(CANCER RESEARCH 50. 1954-1959. March 15. 1990]

Activity of Intrathecal 4-Hydroperoxycyclophosphamide in a Nude Rat Model ofHuman Neoplastia Meningitis1

Herbert E. Fuchs,2 Gerald E. Archer, O. Michael Colvin, Sandra H. Bigner, James M. Schuster, Gregory N. Fuller,

Lawrence H. Muhlbaier, S. Clifford Schold, Jr., Henry S. Friedman, and Darell D. BignerDivision of Neurosurgeiy, Department of Surgery [H. E. F.], and Departments of Pathology [G. E. A., S. H. B., J. M. S., G. N. F., S. C. S., H. S. F., D. D. B./, Pediatrics[H. S. F.], Community and Family Medicine [L. H. M.], and Medicine [S. C. S.], and the Preuss Laboratory for Brain Tumor Research [D. D. B.], Duke UniversityMedicai Center, Durham, North Carolina 27710; and the Johns Hopkins Cancer Center, Baltimore, Maryland 21205 [O. M. C.J

ABSTRACT

Neoplastic meningitis can result from leptomeningeal dissemination ofa variety of cancers. We now report the development of animal modelsof human neoplastic meningitis and activity of intrathecal 4-hydroper-oxycyclophosphamide (4-HC) against the human rhabdomyosarcoma cellline 11 -671 and the human glioma cell line D-54 MG grown in thesubarachnoid space of athymic rats. The injection of 5 x IO" II-67I or

D-54 MG cells resulted in leptomeningeal tumor growth from the baseof the brain to the cauda equina. Daily weights and neurological examinations revealed progressive neurological deficits and weight loss, withdeath occurring between Days 21 and 27 for II,-671 and Days 14 and26 for D-54 MG. 4-HC toxicity in non-tumor-bearing rats was assessedat dose levels of 2.0,10.0,15.0, and 20.0 HIM,with clinical and histológica!evidence of neurotoxicity observed at the 2 highest dose levels. Intrathecaltreatment with 4-HC on Day 8 following injection of TE-671 resulted inan increase in median survival of 20% (P = 0.04) at 1.0 IHM4-HC and41% (P < 0.001) at 2.5 mivi4-HC. Intrathecal treatment with 4-HC (2.5HIM)on Day 5 following injection of D-54 MG resulted in an increase inmedian survival of 23% (P = 0.009). These studies show the usefulnessof the athymic rat model of human neoplastic meningitis and demonstratethe efficacy in vivo of intrathecally administered 4-HC against a humanglioma and a human rhabdomyosarcoma cell line and the lack of toxicityat therapeutic levels of 4-HC in normal athymic rats.

INTRODUCTION

Neoplastic meningitis can result from leptomeningeal dissemination of a variety of cancers, either arising from thecentral nervous system or resulting from invasion by lymphoidneoplasms, carcinoma, sarcoma, or melanoma. The majority ofpatients with leptomeningeal disease due to metastatic carcinoma or melanoma have a dismal prognosis, with minimalresponse to i.t.3 chemotherapy and a mean survival of 2-3months (1-4). Patients with primary neural tumors such asmedulloblastoma and glioma which relapse in the CSF pathways demonstrate a similarly poor outcome with a mean survival of only 12-13 months (5,6). Although many of the tumorsmetastasizing to the leptomeninges are radiosensitive, therapeutic dosages of radiotherapy are often limited by the risk ofcentral nervous system toxicity, particularly radionecrosis. Thisis a particular problem when prior therapy has included neu-raxis irradiation (conventionally used for patients with medulloblastoma or pineoblastoma). Intrathecal administration intothe subarachnoid space of a chemotherapeutic agent mayachieve higher drug levels in the CSF than is possible bysystemic administration with consequently greater antineoplas-

Received 12/2/88; revised 12/8/89.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' Supported by NIH Grants CA 11898. CA 43722. CA 44640, NS 20023, NS00958, and T32-NS07304 and by ACS Grant CH 403.

