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Pharmacological and Toxicological Aspects of 4-Demethoxy-3'-deamino-3'-aziridinyl-4'-methylsulphonyl-daunorubicin (PNU-159548)

A Novel Antineoplastic Agent¹

Departments of Pharmacology [C. G., M. R., L. C., C. Al., M. Ci., R. R.], Toxicology [C. Ar., D. M., P. D. T., M. G. L.], GMIS [F. F.], Chemistry [M. Ca.], and Research and Development Oncology, Pharmacia Corporation, 20014 Nerviano, Milan, Italy, and Molecular Pharmacology Unit, Department of Oncology, Istituto di Ricerche Farmacologiche "Mario Negri," 20157 Milan, Italy [S. M.]

	ABSTRACT

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS DISCUSSION REFERENCES

 
4-demethoxy-3'-deamino-3'-aziridinyl-4'-methylsulphonyl-daunorubicin (PNU-159548) belongs to a novel class of antitumor compounds (termed alkycyclines) and is currently undergoing Phase II clinical trial. In the present study, we investigated the in vitro and in vivo antitumor activity, the pharmacokinetics, and the toxicological profile of this compound. PNU-159548 showed good cytotoxic activity in murine and human cancer cells growing in vitro, with an average concentration for 50% growth inhibition of 15.8 ng/ml. The drug showed strong antitumor efficacy in vivo after i.v. and p.o. administration against rapidly proliferating murine leukemias and slowly growing transplantable human xenografts. At nontoxic doses, PNU-159548 produced complete regression and cures in ovarian, breast, and human small cell lung carcinomas. Fourteen of 16 models studied, including colon, pancreatic, gastric, and renal carcinomas, astrocytoma and melanoma, were found to be sensitive to PNU-159548. In addition, PNU-159548 was effective against intracranially implanted tumors. Toxicological studies revealed myelosuppression as the main toxicity in both mice and dogs. The maximum tolerated doses, after a single administration, were 2.5 mg/kg of body weight in mice, 1.6 mg/kg in rats, and 0.3 mg/kg in dogs. In the cyclic studies, the maximum tolerated doses were 0.18 mg/kg/day (cumulative dose/cycle: 0.54 mg/kg) in rats and 0.05 mg/kg/day (cumulative dose/cycle: 0.15 mg/kg) in dogs. PNU-159548 showed minimal cardiotoxicity, when compared with doxorubicin in the chronic rat model at a dose level inducing similar myelotoxicity. Animal pharmacokinetics, carried out in mice, rats, and dogs, was characterized by high volumes of distribution, plasma clearance of the same order of the hepatic blood flow, and short terminal half-life. These findings support the conclusion that PNU-159548 is an excellent candidate for clinical trials in the treatment of cancer.

	INTRODUCTION

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The search for new antitumor drugs has been directed at finding compounds having a novel mechanism of action, activity against resistant cancers, and a higher therapeutic index. One of the strategies we used was to build a new class of derivatives by modifying the anthracycline structure. Anthracyclines have gained a major place in cancer treatment, with activity demonstrated in a wide spectrum of tumors. Because toxic side effects (mainly the dose-related cardiomyopathy) and primary or secondary resistance (mainly MDR³ -1 or MDR-associated proteins) limit their value as anticancer agents (1, 2, 3, 4, 5, 6, 7, 8) , extensive research has been directed toward means of reducing toxicity and increasing activity. It was shown that substitution in the sugar at position C-3' is critical for the ability of drugs to interfere with DNA topo II (9 , 10) , and that the configuration of C-3'-NH₂ is fundamental for the ability of drugs to overcome MDR (11, 12, 13, 14, 15) .

To generate novel compounds with potentially improved characteristics, alkylating substituents were introduced at position C-3' of the amino sugar of idarubicin, a second-generation anthracycline used in clinical practice (16 , 17) . A hindered withdrawing group, such as a methylsulphonyl substituent, was attached at position C-4' of the sugar moiety to increase lipophilicity and to reduce the chemical reactivity of these molecules. This new class of derivatives were found to alkylate DNA and showed high in vitro and in vivo activity on sensitive and resistant tumor models (18, 19, 20, 21, 22) .

