A new in vivo anti-viral assay using microencapsulated infected cell cultures.

Microencapsulation technology makes it possible to encapsulate virus infected human or animal cells in microcapsules with semipermeable membranes. These may be implanted intraperitoneally into mice which may then be treated with antiviral drugs. The implanted microcapsules may be recovered at various intervals following in vivo treatment and the effect of the drug is evaluated by assaying the virus titers inside the microcapsules. In this paper, the feasibility of this model was tested using microencapsulated human or non-human cells infected with herpes simplex virus type 1. The microcapsules were implanted in the peritoneal cavity of mice, and the effect of systematically administered acyclovir on HSV-1 replication was ascertained. We found that (a) HSV-1 can replicate in both human (A549 and FEMx) and non-human (Vero) cells after they are infected and encapsulated. (b) HSV-1 replication was inhibited by 0.005 microgram/ml to 0.08 mg/ml of acyclovir in the medium when virus producing A549 cells were encapsulated or when they were in monolayers. (c) Acyclovir (20-80 mg/kg), injected twice daily by intraperitoneal, subcutaneous or intravenous routes in mice, significantly inhibited HSV-1 production in encapsulated Vero cells implanted in the peritoneal cavity. The major advantage of this in vivo model is that it can be used to study antivirals in experimental animals in which viruses do not replicate in non-permissive animals. Toxicity, pharmacokinetic and efficacy data may be obtained. It can also be used to test drugs which require activation in vivo to be effective.

Microencapsulated tumor assay: new short-term assay for in vivo evaluation of the effects of anticancer drugs on human tumor cell lines.

A new in vivo has been developed for evaluating the antitumor activity of chemotherapeutic drugs. The assay is based on a microencapsulation technology developed by Damon Biotech, Inc., Boston, MA, which makes it possible to encapsulate human tumor cells in small (about 1 mm in diameter) microcapsules with semipermeable membranes. Microcapsules containing human tumor cells were injected i.p. into nude or C57BL/6 mice and drugs were administered i.v. The microcapsules were recovered at various intervals following treatment and determinations of drug effects were made based on the differences in the number of tumor cells recovered from the treated and nontreated animals. Using this assay we found that (a) encapsulated tumor cells grew better in the in vivo system than in vitro under the conditions tested; (b) drugs crossed the capsular membrane and killed or inhibited the proliferation of tumor cells; and (c) the antitumor effect was consistent with the relative therapeutic efficacy of drugs or level of resistance of tumor cells detected by other in vitro or in vivo tests. The tumor microencapsulation assay offers several properties which make it attractive for use in new drug development: (a) the antitumor activity of drugs can be tested against human tumor cells under conditions which provide for three-dimensional growth and in vivo supply of nutrients; (b) the sensitivity of tumor cells can be assessed following exposure to drugs at concentrations which are achievable in vivo; (c) compounds requiring in vivo metabolic activation can be tested; (d) the effect of each drug injection can be quickly evaluated; (e) inhibition of tumor cell proliferation versus cytoreductive effects of drugs can be discriminated; (f) the test is applicable to virtually all histological types of human tumor cells; and (g) the tumor microencapsulation assay is a short-term, simple, and relatively inexpensive assay.

Effects of ethylene glycol monomethyl (EGME) and monoethyl (EGEE) ethers on the immunocompetence of allogeneic and syngeneic mice bearing L1210 mouse leukemia.