! To whom requests for reprints should be addressed, at Division of Neurosur-

gery. Department of Surgery. Duke University Medical Center, Durham, NC27710.

3The abbreviations used are: i.t., intrathecal; CSF, cerebrospinal fluid; 4-HC,

hydroperoxycyclophosphamide; NM. neoplastic meningitis.

tic activity (and potentially less systemic toxicity). Furthermore,the steep dose-response relationship demonstrated by alkylatingagents suggests that efforts designed to maximize alkylatorlevels in the subarachnoid space of patients with leptomeningealtumor dissemination may result in an improved therapeuticoutcome (7).

Cyclophosphamide is a bifunctional alkylating agent activein a wide spectrum of human malignancies including lym-phoma, leukemia, medulloblastoma, glioma, and breast cancer(8-10). However, the requirement for intrahepatic conversionof Cyclophosphamide (which is a prodrug) into biologicallyactive metabolites precludes its use as an agent for regionalchemotherapy. 4-HC is a preactivated derivative of Cyclophosphamide which spontaneously converts into 4-hydroxycyclo-phosphamide, the principal cytotoxic metabolite of Cyclophosphamide, which itself subsequently converts to phosphoramidemustard, the ultimate alkylating moiety of this prodrug (8). 4-HC is active in vitro against a diverse group of human cell linesincluding medulloblastoma (11), LI210 murine leukemia (8),Burkitt's lymphoma (12), and breast carcinoma (13).

Arndt et al. (14, 15) have recently demonstrated that the i.t.administration of 4-HC may provide higher levels of the activemetabolites than systemic administration of Cyclophosphamidewhile avoiding systemic toxicity. We now report the development of an animal model of human NM using the humanrhabdomyosarcoma cell line TE-671 and the human glioma cellline D-54 MG grown in the subarachnoid space of athymicnude rats, allowing evaluation of the activity and toxicity of i.t.4-HC. The neurotoxicity of i.t. 4-HC was observed only at doselevels of 15.0 and 20.0 mivi. Treatment of TE-671 or D-54 MGNM with 1.0 or 2.5 mivi4-HC resulted in statistically significantincreases in median survival.

MATERIALS AND METHODS

Animal Model

Female athymic nude rats (200-250 g) maintained in the DukeUniversity Animal Laboratory and Isolation Facility were anesthetizedwith ketamine/xylazine (55 mg/ml ketamine, 9 mg/ml xylazine) administered by i.p. injection at a volume of 1 ml/kg. Subarachnoidcatheters were placed using a modification of the technique describedby Kooistra et al. (16). Briefly, rats were placed with the neck flexed 90degrees in a stereotactic frame with tilt adaptor (David Kopf Instruments, Tujunga, CA). A midline sagittal incision was made from theinion to the laminai arch of Cl. The atlantooccipital membrane wasexposed by sharp dissection. The tough, outer membrane and underlying cisterna magna dura were opened using the tip of a 20-gauge needleas a knife, under 5-1 Ox magnification using an operating microscope(Zeiss OPMI 99). A PE-10 catheter (Intramedic, Clay Adams, FranklinLakes, NJ) with a 5-0 stainless steel wire stylet was inserted into thesubarachnoid space and passed along the posterior aspect of the spinalcord to the lumbar region (8.5 cm). A loose knot was tied in the catheterand fixed with dental epoxy (Lang Dental Manufacturing Co., Chicago,IL). The catheter was passed through the skin lateral to the incisionusing a 19-gauge needle. The wound was closed in 3 layers using 6-0

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INTRATHECAL 4-HC IN NEOPLASTIC MENINGITIS

Ethilon (Ethicon Co., Somerville, NJ). After documenting spontaneousflow of CSF, the catheter was occluded using a short length of 2-0stainless steel wire. The animals were allowed to recover for 3-5 days,and only animals showing normal motor and sensory function wereused in experiments (>999c of animals undergoing catheter placement).Injections of tumor cells or drugs were performed through the catheterin volumes of 20-40 /jl using a Hamilton syringe and injector equippedwith a 30-gauge needle. All injections were followed by 20 M'of sterilesaline to rinse the catheter.