The leading compound of this new class is PNU-159548. It was selected for clinical trials because of its outstanding antitumor activity in a number of preclinical tests and its favorable toxicity profile, and it is currently undergoing Phase II clinical evaluation. DNA interaction studies have established that in vitro the primary effect of PNU-159548 is a DNA alkylation with sequence specificity similar to that of conventional nitrogen mustards (20) . Furthermore, despite its anthracycline backbone and its ability to alkylate N⁷ guanine in the DNA, PNU-159548 is highly active in vitro and in vivo against tumors expressing the MDR phenotype and on cells resistant to alkylating agents⁴ . We report here the pattern of in vitro and in vivo antitumor activity of PNU-159548 on murine and human tumors and its preclinical pharmacokinetics and toxicological profile.

	MATERIALS AND METHODS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS DISCUSSION REFERENCES

 
Drugs
PNU-159548 and DX were synthesized by the Pharmacia Corporation (Milan, Italy). The chemical structure of PNU-159548 is reported in Fig. 1 . m-AMSA was purchased from Sigma Chemical Co. (St. Louis, MO). PNU-159548 was dissolved in Cremophor/ethanol 6.5:3.5 v/v or Tween 80 10%, as a powder, or was reconstituted in sterile water as a pharmaceutical formulation (freeze-dried colloidal lipid dispersion formulation). The compounds were dissolved and diluted just before use.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Chemical structure of PNU-159548.

In Vitro Cytotoxicity
In vitro drug sensitivity was determined for HT-29, LoVo, A2780, DU 145, and B16F10 cells according to the sulphorodamine B assay (23) . Exponentially growing cells were seeded 24 h before treatment, exposed to drug for 1 h, and then incubated in drug-free medium for 24 h. Drug effect against CEM, Jurkat, and L1210 cells was evaluated by counting surviving cells on a Coulter ZM Cell Counter (Coulter Electronics, Hialeah, FL). Exponentially growing cells were seeded and exposed to various concentrations of drug for 1 h immediately after seeding, than cells were washed and incubated for 72 h in drug-free medium. The antiproliferative activity of the drug was calculated from dose-response curves and expressed as IC₅₀. All assays were repeated at least three times, and each time consisted of six replicates.

Flow Cytometry
HT-29 cells were treated, 24 h after seeding, with nocodazole (75 ng/ml; SIGMA, St. Louis, MO) and incubated overnight at 37°C. After "mitotic shake off," synchronized (nonadherent) cells were collected. Cells were washed with PBS, seeded (1 x 10⁵ cells/ml) in complete medium, and treated with PNU-159548 and DX. After 8- and 24-h treatment or 8-h drug exposure plus 16 h in drug-free medium, cells were collected, fixed, and processed as described previously (24) . cytofluorometric analysis was performed using a FACScan (Becton Dickinson, San Jose, CA). Cells (10⁴cells/sample) were analyzed, and the fraction of cells in G₀/G₁, S, and G₂-M phases was estimate by using Cell Quest (Becton Dickinson) and Modfit LT (Verity Software House, Inc., Topsham, ME) programs.

Topo II Assay
PNU-159548 was serially diluted and incubated with 250 ng of pRYG plasmid DNA (TopoGEN, Inc.) in the presence of 4 units of purified human DNA topo II. After a 30-min incubation at 37°C, the reaction was stopped by adding SDS (final concentration 10%) and proteinase K (final concentration 50 µg/ml), and the samples were loaded on 1% agarose gel to separate supercoiled from open circular and linear DNA. m-AMSA and DX were used as positive controls.

Animals
Female inbred DBA/2 and BALB/cxDBA/2 (CD2F1) mice 2–3 months of age, weighing 20–22 g, were used for murine leukemias. They were kept under standard laboratory conditions. Female nude Swiss Nu/Nu and HSD:Nu/Nu mice, 4–6 weeks of age and weighing 20–25 g, were used in experiments with human tumors. They were maintained under specific pathogen-free conditions and provided sterile food and water ad libitum. Male and female Crl:CD-1(ICR)Br mice, Crl:CD(SD)Br rats, and beagle dogs were used for pharmacokinetics and toxicological studies. Charles River (Calco, Lecco, Italy) supplied rodents; dogs were purchased from Green Hill (Montichiari, Brescia, Italy). Animal health was routinely tested for the absence of antibodies to a panel of pathogens, including mouse hepatitis virus, Sendai virus, and Mycoplasma pulmonis.

