Potent Inhibition of Hemangioma Formation in Rats by the Acyclic Nucleoside Phosphonate Analogue Cidofovir1

(CANCER RESEARCH 58, 2562-2567. June 15. 1998| Potent Inhibition of Hemangioma Formation in Rats by the Acyclic Nucleoside Phosphonate Analogue Cidofovir1

Sandra Liekens,2 Gracida Andrei, Michel Vandeputte, Erik De Clercq, and Johan Neyts

Rega Institute for Medical Research, Katholieke L/niversiteit Leuven, B-3000 Leuven, Belgium

ABSTRACT genie therapy could improve the life span of hemangioma-bearing children. At present, IFN-a is being used with relative success for the The acyclic nucleoside phosphonate analogue cidofovir elicited a treatment of hemangiomas (7, 8). The mechanism underlying this marked protection against hemangioma growth in newborn rats that had antitumor action is, however, not completely understood. been infected i.p. with a high titer of murine polyomavirus. Untreated, We have developed a novel animal model for the study of strategies infected rats developed cutaneous, Â¡.in.,and cerebral hemangiomas asso for the treatment of hemangiomas. Infection of newborn rats with a ciated with severe hemorrhage and anemia leading to death within 3 high titer of the Marseille strain of mPyV3 was found to induce weeks postinfection (p.i.). s.c. treatment with cidofovir at 25 mg/kg, once a week, resulted in a complete suppression of hemangioma development cutaneous, i.m., and intracerebral hemangiomas with a short latency and associated mortality when treatment was initiated at 3 days p.i. (100% period. The cerebral hemangiomas were associated with hemorrhage, survival compared with 0% for the untreated animals). Cidofovir still resulting in severe anemia and subsequent death of the animals within afforded 40% survival and a significant delay in tumor-associated mor 3 weeks postinfection (p.i.). Because newborn animals are used in this tality when treatment was started at a time at which cerebral hemangio model, only selective inhibitors with low toxicity may cause protec mas were already macroscopically visible (i.e., 9 days p.i.). Infectious virus tion. This model may thus be relevant to the pediatrie clinical setting or viral DNA was undetectable in the brain at different times p.i. as assessed by means of (a) a DNA-DNA hybridization assay and (/;) titration and suitable for the preclinical evaluation of strategies for the treat ment of this disease. of the brain for infectious virus content, indicating that there was no viral replication in murine polyomavirus-infected rats. Moreover, a semiquan Cidofovir is an acyclic nucleoside phosphonate analogue with titative PCR for viral protein 1 DNA revealed that the amount of viral broad-spectrum antiviral activity (9, 10), which has been approved in protein 1 DNA declined with time after infection to become virtually the United States and Europe for the treatment of cytomegalovirus undetectable at 18 days p.i. Therefore, an antitumor or antiangiogenic retinitis in AIDS patients. Cidofovir has also been found to induce effect, rather than inhibition of viral replication, may be the reason for the regression after topical treatment of severe, relapsing penile, perigeni- inhibitory activity of cidofovir in this model. Cidofovir may thus be tal/intra-anal, and cervical/vulvar lesions associated with HPV (11). further explored for the treatment of vascular tumors and, in particular, Furthermore, we have recently shown that cidofovir also has potent life-threatening juvenile hemangiomas. antitumoral activity against NPCs that were grown in athymic, nude mice (12). INTRODUCTION The present study was designed to evaluate the growth of heman giomas and, therewith, associated mortality in rats after systemic Hemangiomas are benign, endothelial tumors that occur in 10% of treatment with cidofovir at clinically