Cell Lines

TE-671 is a well-characterized human cell line initially reported tobe derived from a cerebellar medulloblastoma (17) but now known tobe the subline of a rhabdomyosarcoma cell line. RD, established in thesame laboratory 9 years earlier (18-21). D-54 MG is the Duke University subline of A-172 established by Giard et al. (22) from a humanglioblastoma multiforme. Cell culture techniques for both lines were asdescribed previously (23). Both cell lines grown as s.c. xenografts inathymic mice demonstrate sensitivity to cyclophosphamide (11, 24);additionally, the dose-response relationship of TE-671 in cell cultureto 4-HC has been described previously (11).

Optimization of Tumor Injection Volume

For preliminary studies to determine the optimum volume of tumorinjection, TE-671 cells were harvested in log phase into phosphate-buffered saline at 6.2 x IO6 or 1.25 x IO7 viable cells/ml (as assessedby trypan blue exclusion) and 5 x IO5cells injected into subarachnoid

catheters within l h of harvest. Groups of 5 animals each were giveninjections of 5 x IO5cells in either 20 or 40 ¡A.Animals were followed

until death with daily weights and neurological examinations, includingthe presence or absence of the stepping and placing reflex and theability to negotiate a 60-degree incline ramp. These functions have beenreported to correlate with subarachnoid tumor growth in other systemsin contrast to sensory function tests (16). After death, the spinal columnand skull were removed intact and processed for histology as describedbelow.

Survival Studies

TE-671. Initial survival studies were performed in 25 animals giveninjections of 5 x IO5TE-671 cells in 40 ¡t\.One group of 10 animals

was followed until death as described above. A separate group ofanimals was randomized (prior to tumor inoculation) for histologicalexamination of tumor progression on Days 4, 8, 12, 16, and 20posttumor inoculation (3 animals/day).

D-54 MG. Tumor inoculates for subarachnoid administration of D-54 MG were prepared from xenografts growing s.c. in athymic mice,due to the higher tumorigenicity of the disaggregated xenografts compared to D-54 MG grown in cell culture (data not shown). D-54 MGxenografts were grown s.c. in athymic mice, harvested as describedpreviously (24), cut into fine pieces, and dissociated into a single cellsuspension with 0.8% collagenase in a trypsinization flask for 2 h at37°C.The cells were washed twice in phosphate-buffered saline, dilutedto a final cell count of 1.25 x IO7 viable cells/ml (assessed by trypanblue exclusion), and 5x10* cells in 40 ß\were injected into 25 animals.

One group of 10 animals was followed until death. A separate group of15 animals was randomized (prior to tumor inoculation) for histologicalexamination of tumor progression on Days 3, 6,9, 12, and 15 posttumorinoculation (3 animals/day).

Histology

The entire spine and skull were removed en bloc, fixed in 10%formalin for 7-10 days, and then decalcified using RDO solution (ApexEngineering Products Co., Plainfield, IL). The brain and spinal cordwere cut into 6 sections. Coronal sections of the brain were taken atthe level of the coronal suture and the pituitary gland. Axial sectionsof the spinal cord were taken from the cervical, thoracic, and lumbarcord and the cauda equina. Sections were embedded in paraffin, and 6-nm sections were stained with hematoxylin and eosin and examined bylight microscopy.