Tumor Models
P388 and L1210 murine leukemias and MX1 human mammary carcinoma were from the National Cancer Institute (Frederick, MD). Human lung (N592, A549, and H460), colon (HCT-116, HT-29, and LoVo), and prostatic (DU 145) carcinomas were from American Type Culture Collection (Rockville, MD). Human pancreatic (CAPAN 1) and renal (Caki-2) carcinomas were distributed by Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia (Brescia, Italy). Human astrocytoma (U-87 MG) was purchased from the European Collection of Animal Cell Culture. Human ovarian (A2780 and IGROV 1) and gastric (GTL 16) carcinomas were provided by Dr. Robert F. Ozols, National Cancer Institute, Dr. Jean Bénard, Istitute Gustave Roussy (Villejuif Cedex, France), and Prof. Paolo Comoglio, University of Turin (Turin, Italy), respectively. Human ovarian carcinoma (MRI-H-207) and melanoma (M-14) were provided by Dr. Gabriella Pezzoni (Milan, Italy).

L1210 and P388 leukemias were maintained by weekly i.p. transplants of 10⁵ and 10⁶ cells/mouse, respectively, in DBA/2 mice, according to Geran et al. (25) . For experiments, CD2F1 mice were injected i.v. with 10⁵ (L1210) or 10⁶ (P388) cells/mouse or intracranially with 10⁴ cells/mouse.

The human solid tumors were transplanted s.c. on athymic mice using 15–20 mg of tumor brei or maintained in vitro as continuos cultures. For drug testing, fragments of tumors or 5 x 10⁵ (H460) or 10⁶ (A431, IGROV 1, HCT-116, and DU 145) cells/mouse were implanted s.c. into the left flank of recipient mice. When the tumor was palpable (0.2–0.3 g), animals were divided randomly into test groups consisting at least of six mice each (day 0).

Drug Administration and Testing
Antitumor Activity.
Drug was administered i.v. or p.o. (by stomach tube) in a volume of 10 mg/kg of body weight, from day 0, according to the indicated schedules. Toxicity was evaluated on the basis of weight loss and gross autopsy findings, mainly in terms of reduction in spleen and liver size.

Drug activity for leukemia models was calculated as ILS of treated animals compared with the control group.

For solid tumors, the length (L) and width (W) of the solid tumor mass were measured by caliper twice weekly and the tumor volume (TV) was calculated as: . The tumor volume at day n was expressed as relative tumor volume (RTV) according to the following formula: , where TV_n is the tumor volume at day n and TV₀ is the tumor volume at day 0. The percentage of tumor growth inhibition (T/C%) was determined by calculating RTV as: . According to the National Cancer Institute standards (25 , 26) , a T/C 42% is the minimum level for activity. A T/C <10% is considered a high activity level.

The tumor cell kill was calculated from the following formula: , where T - C is the median time (in days) required for the treatment-group tumors less the median time required for the control-group tumors to reach a predetermined size (usually 1 g), and Td is the tumor volume doubling time, in days, measured from a best-fit straight line of the control group in exponential growth. The compound was considered active when the log cell kill value was >0.7. Tumor-free survivors (90 days after tumor implant) are excluded from these calculations and tabulated separately.

Acute and Chronic Toxicity.
Single- and repeated-dose toxicity studies in rodents and nonrodents were carried out to investigate both the early and the delayed toxicity of PNU-159548 after i.v. administration. Drug toxicity was evaluated after single injections in mice, rats, and dogs; chronic toxicity studies were performed by administering the drug to rats and dogs for 3 consecutive days every 4 weeks for a total of seven cycles. Table 1 summarizes the number of animals and the doses tested for each study. Control groups were given the vehicle. The animals were checked daily for mortality, behavior, and general condition. Clinical observations and laboratory examinations were performed during the study. A postmortem examination was performed on all animals. Organs were collected and examined histologically.

Pharmacokinetics.
The systemic exposure and the drug disposition of PNU-159548 were evaluated after single i.v. administration in mice, rats, and dogs. Blood (0.5 ml) was withdrawn into heparinized syringes at the following times: (a) pre-dose; (b) 5 and 30 min after administration; and (c) 1, 3, 6, 24, 30, and 48 h after administration. Samples were collected in precooled heparinized tubes, placed immediately in an ice/water bath, centrifuged at 1200 x g for 10 min at 4°C, and frozen at -80°C until analysis. PNU-159548 was assayed by HPLC coupled with tandem mass spectrometry using Turbo IonSpray interface (lower limit of quantitation: 0.25 ng/ml; total run time: 6 min). Pharmacokinetic data analysis was carried out using a noncompartmental approach with the aid of the WinNonlin package (Scientific Consulting, Inc.).