achievable doses (13). all newborns and in up to 22% of preterm babies weighing < 1 kg. In contrast to vascular malformations, hemangiomas are not present at MATERIALS AND METHODS birth. However, they appear shortly postpartum and continue to grow very rapidly for the following 6 months. This proliferative phase is Animals. Rats of the inbred Wistar R strain were used throughout the generally followed by regression of the tumor during the next 5-10 experiments. The rats were obtained from the Animal Production Center of the years (1). Approximately 10% of the tumors do not resolve com Katholieke Universiteit Leuven and were housed under germ-free conditions pletely, and 3-5% become extremely large, involve vital organs, and during the experiments. can cause life-threatening complications, such as ulcÃ©ration,infection, Virus. mPyV (strain Marseille) was grown in MEF cultures and titrated hemorrhages, and Kasabach-Merritt syndrome, which may be fatal with the plaque method of Dulbecco and Freeman in MEF cells as described when left untreated. Treatment with steroids has been recommended before (14). Cells. UC1-B cells (MEFs, ATCC 6465-CRL) and MO cells (murine for these alarming hemangiomas. However, the response rate has been C3H/3T3 fibroblasts) were used. Cells were grown in MEM supplemented with variable, and complications have occurred frequently (2). Radiation 10% inactivated PCS, 1% L-glutamine, and 1% sodium bicarbonate. therapy is no longer used because of its malignant potential, and Animal Studies. Four-day-old rats were infected i.p. with either IO97 or surgical therapy (resection or arterial ligation) is only performed as a IO87 pfu of mPyV. Cidofovir [(S)-l(3-hydroxy-2-phosphonylmethoxypropyl) last resort when other therapies have failed (3, 4). cytosine], kindly provided by Gilead Sciences, Foster City, CA was injected In 1987, Folkman and Klagsbrun (5) put forward the concept of s.c. in 200 p.] of MEM, according to a variety of treatment schedules, as "angiogenic diseases," which are characterized by abnormal, unreg described in "Results." Control animals received the same volume of MEM ulated capillary growth. Examples of diseases in which formation of only. Mortality and the appearance of cutaneous hemangiomas were recorded daily for those rats that were infected with IO97 pfu. Those animals that had blood vessels is no longer under stringent control include diabetic been infected with 10s 7 pfu were killed at 4 weeks p.i. The brains, lungs, liver, retinopathy, psoriasis, and hemangiomas (6). Therefore, antiangio- spleen, and kidneys were dissected and fixed in Bouin's fluid and embedded in paraffin, and sections were stained with H&E. Received 11/13/97; accepted 4/15/98. Statistical Analysis. The two-tailed unpaired Student's t test was used to 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 calculate the differences in the MOD and in the mean lesion score. Statistical 18 U.S.C. Section 1734 solely to indicate this fact. significance of differences in the number of rats with cerebral hemangiomas 1This work was supported in part by Belgian Fonds voor Geneeskundig Wetenschap- was evaluated by the %*test with Yates' correction. pelijk Onderzoek Grant 3.0180.95. J. N. is a postdoctoral research assistant from the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen. 2 To whom requests for reprints should be addressed, at Rega Institute for Medical 3 The abbreviations used are: mPyV, murine polyomavirus; HPV, human papilloma- Research, Katholieke UniversitÃ©Â«Leuven.Minderbroedersstraat 10, B-3000 Leuven, Belgium. virus; MDD, mean day of death; MEF, murine embryo fibroblast; NPC, nasopharyngeal Phone: 32-16-337355; Fax: 32-16-337340; E-mail: [email protected]. carcinoma; p.i., postinfection; VP1, viral protein 1; pfu, plaque-forming unit. 2562