4-HC Toxicity Studies

For toxicity studies, non-tumor-bearing rats (groups of 10) weregiven injections of 40 ^1 of a 2.0, 10.0, 15.0, or 20.0 mM solution of 4-HC in 0.9% saline synthesized by the method of Hohorst et al. (25).Immediately following injection, the remaining dose solution was snapfrozen in dry ice-ethanol and stored at —¿�135°Cuntil analysis by the

method of Arndt et al. (14). The animals were sacrificed 6 weeksposttreatment and submitted for histological analysis as above. Theseinjections were chosen to provide estimated final CSF 4-HC concentrations of 200, 1000. 1500, and 2000 ¿<Mbased on the rat CSF volumeof 400 Ml(26).

4-HC Activity Studies

TE-671. 4-HC diluted in 0.9% saline was administered via subarachnoid catheter on Day 8 following injection of TE-671 (5 x 10' cells/

animal). In the first experiment, one group of 7 animals was treatedwith 40 /¿Iof a 1.0 mM solution of 4-HC, with a control group of 10animals receiving 40 ^1 of 0.9% saline. In the second experiment, 1group of 9 animals was treated with 40 ^1 of a 2.5 mivi solution of 4-HC, with a control group of 9 animals receiving 40 ^1 of 0.9% saline.Animals were followed as described above, with histopathology obtained on all animals after death. Statistical analysis was performedusing the Wilcoxon rank sum test. P < 0.05 was considered significant.

D-54 MG. 4-HC diluted in 0.9% saline was administered via sub-arachnoid catheter (40 /il of a 2.5 mM solution) on Day 5 followinginjection of D-54 MG (5 x IO5 cells/animal) to 10 animals, with anequal number of saline-treated control animals. Animals were followedand statistical analysis performed as above.

RESULTS

Tumor Inoculation Volume Response

The effect of altering the volume of inoculated tumor cellswas assessed using a constant dose of 5 x 10' TE-671 cells. Ata 20-fil inoculation volume, animals developed progressive neurological deficits and died at Days 14-18. Histological analysisdocumented leptomeningeal tumor extension from the caudaequina to the low cervical cord. With a 40-^1 injection volume,animals died by Days 21-27, with leptomeningeal tumor extending from the cauda equina to the base of the brain (Fig. 1).For all further studies, a 40-^1 injection volume was used.

Survival StudiesTE-671. Animals given injections of 5 x IO5TE-671 cells in

40 M'developed a progressive quadriparesis, with loss of ramp-

r-

Fig. 1. Histological section of athymic nude rat spine demonstrating growthof TE-671 human rhabdomyosarcoma and position of the subarachnoid catheterin an animal which died 21 days following subarachnoid injection of 5 X 10'cells¡n40,J. H & E, x25.

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INTRATHECAL 4-HC IN NEOPLAST1C MENINGITIS

climbing ability and stepping and placing reflexes by Days 18-24, with death occurring by Days 21-27.

D-54 MG. Animals given injections of 5 x IO5 D-54 MG

cells in 40 ^1 developed a progressive quadriparesis, with lossof ramp-climbing ability and stepping and placing reflexes byDays 12-23, with death occurring by Days 14-26.

Histology

TE-671. On Day 4 no tumor could be demonstrated. By Day8 sections from most animals showed small, focal nests oftumor cells in the subarachnoid space (Fig. 2, A, B). By Day 20sections from most animals showed confluent filling of thesubarachnoid space by tumor cells, which extended from thebase of the brain to the cauda equina (Fig. 2, C, D).

D-54 MG. On Day 6 all animals showed focal aggregates oftumor cells in the subarachnoid space. By Day 15 sections fromall animals demonstrated that the subarachnoid space was filledwith tumor cells from the base of the brain to the cauda equina.