RESULTS
In Vitro Antiproliferative and Cell Cycle Effects
The growth of a panel of human and murine tumor cells in the presence of different concentrations of PNU-159548 or DX is shown in Fig. 2 . PNU-159548 showed dose-dependent antiproliferative activity; the IC₅₀ values of PNU-159548 after 1 h exposure ranged from 1.2 to 81.1 ng/ml. Under these experimental conditions, the compound is more potent than DX (IC₅₀s = 72–1365 ng/ml). PNU-159548 seemed to be more effective against proliferating leukemias (Jurkat, L1210, and CEM) than against human ovarian (A2780), colon (LoVo, HT-29), and prostatic (DU 145) carcinomas and murine melanoma (B16F10) cells.

View larger version (26K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Antiproliferative activity of PNU-159548 (filled circle with solid line) and DX (filled circle with broken line) against a panel of cancer cells. Each cell line was treated for 1 h. Cell growth was determined as described in "Materials and Methods." Values, means ± SE of at least three experiments, each consisting of six replicates.

View larger version (48K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Effects on the cell cycle of HT-29 cells treated with PNU-159548 and DX. HT-29 cells were synchronized with nocodazole as described in "Materials and Methods." Immediately after nocodazole removal, cells were treated with PNU-159548 (5 ng/ml) and DX (200 ng/ml). Effects on the cell cycle were determined after 8- and 24-h treatments and after 8-h treatment and then 16-h recovery in drug-free medium. Percentage of cells in each phase of the cell cycle: , G0/G1; , S; and , G2-M.

View larger version (52K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Effect of PNU-159548 on topo II activity. DNA was incubated with PNU-159548 (1–10 µM) for 30 min in the presence of 4 units of topo II, as described in "Materials and Methods." As positive controls, DX (1–10 µM) and m-AMSA (125 µM) were used.

In Vivo Studies
The initial in vivo evaluation of PNU-159548 was conducted with L1210 leukemia by testing the i.v. and, considering its high lipophilicity, the p.o. administration routes. As shown in Table 2 , comparable antitumor activity was observed after i.v. administration and, although at 3-fold higher doses, after p.o. administration, in accordance with the oral bioavailability of PNU-159548 [F% (absolute oral bioavailability) = 44 ± 14].

Early toxicity.
After single i.v. administration, the MTDs, defined as the dose level inducing a moderate (50–60%) decrease in leukocyte and platelets without other main side effects, were 2.5 mg/kg in mice, 1.6 mg/kg in rats, and 0.3 mg/kg in dogs. In the repeated-dose studies, the MTDs were 0.18 mg/kg/day (cumulative dose/cycle = 0.54 mg/kg) in rats and 0.05 mg/kg/day (cumulative dose/cycle = 0.15 mg/kg) in dogs. In all species, the hemolymphopoietic system was, as expected for a cytotoxic agent, the main target organ; and, in those species in which the compound was given at lethal doses, i.e., mice and rats, myelosuppression was considered to be the most probable cause of death. Peripheral leukocytes and platelets were mainly affected, with variable times of nadir (days 5–7 in mice and rats and day 11 in dogs after a single administration; Table 7 ) and days 10–15 in both rats and dogs in the chronic studies. Erythroid series appeared less sensitive to the cytotoxic effects of PNU-159548 and were marginally affected. The myelotoxicity induced by the compound was transient, and all changes observed proved to be almost completely reversible by 2–4 weeks after treatment. In acute studies, postmortem examination revealed small spleens and thymuses in mice and rats, whereas enlarged spleens were seen in rats after repeated administration. This last finding is to be ascribed to an increased extramedullary hematopoiesis, which is a particularly effective compensatory mechanism in rodents.

Delayed Toxicity (Cardiomyopathy).
The chronic cardiotoxicity study was performed in rats, administering PNU-159548 (weekly for 7 consecutive weeks) and DX as the reference drug (27) . Myocardial lesions were assessed in semithin sections using a qualitative-quantitative score (MTS). At equimyelotoxic doses, the histological examination of the hearts indicated that PNU-159548 was markedly less cardiotoxic than DX, with an MTS of 0.2 for PNU-159548 (0.5 mg/kg/week) and 5 for DX (1 mg/kg/week; Fig. 5 ).