Fig. 1. Effect of cidofovir (25 mg/kg, adminis tered s.c. once a week) initiated at 3, 6, 9, 12, or 16 days p.i. on the incidence of cutaneous hemangiomas in 4-day-old rats infected with 10" pfu of mPyV. The appearance of hemangiomas on paws and tail was recorded daily. S I

2Â«25 2e ,7 â€žâ€ž"~ Days after Infection 27 28 29

Dissection and Processing of Organs. Four-day-old rats were infected i.p. program was run for 28 cycles: denaturation, 94Â°C,45 s; annealing, 57Â°C,45 s; with IO9'7 pfu of mPyV. At different time points after infection (i.e., 4 h or 3, and extension, 72Â°C,1 min. 6, 9, 12, 15, or 18 days p.i.), animals (n = 2) were dissected aseptically, each Determination of the Virus Titers in Brain, Lungs, and Spleen of time using clean scissors and forceps. A parallel group of infected animals mPyV-infected Rats. Titrations of tissue homogenates using 10-fold dilu received s.c. treatment with cidofovir (25 mg/kg, once a week), starting at 3 tions were performed on confluent UCI-B and MO cells grown in 24-well days p.i. These animals were killed at 6, 9, 12, 15, or 18 days p.i., and brain, microtiter plates. The virus stock was used as a positive control. After a 2-h lungs, liver, kidney, and spleen were removed. Individual organs were homog incubation period, residual virus was removed, and the infected cells were enized through a 70-^m nylon cell strainer (Becton Dickinson, Vel, Leuven, further incubated with MEM supplemented with 2% inactivated PCS, 1% Belgium) and were suspended (10% w/v) in MEM containing 2% FCS. Total L-glutamine, and 1% sodium bicarbonate. After 5 days of incubation at 37Â°C, DNA was purified from these tissue suspensions by the QIAamp Tissue Kit the virus-induced cytopathic effect was monitored microscopically. (Qiagen, Westburg, Leusden, the Netherlands). Dot Blot Assay. Five micrograms of denaturated DNA extracted from Determination of VP1 DNA Levels in Different Organs. A semiquanti brains of control and cidofovir-treated animals (dissected at different times p.i. tative PCR was used to detect the presence of VP1 DNA in the different with mPyV) and serial dilutions of denaturated total cellular DNA, obtained organs. The following primers (Gibco, Merelbeke, Belgium) were used for from mPyV-infected MO cell cultures (as positive control), were blotted onto detection of VP1: 5'-GAA AAA GCG GCG TCT CTA AAT G-3' and 5'-ACC a nylon membrane (Hybond-N, Amersham). Subsequently, DNA was UV CCT TCT TCC TTG TAT TTT G-3'. To make quantification of the PCR cross-linked, after which prehybridization was performed for l h at 42Â°C.The products possible, dilutions of the virus stock were made, and PCR products VP1 probe was labeled with digoxigenin-dUTP in a PCR. The probe was gel obtained after different cycles were run on the gel to define the number of purified, and hybridization was performed for 18 h at 42Â°Cwith 30 ng/ml cycles required to obtain a concentration-dependent response (below saturation digoxigenin-11-dUTP-labeled probe. The membrane was washed at high strin level). All PCR reactions were performed using Taq Polymerase (Sphaero Q, gency [2X SSC (1 X SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and Leiden, the Netherlands). For amplification of VP1 DNA, the following 0.1% SDS] for 10 min at room temperature, followed by two washes of 15 min

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Fig. 2. Effect of cidofovir (25 mg/kg, adminis tered s.c. once a week) on hemangioma-associated mortality (n = 5-12) when treatment was started at different times after infection, as indicated. Ther apy was then continued for 6 consecutive weeks.

Days after infection

2563

each in 0.1 XSSC and 0.1% SDS at 65Â°C.After incubation in blocking buffer, the filter was incubated with an anti-digoxigenin antibody conjugated with alkaline phosphatase (anti-digoxigcnin-AP and Fab fragments; Boehringer Mannheim). Detection by means of chemoluminescence was performed by standard methods.

