10 weeks of age, weighing 20-24 g.

A single dose of adriamycin (ADM) or ADM-DNA (6 mg/kg of adriamycin in both cases) was injected intravenously 24 hr after L1210 cells inoculation. At selected intervals, venous blood samples were collected from the inferior vena cava of anesthetized animals. Heparinized pooled blood of 3 groups of 6 mice was centrifuged immediately and the plasma was frozen at -20°C for a maximum of one month. In men, ADM or ADM-DNA was administered by intravenous infusion to 2 groups of 4 patients at the dose of 75 mg/m 2 of body surface area in 1-3 hr. Ten milliliter samples of heparinized venous blood were collected before drug administration and at specified intervals thereafter. The samples were immediately centrifuged, and the plasma

433

434 M. Staquet, M. Rozencwdg, M. Duarte-Karim and 1". Kenis

separated, frozen and kept at -20°C for a maximum of one month.

Total plasma fluorescence assay Total plasma fluorescence was assayed by a

modification of the method of Bachur et al. [6]. Frozen plasma was thawed and sonicated for dispersion of insoluble material. Absolute ethanol (1.5 ml) and hydrochloric acid 2 N (0.45ml) were mixed with each plasma sample (1 ml).

The mixture was allowed to stand at 4°C for 24 hr. The samples were centrifuged at 50,000 x 0 in a Beckman Spinco L265B ultra- centrifuge, for 25 rnin and the fluorescence of the plasma acid alcohol extracts was deter- mined in a Zeiss PMQ3 spectrophotofluori- meter.

An activation wavelength of 465 m/~ and an emission wavelength of 595 m/J were used. The concentration of adriamycin equivalents in the plasma samples were determined by direct comparison of the relative fluorescence intensity of the plasma extracts with a calibra- tion curve.

A standard curve was performed for each set of estimations. A corresponding blank containing no drug was aiso carried through the above procedure, and the relative fluores- cence intensities were corrected by subtracting the value obtained for the blank (endogenous fluorescing materials) from that of each of the extracts. All samples were analyzed in triplicate. All results are given in adriamycin equiva- lents.

RESULTS

l . Recovery studies Standard curves obtained when the ADM

and ADM-DNA were added to plasma (0.01-5/~g]ml) were identical with curves obtained by adding adriamycin directly to plasma supernatant obtained after protein precipitation. Acid-alcohol extraction of standard solutions of ADM and ADM-DNA in plasma resulted in a 90-100% recovery of both drugs above 0.01 gg/ml concentration in plasma. The relationship between the relative fluorescence intensity and the concentration of ADM and ADM-DNA in the standard solutions is linear in the range of experimental data.

2. Pharmacokinetics in mice with L 1210 leukemia Plasma concentrations of ADM equivalent

are displayed in Fig. 1. There is a large differ- ence between plasma concentration during the first two hours after drug injection. How-

\ x

10 A

0.1 HOURS i ~ § ~.

Fig. 1. Plasma concentrations of A D M equivalents in DBA/2 mice with L1210 leukemia after i.v. injection of 6 mg/kg A D M or ADM-DNA. Least square curves and

means of experimental values.

50

,~g/mt

I0

O.t HOURS

Fig. 2. Plasma concentrations of A D M equivalents in men after i.v. infusion of 70 mg/kg. Least square curve and means

of experimental values.

ever, after the third hour, both drugs give the same plasma concentrations.

3. Pharmacokinetics in men with various neoplasia Plasma levels of ADM equivalents after

cessation of the infusion are shown in Fig. 2. ADM-DNA in spite of a longer infusion time,

Pharmacokinetics in L1210 Mice and Men 435

gives plasma concentrations of ADM equi- valents about 8 times higher than ADM just after cessation of the infusion. At the 10th hr, the same plasma concentration of ADM equivalent is seen with both forms of the drug.

4. Toxicity and therapeutic effwacy It has been shown that ADM-DNA is less

toxic than ADM in mice, and an increased activity in some sublines of leukemia L1210 has been demonstrated [7]. In solid tumors, the complex, when given at the dose of 6 rag/ day for 5 days, was more active than adria- mycin alone in Melanoma B16 when adminis- tered i.p. In Lewis Lung carcinoma and L1210 transplanted s.c., adriamycin was shown to be superior to the complex [8, 9].

