Efficacy of HMAF (MGI-114) in the MV522 metastatic ... - Springer Link

InvestigationalNewDrugs 14: 161-167, 1996. 9 1996 KluwerAcademic Publishers. Printed in the Netherlands.

Efficacy of HMAF (MGI-114) in the MV522 metastatic lung carcinoma xenograft model nonresponsive to traditional anticancer agents

Michael J. Kelner 1, Trevor C. McMorris 2, Leita Estes 1, Wen Wang 2, Kyra M. Samson 1 and Raymond Taetle 3

1Department of Pathology, UCSD, San Diego, 92103; 2Department of Chemistry, UCSD, San Diego, 92093; 3Arizona Cancer Center, University of Arizona, Tucson, AZ 85724, USA Key words: illudin, HMAF, xenograft, adenocarcinoma

Summary Illudin analogs are cytotoxic to a variety of multidrug resistant cell lines, and display an unusual toxicity towards DNA helicase-deficient cell lines. Earlier illudin analogs demonstrated efficacy in several xenograft models, including a metastatic MV522 lung cancer model, resistant to conventional anticancer agents. These illudin analogs prolonged life span as compared to conventional agents, but did not induce complete remission of primary tumors. In vitro screening studies identified a semisynthetic derivative, hydroxymethylacylfulvene (HMAF, MGI-114), with increased selective cytotoxicity towards carcinoma cells. The HMAF analog was markedly effective in the experimental MV522 metastasizing lung carcinoma xenograft system, a model refractory to treatment with existing anticancer agents. Treatment with paclitaxel, doxorubicin, or cisplatin failed to significantly inhibit primary tumor growth or prolong life span of MV522 tumor-bearing animals. Treatment with mitomycin C at the LD20 increased life span in surviving animals up to 61% (p = 0.04). Treatment with HMAF induced primary tumor regression in all animals and increased life span greater than 150% (p < 0.001). Thus, administration of HMAF inhibited development of lung metastasis in a model refractory to treatment with conventional anticancer agents. These results support further evaluation of HMAF as a therapeutic agent for treatment of solid tumors such as adenocarcinoma of the lung.

Introduction The proportion of lung cancers classified as adenocarcinoma of the lung (ACL) is increasing and outnumbers all other histologic types [1, 2]. In contrast to therapy for small cell lung cancer which is clinically effective [3], systemic chemotherapy for ACL has limited success. The majority of ACL patients have metastases at presentation, and patients with localized disease usually develop systemic disease [4, 5]. Reviews of single agent and combination chemotherapy for ACL concluded that no single agent was active, and no combination of agents was superior to single agent treatment [6, 7]. Sev-

eral studies reported limited success with combination chemotherapy in selected patients [8-12]. A recent meta-analysis of chemotherapy trials for non-small cell lung cancer (NSCLC) concluded chemotherapy in patients with limited disease may prolong life, but only by several months [13]. Thus, for the majority of ACL patients, chemotherapy does not increase survival or quality of life. The difficulty in treating ACL patients is due to development of metastases and development of multidrug resistant (mdr) phenotypes [14]. We identified a class of compounds called illudins which are preferentially cytotoxic in vitro using short exposure times to a variety of carcinomas, including ACL [15-19]. This preferential

162 cytotoxicity for carcinoma tumor cells appears due to rapid intracellular accumulation of toxin by an energy-dependent process [16]. The illudins were also cytotoxic in vitro to a variety of mdr cell lines resistant due to abnormal gpl70/mdrl, gpl80/ MRP, glutathione-S-transferase (pi isoenzyme), cellular thiol content, topoisomerase I, and topoisomerase II expression [20]. Illudins displayed a second unusual property. In contrast to DNA damage induced by other DNA reactive chemotherapeutic agents (such as mitomycin C, BCNU, cisplatin), the repair of illudininduced DNA damage requires the action of the ERCC2 and ERCC3 DNA repair enzymes (also known as DNA helicases of the TFIIH complex), before repair of the damaged DNA can proceed [21]. Due to these unusual properties of illudins, we investigated the ability of illudins to inhibit ACL tumor cell growth in vitro and in vivo [20, 22]. We previously developed a metastatic xenograft model in athymic nude mice utilizing the human lung carcinoma cell line MV522 [22]. Of 10 conventional agents tested against this line in vivo, only mitomycin C at a toxic dose (LD20) caused a mild increase in median life span [22]. The MV522 model was resistant to BCNU, 5FU, cisplatin, doxorubicin, methotrexate, vincristine, vinblastine, paclitaxel, cyclophosphamide, Navelbine and VP-16 as these compounds did not prolong life span in the MV522 model. As described here, we developed an illudin analog which induces significant tumor regressions and increased life span in MV522-bearing animals.