4-HC Toxicity Studies

Actual dose concentrations of 4-HC, as measured by highpressure liquid chromatography, were 10.73 DIMfor 10.0 mivi,15.49 mivi for 15.0 mivi, and 21.56 mivi for 20.0 mM. There wasno neurological dysfunction noted in the animals treated with40 n\ of 2.0 or 10.0 HIM 4-HC dose solutions. One animaltreated with 15.0 mivi 4-HC showed a transient loss of inclineramp function; of animals treated with 20.0 mM 4-HC, 3exhibited loss of ramp function and 5 showed a loss of thestepping and placing reflex (Table 1). Histological examinationof the brains and spinal cords from animals treated with 2.0and 10.0 mM revealed no abnormalities, while animals treated

with 15.0 or 20.0 mM showed myelin degeneration with lipid-laden macrophage accumulation in the peripheral white matterof the spinal cord (Table 2). The cervical and thoracic levelswere most frequently affected; lumbar involvement was seenonly in the 20.0 mM dosage group. The lateral white columnwas most often involved, followed by the posterior and anteriorcolumns. The 20.0 mM dosage group showed more frequentinvolvement of multiple spinal cord levels and multiple whitematter columns. In all cases, myelin degeneration was locatedat the periphery of the white matter columns (Fig. 3). Only oneanimal in the toxicity studies died. This animal was in the grouptreated with a 4-HC dose of 2.0 mM and did not exhibit anyneurological dysfunction prior to death or histológica! evidenceof neurotoxicity.

4-HC Activity Studies

TE-671. Treatment with i.t. 4-HC (40 n\ of a 1.0 mM solution)resulted in a delay in the loss of antigravity strength (P = 0.035)and an increase in median survival of 20% (P = 0.04) (Fig. 4).Increase in the dose of 4-HC (40 ¿¿1of 2.5 mM solution) resultedin a further delay in the loss of antigravity strength (P< 0.001)and an increase in median survival of 41% (P< 0.001) (Fig. 5).There was no clinical evidence of toxicity with either dose. Inboth treatment studies, loss of stepping and placing reflexesand ramp-climbing ability preceded death by approximately 3days. Sections from all animals in both the 4-HC treatment andsaline-treated control groups showed a diffuse subarachnoidinfiltrate of tumor cells extending from the base of the brain tothe cauda equina.

D-54 MG. Treatment with i.t. 4-HC (40 n\ of a 2.5 mMsolution) resulted in a delay in the loss of antigravity strength

Fig. 2. Histológica) sections of athymic nude ratspine documenting extent of tumor progression following subarachnoid injection of TE-671 human rhab-domyosarcoma cells on Day 0. A, isolated nest oftumor cells adjacent to the basilar artery at the levelof the base of the brain on Day 8. This was the onlytumor seen in this section. B, isolated nest of tumorcells in the lumbar region on Day 8. C and l>.completefilling of the subarachnoid space by tumor on Day 20at the base of the brain and in the lumbar region,respectively. H & E, x400.

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INTRATHECAL 4-HC IN NEOPLASTIC MENINGITIS

Table 1 Summary of neurological findingsConcentration of 4-HC dose solution (HIM)

Neurological criterion 2.0 10.0 15.0 20.0No. of animals that died 1/10° 0/10 0/10 0/10No. of animals* with a Iran- 0/10 0/10 0/10 5/10

sient loss of stepping andplacing reflex

No. of animals* with a Iran- 0/10 0/10 1/10 3/10

sient loss of incline rampfunction

Total no. of animals'demon- 0/10 0/10 1/10 5/10

strating a loss of either stepping and placing, or rampfunction°This animal survived for 43 days and showed no histological evidence of

toxicity.* Loss of stepping and placing reflex or ramp function was defined as a plus/

minus or minus result on 3 consecutive days.' Some animals exhibited a loss of both neurological parameters.

Table 2 Histological findingsConcentration of 4-HC dose solution (mM)

Histological criterion 2.0 10.0 15.0 20.0

No. of animals with myelin 0/10 0/10 5/10 9/10degeneration/macrophageaccumulation"

No. of animals having neu- 0/10 0/10 1/10 0/10roñalloss with gliosis

Total no. of animals exhibiting 0/10 0/10 5/10 9/10any of the above pathological changes'

' Assessed on sections stained with hematoxylin and eosin.