RESULTS

Effect of Cidofovir on Tumor Incidence and Associated Mor tality after Infection of Rats with a High Virus Load. i.p. infection of 4-day-old rats with IO97 pfu of mPyV resulted in the development of cerebral hemangiomas that appeared macroscopically at 9 days p.i., followed by the appearance of cutaneous, i.m., and peritoneal heman giomas at 14 days p.i. Rapid growth of the cerebral hemangiomas associated with severe hemorrhages and anemia resulted in death of the rats within 3 weeks p.i. When treatment with cidofovir (25 mg/kg, S.C.,once a week) was initiated at 3, 6, or 9 days p.i., a complete (days 3 and 9) or almost complete (day 6) suppression of development of cutaneous lesions was observed (Fig. 1). Furthermore, all animals in which treatment was initiated at 3 days p.i. survived, and 25 or 40% survival was observed when treatment was initiated at 6 or 9 days p.i., respectively (Figs. 2 and 3). Survivors were kept for 6 months, at which time autopsy (and histological examination) revealed that the animals were tumor free. A marked delay in tumor-associated mortality was observed for those animals in the day 6 and day 9 group that ultimately succumbed [i.e., MOD, 23.0 Â± 3.8 for the control group compared with 37.7 Â±4.4 (P < 0.0005) and 28.0 Â±1.0 (P < 0.005) for the day 6 and day 9 group, respectively (Fig. 2)]. Even at a dose as low as 5 mg/kg once a week. s.c. treatment with cidofovir resulted in a marked protection (i.e., delay in mortality; Fig. 4). When the start of treatment was delayed until 9 days p.i., at which time cerebral hemangiomas were already clearly visible (as determined after dissection of a parallel control group), a dose of 5 mg/kg cidofovir once weekly still caused a 5-day delay in tumor-associated mortality (MOD, 23.2 Â±1.8 compared with 18.5 Â±0.6 for the control group; P < 0.05). Further more, although hemangiomas were observed in the brains of those animals that succumbed after this delayed low-dose cidofovir therapy, their number was markedly lower than in the control animals, and almost no hemorrhages were noted in the brains of the treated animals. Effect of Cidofovir on Incidence and Development of Heman giomas. To study the effect of cidofovir in a less-aggressive model for cerebral hemangiomas, 4-day-old rats were infected with a 10-fold lower virus load than was the case for the mortality experiments described above. In rats inoculated with this lower virus dose, cerebral hemangiomas developed within 14 days p.i., and cutaneous or i.m. lesions were not observed. Treatment with cidofovir (25 mg/kg, s.c., once a week) was started at different times after infection, and all animals were killed at 4 weeks p.i. Their organs (brain, lungs, kidneys, spleen, and liver) were examined macroscopically and histologically for the appearance of hemangiomas. Macroscopically detectable cer ebral hemangiomas were observed in all control animals. No heman giomas or other tumors were detectable in any of the other organs. Cidofovir therapy, started before the appearance of brain tumors (as revealed by the dissection of a parallel group of control rats at different days p.i.), inhibited the incidence of hemangiomas; i.e., compared with 100% in the control group, none of the rats developed cerebral hemangiomas when treatment was started at 3 days p.i. and only 44% of the rats developed cerebral hemangiomas when treatment Fig. 3. Macroscopic view of cerebral hemangiomas at 18 days p.i., after infection of newborn rats with IO97 pfu of mPyV (a). Cidofovir treatment, initiated at 9 days (fc) or was started at 9 days p.i. (Table 1). In addition, cidofovir was found 6 days (c) p.i., resulted in a significant decrease in number and si/e of the cerebral to cause stabilization or even regression of already established he hemangiomas. whereas treatment started at 3 days p.i. completely protected against mangiomas. Indeed, cidofovir treatment, initiated at 14 days p.i. (at a formation of the lesions (data not shown). time when brain lesions were detectable in the parallel control group), 2564

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0 Â«5 Fig. 4. Effect of 5 mg/kg cidofovir on hemangioma-associated mortality (n = 5-7) when treatment was started at different times after infection, as indi _> cated. The compound was administered s.c. once a > -- control week until the end of the experiment. a 30 day 3 day 6 20 day 9 day 12 10