Materials and methods

Cell culture

The MV522 lung carcinoma line used for xenograft studies was derived as described previously [22-24] and maintained in antibiotic-free RPMI 1640 media (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum and 2 mM glutamine in 37~ humidified 5% carbon dioxide incubators. Other cell lines in this study included the myeloid leukemia cell line HL60 [25] and the human B-cell-derived leukemia/lymphoma cell

line 8392 [26]. Colony-forming assay (CFA) and trypan blue exclusion assay for cytotoxicity were performed as previously described [15, 16].

Animals

Balb/c nu/nu 4-week-old female mice weighing 18-22 grams were obtained from Simonsen, Inc. (Gilroy, CA) and maintained in the athymic mouse colony at the University of California, San Diego, under pathogen free conditions using HEPA filter hood. Mice were provided with sterilized food and water ad libitum in groups of 4-5 in plastic cages banded with polyester fiber filter covers. Clean, sterilized gowns, gloves, face masks, shoe and hood covers were worn by all personnel handling the animals. All studies were conducted in accordance with guidelines of the NIH Guide for Care and Use of Animals and approved by the University Animal Care and Use Committee (protocol 3006-2).

Experimental lung metastasis model

Mice were randomized into treatment groups of 4 5 animals for initial studies and groups of 16-20 animals to confirm analog efficacy. Each animal was earmarked and followed individually throughout experiments. Mice received subcutaneous injections of 107 parental cell line MV522 per inoculation over the shoulder. Ten days after subcutaneous implantation, when s.c. tumors were approximately 5 mm x 5 mm, the animals received the stated drugs. Primary s.c. tumor growth was monitored starting on the first day of treatment and at weekly intervals thereafter. Tumor size was measured in two perpendicular diameters. Tumor weights were estimated according to the formula W = [(width) 2 x length/2] [30]. Relative weights (RW) were calculated to standardized variability in tumor size amongst the test groups at initiation of treatment using the formula RW = Wt/Wi where Wi is the tumor weight for a given animal at the beginning of drug treatment and Wt is tumor weight at a subsequent time [30]. Selected animals were necropsied and organs examined for evidence of metastases.

163 Table 1. Drug cytotoxicity (ICs0) in select cell lines Cell line MV522

HL60

8392

Drug

2 hr ~ nM

48 hr aM

2 hr aM

48 hr aM

2 hr nM

48 hr nM

Illudin S Dehydroilludin M Acylfulvene HMAF Cisplatin Mitomycin C Doxorubicin

79 • 11 b 8300 4- 700 2200 4- 100 1200 4- 100 6100 4- 500 1800 4- 40 7704-150

4 4- 1 310 4- 20 350 4- 20 73 • 8 3000 4- 500 720 4- 30 230•

11 4- 4 1700 4- 200 2100 4- 100 830 4- 90 1900 4- 300 820 • 100 2204-20

3 4- 1 300 4- 70 360 • 20 73 4- 8 1200 4- 200 400 4- 90 404-5

363 • 20 56,900 4- 8100 81,900 4- 2700 26,000 • 4500 3700 4- 400 500 4- 27 110+20

8 4- 2 370 • 100 830 4- 150 76 4- 4 340 4- 50 620 4- 60 110+10

"The 2 hour exposure cytotoxicity assay was by CFA and 48 hour exposure cytotoxicity assay was by trypan blue (see Methodology). bThe ICs0 value is reported as the m e a n 4- standard deviation for 3 to 5 experiments.

Conventional anticancer drugs used as control agents for the xenograft studies were obtained from the UCSD Medical Center Pharmacy and formulated with diluent according to directions provided by the manufacturer. Drugs included cisplatin (Platinol, Bristol-Myers-Squibb, Princeton, N J), mitomycin C (Mutamycin, Bristol-MyersSquibb), paclitaxel (Taxol, Bristol-Myers-Squibb), and doxorubicin (Adriamycin-PFS, Adria Laboratories, Dublin, OH).

HMAF isolation and analog synthesis

The precursor compound, illudin S, was isolated from corn steep broth as previously described [27, 28]. The acylfulvene analog was prepared from illudin S by reacting 2 M H 2 S O 4 a s previously described [29]. Synthesis of HMAF from the acylfulvene analog and spectroscopic data for HMAF will be described in detail elsewhere.