In men, a phase I study suggested that the toxicity induced by the complex was similar to that observed with the free drug [10].

DISCUSSION

In mice and men, plasma levels of ADM and its metabolites are strikingly different for both forms of the drug. The most important factor

playing a role in these differences seems to be the size of the macromolecule of ADM-DNA which hampers the passage through the capillary barrier. After a few hours, plasma levels of ADM equivalents are the same for both drugs in mice and men, as if the complex ADM-DNA is dissociated either by pino- cytosis or by the action of circulating desoxy- ribonuclease.

Therapeutic results observed in mice [8, 9] have indicated that ADM-DNA is superior to ADM in intraperitoneaUy transplanted malignancies whereas the contrary is seen in solid tumors, although the optimal schedule was not used for ADM. This can be interpreted as due to the slow diffusion of ADM-DNA from well-perfnsed compartment to the tissues and the tumors. This hypothesis has been confirmed in the case of the isolated perfused heart of the rat where ADM-DNA was shown to be less toxic than ADM [11].

In men, no controlled therapeutic trial has been conducted so far. A recent phase I study [10] suggested that hematologic toxicity is identical with both drugs in spite of a higher C × t for ADM-DNA.

REFERENCES

1. R.H. BLUM and S. K. C~mTER, Adriamycin, a new anticancer drug with signi- ficant clinical activity. Ann. int. Med. 80, 249-259 (1974).

2. S . K . CARTER, A. DI M~RCO and M. GHIONE, International Symposium on Adriamycin. Springer, New York (1972).

3. M. STAQUET, H. TAONON, Y. KEms, G. BONADONNA, S. K. CARTER, G. SOKAL, A. TROUET, M. GmoNE, C. PRAOA, L. LENAZ and O. ~.xtM, Adriamycin Review. E.O.R.T.C. International Symposium. European Press Medikon, Ghent (1975).

4. A. TROUET, D. DEPFa~Z-Dr CAMPEm~E~ and C. DE Duw, Chemotherapy through lysosomes with DNA-Daunorubicin complex. Nature New Biol. 239~ 110 (1972).

5. A. TROUET, D. DEPREZ-DE CAMPENEERE, M. DE SMEDT-MALENGKAUX and G. ATASSI, Experimental leukemia chemotherapy with a "lysosomotropic" adriamycin DNA complex. Europ. J. Cancer 10, 405 (1974).

6. N.R. BACHUR, A. L. Moo~ , J. G. BE~STEm and A. LIu, Tissue distribution and disposition of daunomycin in mice: fluorometric and isotopic methods. Cancer Cheraother. Rep. 54, 89 (1970).

7. G. ATASSI, H.J. TAONON and A. TROtr~T, Comparison of adriamycin with the DNA-adriamycin complex in chemotherapy of L1210 leukemia. Europ. J. Cancer 10, 399 (1974).

8. G. ATAssx, M. DUARTE-I~RIM and H. J. TAONON, Comparison of adriamycin with DNA-adriamycin complex in chemotherapy of experimental tumors and metastasis. Europ. J. Cancer 11~ 309 (1975).

9. G. ATmsx, H. J. TAONON and M. STAQtn~T, Comparison of adriamycin (NSC-123127) and DNA-adriamycin complex in chemotherapy of experi- mental tumors and metastases. In Adriamycin Review (Edited by M. Staquet) European Press Medikon, Ghent (1975).

10. M. ROZENCWEIO, Y. KENIS, G. ATASSI, M. STA~UET and M. Du,,a~aTs-K~M, DNA-adriamycm complex: preliminary results in animals and man, Cancer Chemother. Rep. 6, 131 (1975).

11. A. LANGSLET, I. OYE and S. O. Lm, Reduction of the immediate cardiac toxicity of adriamycin and daunorubicin when bound to DNA. In STA~UET et al., Adriamycin Review, Edited by M. STAQVET, Ghent, European Press Medikon (1975).