Some animals showed more than one pathological change. In addition to thepathological findings listed, all animals exhibited a catheter reaction consistingof fibrosis and occasionally acute and/or chronic inflammation. Additionally, 6of 10 animals in the 2000 ^M dosage group showed focal periventricular and/orbasilar pontine mineralization and vascular changes in the anterior spinal arteryconsisting of inflammation or hyaline thickening of the media.

(P = 0.004) and an increase in median survival of 23% (P =0.009) (Fig. 6). Sections in both the 4-HC treatment and saline-treated control groups showed a diffuse subarachnoid infiltrateof tumor cells similar to that seen with TE-671, with extensionfrom the base of the brain to the cauda equina. In some regionsthere was infarction of the underlying brain or spinal cord.Sections from some animals also showed invasion and replacement of portions of the brain and spinal cord with masses oftumor cells.

DISCUSSION

Diagnosis of neoplastic meningitis portends a dismal clinicaloutcome, with the majority of patients surviving less than 1year from initiation of treatment (1-4). Although many of themalignancies that metastasize to the subarachnoid space areradiosensitive, limitations in the radiotolerance of the neuraxispreclude treatment at potentially curative dosages. Those chem-otherapeutic agents that have been administered into the CSF(such as l-/3-D-arabinofuranosylcytosine and methotrexate) arelimited either by marginal activity against the tumors that aremost frequently metastatic to the leptomeninges or by a propensity to produce a leukoencephalopathy that can be clinicallydevastating (27). Identification of drugs with a higher therapeutic index after i.t. administration would be a major therapeuticadvance, particularly in the treatment of solid tumors metastaticto the leptomeninges.

Cyclophosphamide is a bifunctional alkylating agent activeagainst a very broad spectrum of malignancies, including lym-phoma, leukemia, medulloblastoma, glioma, and breast cancer(8, 9). The use of the preactivated derivative of cyclophospha-mide, 4-hydroperoxycyclophosphamide, bypasses the need for

Fig. 3. A, whole mount of thoracic spinal cord exhibiting peripheral whitematter demyelination (arrows). Luxol-fast-blue/H & E, x32. B, high power viewof zone of demyelination. Right half, normal white matter: left half, replacementof white matter with macrophages laden with phagocytized myelin debris (arrows).Luxol-fast-blue/H & E, x520.

intrahepatic conversion of Cyclophosphamide into biologicallyactive metabolites and provides the rationale for regionalchemotherapy. Furthermore, the steep dose-response relationships demonstrated by alkylating agents such as Cyclophosphamide suggest that efforts designed to maximize drug levels mayresult in improvement of the therapeutic outcome (7). Arndt etal. (14) have recently demonstrated the CSF pharmacokineticsof 4-HC following intraventricular administration to rhesusmonkeys and suggested that direct injection of this agent intothe CSF will provide higher peak drug levels in the subarachnoid space than is seen following systemic therapy (thus maximizing therapeutic activity without an increase in systemictoxicity). They demonstrated peak CSF drug levels of 100 MMfollowing intraventricular administration of 4-HC (0.4 mg), alevel that is not achievable with the i.v. administration ofCyclophosphamide at a dose (1 g/m2) associated with markedsystemic toxicity (15). Blood levels of 4-HC following i.v.administration of this agent were undetectable. No acute orchronic neurological or systemic toxicity was seen followingthe intraventricular treatment with 4-HC.

Our studies were designed to develop an animal model ofleptomeningeal dissemination of human neoplasms and evaluate the therapeutic benefit seen following i.t. administration of

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INTRATHECAL 4-HC IN NEOPLASTIC MENINGITIS

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Fig. 5. Neurological function and survival for groups of randomly assignedathymic nude rats with subarachnoid TE-671 treated on Day 8 following tumorinoculation with i.t. 4-HC (40 n\ of 2.5 m\i solution) (n = 9) or saline (n = 9).