The effect of ethylene glycol monomethyl ether (EGME) and ethylene glycol monoethyl ether (EGEE) on cell-mediated immunity was evaluated by an allograft rejection assay. Allogeneic B6C3F1 (C57BL/6 X C3H) mice were given oral doses of 600, 1200, or 2400 mg/kg/administration of EGEE or 300, 600, 1200 mg/kg/administration of EGME on days -12 through -8 or cyclophosphamide (Cy) at 180 mg/kg by the IP route on day -1. Untreated controls were given oral doses of water on days -12 through -8 and -5 through -1. On day 0, the mice were challenged with 1 X 10(2), 3 X 10(3), and 1 X 10(5) or 3 X 10(6) L1210 cells by the IP route. Syngeneic CD2F1 (Balb/c X DBA/2) mice were challenged with 1 X 10(5) L1210 cells on day 0 and were treated on days 1 to 5 and 8 to 12 with the same dosages of EGME and EGEE used for the B6C3F1 mice. Water-treated syngeneic mice died with a median survival time (MST) of 8.0 days. There was no effect on the MST of syngeneic mice treated with either EGME or EGEE, indicating no direct antitumor effect of the compounds. All allogeneic mice receiving either water or Cy and challenged with 3 X 10(6) tumor cells, died with ascites. However, when mice were treated with EGME or EGEE and challenged with 3 X 10(6) tumor cells, no more than one animal per group died. This would indicate that there was a prophylactic action of the compounds or that the immune system was stimulated. Blood smears of allogeneic mice were made for differential counts the last day of drug dosing, the day of death where possible, and on survivors at day 43 post-tumor implantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Human brain tumor xenografts in nude mice as a chemotherapy model.

Two human brain tumors which were previously established in nude mice were used to determine antitumor efficacy of various therapeutic agents. These tumors were a medulloblastoma (TE-671) and a glioma (U-251) with mass doubling times of 3.5 and 5.5 days respectively as subcutaneous implants in nude mice. Intracranial (i.c.) tumor challenge was accomplished by inoculating tissue culture-grown cells of either tumor into the right cerebral hemisphere to a depth of 3 mm. Median survival time (MST) in untreated mice with 10(5) i.c. injected TE-671 cells was approximately 30 days and 53 days in the U-251 tumor. With 2 X 10(5) U-251 tumor cells the MST was 27-31 days. Groups of mice which had been inoculated with tumor were treated with various doses and schedules of antineoplastic compounds by the i.p. route. The TE-671 tumor responded to AZQ treatment with an increase in life span (ILS) of 37% compared to untreated controls and an ILS of 30% with CCNU treatment. BCNU and PCNU were ineffective. With the U-251 tumor BCNU produced an ILS of greater than 60%, with 75% cures, greater than 112% ILS with PCNU and 49% ILS with CCNU. Neither tumor responded to procarbazine, PALA, dianhydrogalactitol, D-O-norleucine or dibromodulcitol. The U-251 tumor was treated on various schedules and doses with BCNU and found to respond well on late as well as early treatment. A new drug (rapamycin) being investigated by the NCI was found to be very effective against the U-251 tumor. This model system should prove valuable in assessing the effects of various chemotherapeutic modalities against brain tumors.

Ultrasonic hyperthermia and drugs as therapy for human tumor xenografts.

Ultrasonic energy can generate controlled, local hyperthermia (42 degrees C--43 degrees C), and ultrasonic hyperthermia results in increased cytotoxicity of chemotherapeutic drugs for the treatment of cancer. Human breast (MX-1) and lung (LX-1) tumors implanted subcutaneously in athymic nude mice were treated with a single application of ultrasonic (670 kHz, peak intensity of 5 watts/cm2) hyperthermia (43.0 degrees C +/- 0.5 degrees C) for 30 minutes in combination with suboptimal doses of cyclophosphamide, melphalan, or procarbazine. Delays in the tumor growth rate and temporary tumor regression were observed. Comparison of the tumor growth rate with single modalities, ie, drug or hyperthermia alone, shows evidence of synergistic effects of the combination of ultrasonic hyperthermia and melphalan on MX-1 and of hyperthermia and procarbazine on LX-1 tumors.

Combined modality treatment using radiation and/or chemotherapy in an athymic nude mouse-human medulloblastoma and glioblastoma xenograft model.