Days after infection resulted in significantly fewer hemangiomas, which were smaller, extracted from the brains of both control and cidofovir-treated animals than the control tumors (Table 1). (dissected at different times p.i.). This method revealed that there was Histopathology. Tumors had the histopathological features con no detectable viral VP1 (i.e., viral capsid protein, which is produced sistent with cavernous hemangioma, showing large, blood-filled in a lytic virus infection) DNA. In contrast, VP1 DNA was readily spaces lined by endothelial cells and the presence of hemosiderin- detectable in 1 ng of total cellular DNA extracted from mPyV- containing macrophages (Fig. 5). infected MO cell cultures (data not shown). Therefore, the amount of Effect of Cidofovir on Body Weight. The effect of cidofovir (25 viral DNA in 25 mg of brain tissue must be well < 1 ng, indicating that mg/kg, s.c., once a week) on the growth of uninfected rats was there is no, or no detectable, viral replication taking place. In addition, examined. Administration of the compound resulted in a minor delay a semiquantitative PCR for VP1 was performed on brain, lungs, liver, in body weight gain (body weight of the cidofovir-treated animals at spleen, and kidney, collected at different times after infection (Fig. 6). the end of the experiment was 88% of that of the control animals), Serial 10-fold dilutions of the virus stock were used as standards. The when treatment was initiated at the age of 7 days (this would be 3 days amount of VP1 DNA decreased with time after infection, and, with p.i. in the virus challenge experiments). This delay was less pro the exception of the brain, in which a weak signal was still detectable nounced when treatment was started at a later age or when a dose of 18 days p.i., no signal was obtained in the other organs collected at 18 5 mg/kg was used (data not shown). days p.i. As in the untreated animals, a comparable decrease in VP1 Detection of Viral DNA in Different Organs. It has been shown DNA load over time was detected in the organs of those rats that had that mPyV causes an abortive infection in rats; i.e., little, if any, been treated with cidofovir; i.e., at 18 days p.i., only a weak signal replicative virus is produced (14). To confirm these findings, a titra- could be observed in spleen and lungs. tion for infectious virus, of tissue homogenates from brains dissected at different times p.i., was performed. No infectious virus could be detected in any of these organs (data not shown). Next, a dot blot DISCUSSION DNA-DNA hybridization assay was performed using target DNA Neoplastic processes can directly affect the cellular components of the vessel wall, giving rise to hemangiomas, hemangiosarcomas, and Table 1 Effect of cidofovir on tumor incidence and severity in mPyV-infected Kaposi's sarcoma. Hemangiomas are benign, vascular tumors char (IO* 7 pfu) newborn rats acterized by an increased endothelial cell turnover. They appear Startoftreatment(days withcerebralhemangiomas9/9(100%)0/12(0%)4/9 shortly after birth in 10% of all newborns and tend to regress spon lesionbscore3.6 taneously. However, a small percentage of these tumors do not follow p.i.)Control391414Dose(mg/kg)25"25"25"25'Number this course. Instead, they may develop into life-threatening hemangi Â±0.90Â±01.2 omas by obstructing vital organs or may cause hemorrhage, anemia, (44%)6/8 Â±1.51.8 infections, or Kasabach-Merritt syndrome, causing death of the child (75%)6/10(60%)P"<0.001<0.005NSNSMeanÂ±1.51.5 if left untreated (15). With the exception of IFN-a, there is at present Â±1.7Ffâ€¢C0.001<0.005<0.05<0.05 " Statistical significance was evaluated by the x2 test with Yates' correction. Treatment no treatment for these complex juvenile hemangiomas (7, 8). was continued until 4 weeks p.i., at which time all animals were killed. Here we propose a novel in vivo model for exploring new strategies * Mean lesion standard or cerebral hemangioma development was estimated by the for the treatment of hemangiomas; i.p. infection of newborn rats with following standard: 4, large hemangiomas accompanied by multiple hemangiomas of a high titer of the Marseille strain of mPy V results in the development moderate and small size; 3, large hemangioma and multiple smaller hemangiomas; 2, several small hemangiomas or one hemangioma of moderate size; and 1. a single, small of multiple cutaneous, peritoneal, and cerebral hemangiomas associ hemangioma or hemangiomas only detectable by means of histolÃ³gica! examination. ' Statistical significance was evaluated by the two-tailed, unpaired Student's i test. ated with severe hemorrhage and anemia, causing death of the animals Cidofovir was administered once a week, starting at the indicated time after infection. within 3 weeks p.i. We provide evidence that cidofovir is highly e Cidofovir was administered five times a week. effective in this hemangioma model. After inoculation of the rats with 2565

Fig. 5. HistolÃ³gica! section of a cerebral hemangioma showing endothelial cells (open arrowsi sur rounding a hemorrhagic area filled with blood and infiltration of hemosidcrin-containing macrophages (closed arrow]. Magnification, X75.