Statistical analysis

Comparisons of survival curves were performed using the method of Kaplan and Meier [31]. For comparison of relative tumor weights between multiple groups of animals, ordinary ANOVA followed by Tukey-Kramer Multiple Comparison Post-ANOVA analysis was performed. Probability values less than 0.05 were considered statistically

significant. The life span and relative tumor weight were analyzed using Prism version 1.0 and Instat version 2.02 software, respectively (Graph Pad, Inc., La Jolla, CA).

Results

In vitro toxicity studies

HMAF (hydroxymethylacetylfulvene, MGI-114; see note for patent coverage) was tested in vitro using 3 cell lines representing illudin sensitive and resistant cell lines. MV522 lung carcinoma and HL60 myeloid leukemia cell lines are considered sensitive by virtue of energy-dependent cellular accumulation and in vitro toxicity of illudins [15, 16], and the 8392 B-cell lymphoma/leukemia cell line is considered resistant due to a lack of the energy-dependent accumulation process [ 15]. Results of the in vitro studies revealed the HMAF analog was more cytotoxic to MV522 and HL60 cells with a short exposure time of 2 hours, whereas it was relatively nontoxic at 2 hours to the 8392 cells (Table 1). The HMAF analog was toxic to all 3 cell lines at 48 hours. Due to the short in vivo half-life (tl/2 < 15 minutes) of acylfulvene [32, 33], these results suggest the HMAF analog could possess in vivo anticancer activity. A comparison of HMAF to 3 conventional anticancer agents was performed in vitro (Table 1).

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Figure 1. Efficacy of the HMAF analog against the MV522 xenograft. Controls 20% DMSO/saline (O-O), paclitaxel 4 rag/ kg 5x/wk for 3 wks (*-*), doxorubicin 2.6 mg/kg 3x/wk for 3 wks (x-x), cisplatin 3.2 mg/kg 3x/wk for 3 wks (O-O), mitomycinC 2.4 mg/kg 3x/wk for 3 wks (A-A), HMAF 10 mg/ kg 3x/wk for 3 wks (m-IN). Data points indicate the mean 4standard error of 5 animals.

Figure 2. Dose response study of the efficacy of HMAF analog against the MV522 xenograft. Controls 20% DMSO/saline ((DO), paclitaxel 4 mg/kg 5x/wk for 3 wks (*-*), illudin S 0.25 mg/kg 3x/wk for 3 wks (x_x), HMAF 1 mg/kg 3x/wk for 3 wks (O-O), HMAF 5 mg/kg 3x/wk for 3 wks (A-A), HMAF 10 mg/ kg 3x/wk for 3 wks (BN). Data points indicate the mean 4standard error of 5 animals.

Table 2. Efficacy of HMAF in the MV522 lung carcinoma xenograft model

Xenografi studies

Drug

The efficacy o f H M A F was tested in the MV522 lung carcinoma xenograft model [22]. In the first study (Experiment A), the H M A F analog actually induced tumor regressions in 5 o f 5 animals (Figure 1). Although tumors eventually regrew, there was an increase in life span (Table 2). There was no significant ILS in the animals receiving any o f the 4 other chemotherapeutic agents tested. In a second study (Experiment B), the m a x i m u m tolerated dose of H M A F was determined. A dose-response effect was noted with inhibition o f primary tumor growth (Figure 2) and ILS (Table 2). The m a x i m u m tolerated dose of H M A F was determined to be 10 mg/kg when administered 3 times per week for 3 weeks. Animals receiving lower doses o f H M A F demonstrated an increased survival time without tumor shrinkage (Table 2 and Figure 2). In a third experiment (Experiment C), treatment o f MV522-bearing mice was delayed until day 14. This allowed the primary tumor to enlarge in size. In this study, paclitaxel was also administered subcutaneously based on a previous report of increased efficacy in another xenograft when administered by this route [35]. Again, H M A F markedly increased life span (Table 2), while paclitaxel had no significant effect by either route o f administration.