A human medulloblastoma (BN-2) and a glioblastoma (BN-3) which were previously established in nude mice were used to determine the effect of combined modality therapy with gamma-radiation, and three chemotherapeutic agents, procarbazine, 1,4-cyclohexadiene-1,4-dicarbamic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo diethylester (AZQ), and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The tumor cells were grown in tissue culture and implanted intracranially in the right cerebral hemisphere of NIH Swiss nude mice to a depth of 3 mm. The mice were randomized, and treatment was started 3 days after tumor implantation. Procarbazine and AZQ were injected i.p. every 5 days for three treatments. BCNU was injected one time for a single treatment. Radiation was localized to the head. A 60Co unit was used for irradiation at the rate of 125 rads/min 3 days after tumor implantation. Ten experiments were performed using six to nine mice per group and different drug-radiation dose combinations. The drug dose ranged from 400 to 500 mg/kg/injection for procarbazine, 7.5 mg/kg/injection for AZQ, and 10 to 20 mg/kg/injection for BCNU. The radiation dose ranged from 320 to 1050 rads/mouse (whole head). The day of death was recorded for each animal, and the mean of each treatment group was used to calculate the percentage increase in life span (ILS) compared to the untreated control group. Chemotherapy alone produced a minimal effect, while radiation alone produced minimal effects at 320 to 640 rads with progressively positive effects at 800 and 1050 rads. When the combination treatment of the human medulloblastoma xenograft with procarbazine was used, the ILS was significantly increased in all four experiments, ranging from 25 to 41%, and was superior to single-modality treatment in all but the 1050-rad treatment, where it showed an equal effect. The combination treatment using AZQ and BCNU showed no ILS for the medulloblastoma tumor. Combination treatment of the human glioblastoma xenograft using BCNU produced significant ILSs of 105 and 119% and was superior to single-modality treatment with a drug dose of 10 mg/kg and radiation doses of 540 and 800 rads, respectively. The nude mouse-human tumor xenograft model was found to be useful for combined modality studies and should give valuable information for the experimental design of pilot Phase III clinical studies against a variety of brain tumors.

Collagenase inhibitors retarding invasion of a human tumor in nude mice.

Tumor invasion has been correlated with the ability of tumor cells to produce collagenolytic enzymes which are capable of degrading normal host tissues. However, the human small cell carcinoma implanted subcutanouesly and growing progressively in athymic (nude) mice produced large quantities of collagenase but did not appear to significantly infultrate adjacent host tissue. In comparison, subcutaneously implanted murine Lewis lung tumors produced similar quantities of collagenase and were locally invasive. The human tumors were surrounded by a compact layer of fibroblast cells in a fibrous matrix. This fibrous sheath exhibited anticollagenase activity and indicated a mechanism of host tissue resistance to invasion via the formation of inhibitors to degradative enzymes produced by tumor cells.

Efficacy of 6-diazo-5-oxo-L-norleucine and N-[N-gamma-glutamyl-6-diazo-5-oxo-norleucinyl]-6-diazo-5-oxo-norleucine against experimental tumors in conventional and nude mice.

The chemotherapeutic effects of 6-diazo-5-oxo-L-norleucine (DON) and N-[N-gamma-glutamyl-6-diazo-5-oxo-norleucinyl]6-diazo-5-oxo-norleucine (azotomycin) were evaluated in a spectrum of transplantable experimental tumor systems including xenografts of human tumors in athymic mice. Both drugs displayed remarkable activity against the murine leukemia L1210 and P388, the Colon Tumors C26 and C38 and the CD8F1 mammary tumor. No significant activity was observed against Lewis lung carcinoma, B16 carcinoma, B16 melanoma, and intracranial ependymoblastoma. DON and azotomycin also exhibited striking inhibitory effects on the growth of s.c. human tumor (MX-1 mammary, LX-1 lung and CX-1 and CX-2 colon) xenografts in athymic mice. With the exception of one colon xenograft (CX-1), all tumor lines were markedly responsive to both drugs. Tumor regressions below the initial tumor sizes of 100 to 300 mg, albeit temporary, were brought about by one course of treatment every 4 days for 3 doses (at optimal dose) with either drug. Although these drugs have been tested previously in the clinic and have shown only limited therapeutic effectiveness, they seem to worthy of a second and closer look in light of the recent laboratory results.

Therapy for mouse tumors and human tumor xenografts with the antitumor antibiotic AT-125.