a high virus load, s.c. cidofovir treatment started at 3 days p.i. resulted was, except for a weak signal in spleen, lungs (cidofovir-treated in complete protection against hemangioma development. When the animals), and brain (control animals), no longer detectable at 18 days p.i. start of treatment was delayed until cerebral hemangiomas were Polyoma virus induces a variety of tumors in rodents and is capable already macroscopically detectable (as evaluated by dissection of a of transforming cell cultures in vitro (18, 19). Three early viral parallel control group of rats), still a marked effect on survival and proteins are assumed to be responsible for this behavior: small, severity of the lesions was observed. middle, and large T (tumor) antigen (20), middle T carrying the Although cidofovir has also been shown to inhibit the in vitro cell-transforming activity. Middle T is predominantly associated with replication of many viruses, including mPyV (16), it is unlikely that the plasma membrane (21) and binds and modulates the activity of this anti-polyomavirus activity accounts for the potent inhibitory several molecules involved in signal transduction, such as the Src effect of the compound on tumor growth, as seen in the present model. family of tyrosine kinases, namely Src (22), Fyn (23), and Yes (24, Earlier studies (17), based on titration of homogenized organs, have 25), SHC (26), and the p85 regulatory subunit of phosphatidylinositol shown that no (detectable) replicative virus is produced in different 3-kinase (27), resulting in constitutively activated tyrosine kinase organs of rats infected with mPyV. To confirm this, in the present receptors. From studies with several transgenic and chimeric mouse study we performed (a) a titration of tissue homogenates for infectious model systems (28, 29), it is evident that part of the tumorigenic virus content, (b) a dot blot DNA-DNA hybridization assay, and (c) a potential of polyomavirus resides in the viral early region, and that semiquantitative PCR for VP1 (i.e., a marker for replicating virus) on polyomavirus middle T antigen-mediated tumorigenesis does not need rat organs collected at different times p.i. Our results indicate that no viral replication or the whole viral life cycle. The rapid kinetics by viral replication takes place in these rat organs after infection with which endothelial cells are transformed by the polyomavirus middle T mPyV, because no viral DNA could be detected by means of titration antigen (18. 28) may account for the fact that only endothelial tumors or dot blot assay, and the amount of VP1 DNA in these organs (as (i.e., hemangiomas) are observed upon infection with the high virus assessed by PCR) gradually declined with time after infection and titer used in this study.

s k li lub s k li lu b s k li lu b s k li lu b s k li lu b

control

Fig. 6. PCR analysis of VPl DNA in spleen (s). kidney (k), liver (//'), lungs (lu), and brain (b) ob tained at different times after infection of 4-day-old rats with l()y7 pfu of mPyV. Cidofovir treatment (25 mg/kg, once a week) was initiated at 3 days p.i.