View larger version (112K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Absence of vacuolar degeneration of myocytes after chronic PNU-159548 administration. Several groups of altered myocytes were found after DX (1 mg/kg) treatment (A); conversely, only sporadic single cells were affected by PNU-159548 administration (0.5 mg/kg; B). Semithin section; toluidine blue, x40.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS DISCUSSION REFERENCES

In the studies presented here, we evaluated the antitumor activity of PNU-159548 by an in vitro and in vivo cancer cell line panel and investigated its mode of action; furthermore, toxicity and pharmacokinetic studies were performed to support its clinical evaluation.

PNU-159548 was found to be cytotoxic against both murine and human tumor cell lines in vitro. The median concentration of PNU-159548 required to reduce cell survival by 50% is 15 ng/ml, which is several times lower than DX (493 ng/ml). This difference is probably attributable to the high lipophilicity of PNU-159548, which accumulated more quickly in tumor cells.⁴ Cell cycle kinetic studies with PNU-159548 clearly indicate an S-phase blockage, which is an uncommon feature on the anthracycline mechanism of action. This last finding supports further the identification of PNU-159548 as the lead compound of a novel class of antitumor agents and opens the possibility to investigate innovative combinations with other drugs. As regards the mechanism of action of PNU-159548, in respect to classical anthracyclines, the lack of inhibition of topo II activity is a predictable finding. Previous studies have shown that the presence of bulky substituents at the C-3' position of the amino sugar prevents drug stimulation of DNA topo II cleavage (10) , and, as reported, PNU-159548 is effective on cells presenting the topo II-related MDR.⁴

The most remarkable feature of PNU-159548 was its in vivo antitumor efficacy. PNU-159548 possesses a wide spectrum antitumor activity against rapidly proliferating murine leukemias and on slowly growing transplantable human tumor xenografts. The high lipophilicity of PNU-159548 plays a role in allowing the molecule to cross the blood-brain barrier (efficacy against intracranially implanted tumors has been observed) and to maintain comparable activity after i.v. and p.o. administration routes. These results supported the selection of PNU-159548 as a candidate for clinical investigation.

The toxicological profile of PNU-159548 was defined in mice, rats, and dogs, and target organs were identified after single- and repeated-cyclic-dose i.v. administrations. The main effects of PNU-159548, as expected with a cytotoxic antitumor agent that binds covalently to DNA, are most evident in tissues with high cell turnover. The toxic effects consist mainly of myelosuppression, lymphoid organ cell depletion, and intestinal toxicity. These toxic effects are dose related and reversible. One of the most important problems that has arisen during the widespread use of DX in cancer chemotherapy is related to its cardiotoxicity. The risk of congestive heart failure limits the cumulative dose of DX that can be administered (6) . The cardiotoxicity of anthracyclines observed in most of the species tested in toxicological studies, and mainly in a chronic rat model, has been considered predictive for humans (27 , 29) . PNU-159548 cardiotoxicity, tested in comparison with DX, proved to be negligible.

The pharmacokinetic profile of PNU-159548 is quite different from that of DX (30) , showing a lower volume of distribution and s higher plasma clearance. These results could, at least partially, explain the lack of cardiotoxicity of this new derivative.

Although the mechanism of action of PNU-159548 is not yet fully elucidated, the DNA-interacting characteristics, the effect on cell cycle, and the activity against tumors resistant to alkylating agents and MDR-related drugs, support the consideration of PNU-159548 as a novel cytotoxic antitumor compound with a distinct mode of action.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ S. M. is the recipient of a fellowship from the Italian Federation for Cancer Research (Federazione Italiana Ricerca Cancro).

² To whom requests for reprints should be addressed, at Pharmacology Department, Discovery Research Oncology, Pharmacia Corporation, Viale Pasteur 10, 20014 Nerviano, Milan, Italy. Fax: 39-02-48383398; E-mail: cristina.geroni{at}eu.pnu.com

³ The abbreviations used are: MDR, multidrug resistance; PNU-159548, 4-demethoxy-3'-deamino-3'-aziridinyl-4'-methylsulphonyl-daunorubicin; DX, doxorubicin; m-AMSA, amsacrine; topo II, topoisomerase II; ILS%, percent increase in life span [ILS% = ([median survival time (MST) of treated group/MST of control group] x 100) -100]; MTD, maximum tolerated dose; MTS, mean total score.

⁴ S. Marchini, G. Damia, M. Broggini, G. Pennella, M. Ripamonti, A. Marsiglio, and C. Geroni. PNU-159548, a novel anticancer agent active against tumor cell lines with different resistance mechanisms, submitted for publication.

Received 7/ 5/00. Accepted 1/ 2/01.

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