The antimetabolite antibiotic L-(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) showed significant antitumor activity against L1210 and P388 mouse leukemias and the M5076 mouse ovarian tumor. Depending on the schedule of administration, increases in lifespan of greater than 100% were observed. Activity was observed after ip, oral, or sc inoculation of AT-125 in mice inoculated with L1210 by the ip route. Lewis lung carcinoma and B16 melanoma were not affected by AT-125. The compound was used to treat human tumor xenografts in athymic (nude) mice. The MX-1 mammary tumor regressed when treated with either 8 or 16 mg/kg/day for 10 days, while a dose of 32 mg/kg was toxic. On an every-4-days x 3 schedule there was a marked slowing of MX-1 tumor growth at 50, 100, and 200 mg/kg. The LX-1 lung tumor xenograft growth was slowed significantly by a dose of 32 mg/kg. Growth of colon tumors, CX-1, CX-2, and CX-3, was not affected by AT-125.

Chemotherapy of human tumor xenografts in genetically athymic mice.

A series of studies were undertaken to evaluate the chemotherapeutic response to various antineoplastic drugs of human breast (MX-1) or colon (CX-2) tumor xenografts growing in genetically athymic (nude) mice. Fragments (2mm3) of either tumor type were implanted subcutaneously into the subaxillary region of NIH Swiss nude mice, and single drug therapy was started when tumors became palpable and were growing progressively. 5-Fluorouracil (5-FU) administered on a Q7DX3 schedule starting on Day 21 post tumor implantation elicited significant retardation in the growth rate of CX-2 tumor. A single treatment with methyl-CCNU induced temporary tumor regression. Against MX-1 tumor, both cyclophosphamide and melphalan induced tumor regressions with no recurrence. 5-FU slowed but did not arrest growth of MX-1 tumor. These tumor systems grown in nude mice appear to be suitable models for in vivo screening of anticancer agents that would prove clinically active.

Comparison of the drug metabolizing enzymes in the liver and kidneys from homozygous nude swiss, heterozygous normal swiss, homozygous normal Swiss and DBA/2 mice.

The hepatic and renal microsomal drug metabolizing enzyme systems were isolated from homozygous nude Swiss (nu/nu), heterozygous normal Swiss (nu/+), homozygous normal Swiss (+/+) and DBA/2 mice. Microsomal protein and cytochrome P-450 concentrations were measured and the activity of ethylmorphine demethylase, aniline hydroxylase, aryl hydrocarbon hydroxylase and UDP-glucuronyl transferase were determined. Hepatic microsomes from both experimental groups carrying the nude gene were able to metabolize aniline and ethylmorphine more rapidly (20% and 36%, respectively) than the DBA/2 or Swiss homozygous normal mice. No difference between test groups was observed for hepatic aryl hydrocarbon hydroxylase or UDP-glucuronyl transferase activity. Kidney microsomes from mice carrying the nude gene had approximately twice the aryl hydrocarbon hydroxylase activity of the other two experimental groups. Renal mixed-function oxidase pathways measured for the homozygous nude mouse showed a higher overall rate of activity than the other three experimental groups. No significant difference in renal UDP-glucuronyl transferase was observed between mouse groups.

Effects of Corynebacterium parvum alone and in combination with adriamycin in experimental tumor systems.

Corynebacterium parvum (C. parvum) was used in antitumor tests against four murine tumor models in B6D2F1 mice. The C. parvum was effective at all doses and schedules tested against P388 leukemia, B16 melanoma, and Lewis lung carcinoma but was ineffective against L1210 leukemia. Combination immunochemotherapy of P388 leukemia and Lewis lung carcinoma with C. parvum and adriamycin was better than either regimen alone in increasing the lifespan of mice with tumors. The results show that the effects of C. parvum are due to nonspecific stimulation of the host rather than direct cytotoxic action on tumor cells. C. parvum protected the mice when given before as well as after tumor challenge. In vitro 51Cr-release assay showed that the peritoneal cells were cytotoxic to P388 tumor cells but spleen cells were not. While the C. parvum was effective against P388 in conventional mice, it was ineffective against P388 growing in athymic (nude) mice. Thus, the antitumor effect in this tumor system is T-cell dependent.