cidofovir

day 15 day 18

Moreover, cidofovir was found to cause protection against heman- 7. Ezekowitz, A., Mulliken. J. B.. and Folkman. J. Interferon a-2a therapy for life- giomas when treatment was initiated at a time when brain tumors were threatening hemangiomas of infancy. N. Engl. J. Med., 326: 1456-1463. 1992. 8. White, C. W., Sondheimer. H. M., Crouch, E. C., Wilson. H., and Fan, L. L. already macroscopically visible. This activity may be attributable to a Treatment of pulmonary hemangiomatosis with rccombinant Â¡nlerferona-2a. N. Engl. direct antitumor action of the compound (i.e., through apoptosis) or to J. Med.. 320: 1197-1200. 1989. 9. De Clercq, E.. Holy, A., Rosenberg. I., Sakuma. T.. Baizarini. J.. and Maudgal, P. C. an antiangiogenic effect of cidofovir. Recently, it has been shown that A novel selective broad-spectrum anti-DNA virus agent. Nature (Lond.). 323: 464- cidofovir elicits antitumoral activity by inducing apoptosis in both 467, 1986. HPV-carrying and HPV-negative tumor cell lines but not in control 10. De Clercq, E.. Sakuma. T.. Baba, M.. Pauwels. R.. Baizarini. J.. Rosenberg. 1., and Holy, A. Antiviral activity of phosphonylmethoxyalkyl derivatives of purine and cells (30). Also, intratumoral treatment with cidofovir resulted in the pyrimidines. Antiviral Res.. 8: 261-272, 1987. regression of established NPCs grafted to nude mice, and systemic 11. Snoeck, R., Van Ranst, M., Andrei. G.. De Clercq, E., De Wit, S.. Poncin. M., and treatment with cidofovir led to an arrest of NPC growth. Apoptosis Clumeck. N. Treatmenl of anogenilal papillomavirus infections with an acyclic nucleoside phosphonate analogue. N. Engl. J. Med.. 333: 943-944, 1995. appeared to be responsible for this effect (12). 12. Neyts. J., Sadler, R., De Clercq. E., Raab-Traub. N., and Pagano, J. S. The antiviral At present, the stimulus for endothelial cell proliferation leading to agent cidofovir j(S)-l(3-hydroxy-2-phosphonyl-methoxypropyl)-cytosine] has pro hemangioma development in children is not known. There is no nounced activity against nasopharyngeal carcinoma grown in nude mice. Cancer Res., 58: 384-388, 1998. evidence that transformation of endothelial cells is taking place in 13. Naesens. L., Snoeck, R.. Andrei. O.. Balzarmi, J., Neyts, !.. and De Clercq, E. juvenile hemangiomas, because spontaneous involution occurs in 90% HPMPC (cidofovir). PMEA (adefovir). and related acyclic nucleoside phosphonate of the cases. Nonetheless, our rat model does serve as a good func analogues: a review of their pharmacology and clinical potential in the treatment of viral infections. Antivir. Chem. Chemother.. 8: 1-23. 1997. tional model of hemangiomas for the following reasons: (a) the 14. Vandeputte, M., Meyer, G.. and Sobis, H. Vascular tumors induced by polyoma virus tumors are of the endothelial cell type: (b) there is involvement of in pregnant rats. J. Nati. Cancer Inst., 56; 517-521. 1976. vital organs (i.e., brain); (c) rats develop many of the side effects 15. Fishman. S. J.. and Mulliken. J. B. Hemangiomas and vascular malformations of infancy and childhood. Pedialr. Surg.. 40: 1177-1200. 1993. observed in hemungioma-bearing infants (i.e., hemorrhages, severe 16. Andrei, G., Snoeck. R.. Vandeputte. M., and De Clercq, E. Activities of various anemia, and splenomegaly); and (d) akin to the situation in humans, compounds against murine and primate polyomaviruses. Antimicrob. Agents Chemother.. 41: 587-593, 1997. there is macrophage infiltration in the hemangiomas. In addition, 17. Vandeputte, M. Antilymphocytic serum and polyoma oncogenesis in rats. Transplant. because newborn animals are used, only compounds with sufficient Proc.. /: 100-105, 1969. selectivity (i.e., good activity and low toxicity) will lead to protection. 18. BrUuela. L., Olcese, L. M., and Courtneidge. S. A. Transformation by middle T antigens. Semin. Virol., 5: 381-389. 1994. Therefore, the model presented here may be useful for the in vivo 19. Kiefer. F.. Courtneidge. S. A., and Wagner. E. F. Oncogenic properties of the middle identification of novel inhibitors of vascular tumor growth. T antigens of polyomavirus. Adv. Cancer Res., 64: 125-157, 1994. In this study, we have shown that clinically achievable doses of 20. Rassoulzadegan. M.. Cowie, A., Carr, A.. Glaichenhaus. N.. Kamen. R., and Cuzin. F. The roles of individual polyoma virus early proteins in oncogenic transformation. cidofovir (13) potently inhibit the growth of cutaneous and cerebral Nature (Lond.). 300: 713-718, 1982. hemangiomas in newborn rats. This potent antitumor effect was 21. 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Sandra Liekens, Graciela Andrei, Michel Vandeputte, et al.

Cancer Res 1998;58:2562-2567.

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