4-HC. The human rhabdomyosarcoma cell line TE-671 and thehuman glioma cell line D-54 MG were implanted into thesubarachnoid space of athymic nude rats via a chronic indwelling subarachnoid catheter. The growth of TE-671 and D-54

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Fig. 6. Neurological function and survival for groups of randomly assignedathymic nude rats with subarachnoid D-S4 MG treated on Day S following tumorinoculation with i.t. 4-HC (40 t>\of 2.5 HIMsolution) (n - 10) or saline (n = 10).

MG in the subarachnoid space of athymic nude rats was veryreproducible, with a well-defined progression of neurologicaldysfunction leading to death. Histological analysis demonstrated a progression from nests of tumor cells through theneuraxis to virtual tumor replacement of the subarachnoidspace. Toxicity studies in non-tumor-bearing animals showedneither clinical nor histológica! evidence of neurotoxicity withdoses of 2.0 or 10.0 mM 4-HC. Animals treated with 15.0 or20.0 mM demonstrated a dose-dependent loss of neurologicalfunction, and histológica! analysis demonstrated myelin degeneration of the spinal cord. (The one death in the 2.0 IHM4-HCdose group cannot be attributed to 4-HC toxicity since therewas no evidence of neurological dysfunction prior to death andno histological evidence of neurotoxicity.) Doses of 4-HC (1.0and 2.5 HIM)designed to produce peak CSF drug levels of 100or 250 /¿Mwere chosen for our initial therapeutic studies,reflecting previous measurement of the in vifro 90% inhibitorydose (against TE-671) of 12.8 MM(11). This therapy producedan increase in median survival of 20% with 1.0 HIM4-HC and41% with 2.5 HIM 4-HC in rats bearing leptomeningeal TE-671. Similarly, treatment of rats bearing leptomeningeal D-54MG with 2.5 mM 4-HC produced an increase in median survivalof 23%.

These studies demonstrate the activity of 4-HC in the treatment of human neoplasms metastatic to the subarachnoid spaceand justify further efforts to define an optimal dose scheduleregimen. Kooistra et al. (16, 28) have developed experimentalmodels for meningeal neoplasia using i.t. injections of 9Lgliosarcoma or Walker 256 carcinosarcoma into Sprague-Daw-ley rats, allowing a precise evaluation of the effect of i.t.chemotherapy dose and schedule modulations on therapeuticoutcome. Multiple dose regimens of i.t. diaziquone or mito-mycin C produced significant increases in median survivalcompared to those produced by single-dose regimens of theseagents, suggesting that similar studies with 4-HC in the treatment of leptomeningeal TE-671 or D-54 MG will enable definition of a regimen maximizing therapeutic activity while minimizing toxicity.

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INTRATHECAL 4-HC IN NEOPLASTIC MENINGITIS

These studies, demonstrating the antineoplastic activity ofi.t. 4-HC against human rhabdomyosarcoma and glioblastomacell lines growing in the subarachnoid space of athymic ratsand the lack of toxicity of i.t. 4-HC (2.0-10.0 HIM) in non-tumor-bearing athymic rats, provide the basis for evaluating therole of the i.t. administration of 4-HC in patients with cyclo-phosphamide-sensitive malignancies metastatic to the sub-arachnoid space and primary central nervous system tumorsthat recur or present with NM. Studies in progress will definethe maximal tolerated dose and optimal therapeutic regimenfor i.t. 4-HC as a prelude to initiating a Phase I trial for patientswith recurrent NM from metastatic systemic extraneural neoplasms and from primary brain tumors that seed the neuraxis.

ACKNOWLEDGMENTS

The authors thank Jim Fangmeier and Charles Pegram for technicalassistance and Andrea Tillotson and Ann Tamariz for assistance in thepreparation of the manuscript.

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1959

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1990;50:1954-1959. Cancer Res   Herbert E. Fuchs, Gerald E. Archer, O. Michael Colvin, et al.   Nude Rat Model of Human Neoplastic MeningitisActivity of Intrathecal 4-Hydroperoxycyclophosphamide in a

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