Experiment Aa Doxorubicin Mitomycin C Cisplatin Paclitaxel HMAF Experiment B Paclitaxel Illudin S HMAF

Experiment C Mitomycin C Doxorubicin Paclitaxel Paclitaxel s.c.b HMAF

Dosage (mg/kg)

% ILS (median)

2.6 2.4 3.2 4.0 10

-9 +14 -9 -9 +125 (i0=.015)

4 0.25 1 5 10 15 20

+37 -2 +42(p=.055) +78(p=.020) +156(p=.008) -47 -53

2.4 2.6 6 20 10

+61 (p=0.04) +7 -9 +26 +163 (p=0.015)

~Alldrugs administered3x/weekfor 3 weeks exceptfor paclitaxel which was administered 5• for 3 weeks. All groups consisted of 5 animals. Median survival day for ExperimentA is 55 days, Experiment B is 43 days, Experiment C is 44 days. UThisgroup of animals received paclitaxel s.c. due to a report of increased efficacy in other xenograft models when administered by this route [35].

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Figure 3. Efficacy of HMAF on prolonging life span. Control animals (N=I6) receiving 20% DMSO/saline ( - - ) ; mitomycin C (N=16) at MTD of 2.0 mg/kg ( ...... ); acylfulvene (N=16) at 13 mg/kg ( ....... ); HMAF (N=16) at MTD of 10 mg/kg ( . . . . . ).

A larger study with 16 animals per group was performed to compare HMAF to mitomycin C and the acylfulvene analog at their respective MTD. When directly compared to mitomycin C and the acylfulvene, HMAF was superior at increasing life span (p > 0.001) and one animal was tumor free five months after the start of the experiment (Figure 3). This animal survived for an additional 3 months without evidence of recurrence of tumor. At necropsy there was no evidence of a s.c. tumor or pulmonary metastases in this mouse. Six nonsurviving mice in each group were necropsied and all mice had visible pulmonary metastasis.

Discussion

Natural products, such as illudins, have been a rich source of active anti-cancer agents and lead compounds for cancer therapy. In previous studies, we showed this class of potent cytotoxic agents had unusual properties which mitigated its cytotoxicity in vitro, including differential rates of uptake [16]. The parent compound, illudin S, was tested in the NCI DTP in vitro and in vivo drug screens and found to be too toxic for clinical use [15]. We have synthesized and tested several analogs which show a superior therapeutic index [20, 34, 36]. The HMAF analog described is the most promising to date.

We developed the MV522 metastatic lung cancer model as a stringent test of in vivo illudin efficacy. Unlike most other xenograft models, this tumor undergoes spontaneous metastases which appear responsible for mortality. Similar to results obtained in patients, this line is resistant in vivo to nearly all conventional chemotherapeutic agents. The exception is mitomycin C which occasionally increases life span at a toxic dose (LD20) and also shows activity against lung cancer in patients [37]. The HMAF analog described here markedly increased life span in the metastatic human carcinoma MV522 xenograft model, and produced a low rate of cure. This analog is the only compound we have tested to date in this model which induces tumor regression in 100% of the animals tested. This is in contrast to the limited efficacy of standard chemotherapeutic agents against MV522 xenograft, which with the exception of mitomycin C, do not inhibit primary tumor growth or prolong life span [20, 22, 34]. Mitomycin C at the MTD dose of 2.0 mg/kg mildly inhibited primary tumor growth, but has minimal effect on the life span of tumorbearing animals. Mitomycin C at a toxic dose (LD20) of 2.4 mg/kg produced partial primary tumor regression in some animals and increased life span by 61% (Table 2). We are now testing HMAF against other tumor xenografts. Few other models are likely to be as relevant as the MV522 model due to the marked resistance of this xenograft to numerous chemotherapeutic agents [22, 34] which reflects clinical experience with carcinoma of the lung in humans. The present and previous studies indicate that illudins are interesting and potentially useful lead compounds for developing chemotherapeutic agents. Their spectrum of activity may be determined by their unusual uptake properties or metabolism, and the present studies suggest compounds such as HMAF merit clinical investigation as therapeutic agents for solid tumors.

Notes

Supported by funds provided by cigarette and tobacco tax fund of the State of California through the Tobacco-Related Disease Research Program of the University of California Award 4RT-0226

166 (MJK) and funds provided by MGI PHARMA, Minneapolis, MN (MJK and TCM). The hydroxymethylacylfulvene (HMAF, MGI-114) analog is covered by U.S. Patent 5,439,936, pending U.S. patent application claims, and pending foreign patent application claims.

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Address for offprints: Dr. Michael J. Kelner, Department of Pathology 8320, University of California, San Diego, Medical Center, 200 WestArbor Drive, San Diego, CA 92103-8320, USA. Fax: (619)543-3730