Study of Lymphotropic Targeting and Macrofilaricidal Activity of a Melphalan Prodrug on the Molinema dessetae Model

Summary

Thia Mudv deals with the design of a new macrofilaricidal drug derived from melphalan and having a lymphotropism to avoid the hepatic first pass effect and enhance bioavailability after oral administration. Melphalan was linked to a ligand leading to a prodrug called 1,3-dp-melphalan which has structural analogy to triglycerides. The Molinema dessetae / Proechimys oris model was used for antiparasitic evaluation. Melphalan was macrofilaricidal in vitro against Molinema dessetae at 1 mM, inactive in vivo after an oral single dose at 164 μmol/kg while the prodrug 1,3-dp-melphalan was active against adult worms after a single dose at 82 μmol/kg. After an oral administration of the prodrug to rats, the maximum concentration and the cumulated quantities of melphalan in lymph were about 45-fold higher than those observed with the free drug under the same conditions. Moreover, the plasma concentration of melphalan was 2-fold higher than those observed after the administration of the free drug. These results are in favor of lymphotropic targeting as a novel approach to develop new orally active macrofilaricides.     

Experimental chronotherapy of mouse mammary adenocarcinoma MA13/C with docetaxel and doxorubicin as single agents and in combination

The therapeutic index of docetaxel, doxorubicin and their combination may be improved by an adequate selection of the circadian time of administration. The present study constitutes a prerequisite to testing the clinical relevance of chronotherapy in human breast cancer. Three experiments were performed in C3H/HeN mice. Each treatment modality was administered i.v. once a week for 3 weeks at one of six circadian stages, during the light span, when the mice were resting: 3, 7, and 11 h after light onset (HALO), or during darkness, when the mice were active: 15, 19, and 23 HALO. The circadian time dependency of single agent tolerability was investigated in healthy mice using four dose levels for docetaxel (38.8, 23.3, 14, and 8.4 mg/kg/injection) and for doxorubicin (13.8, 8.3, 5 and 3 mg/kg/injection; experiment 1). The circadian time dependency of each single agent efficacy was studied in MA13/C-bearing mice, using two dose levels of docetaxel (38.8 or 23.3 mg/kg/injection) or doxorubicin (8.3 or 5 mg/kg/injection; experiment 2). The toxicity and the efficacy of the simultaneous docetaxel-doxorubicin combination were assessed as a function of dosing time in experiment 3. Two combinations were tested (A, 16.3 mg/kg/injection of docetaxel and 2.5 mg/kg/injection of doxorubicin; and B, 11.6 and 3.5 mg/kg/injection, respectively) at each of the above six circadian times. Mortality, body weight change, and tumor size were recorded for 60-70 days in each experiment. Single agent docetaxel or doxorubicin was significantly best tolerated near the middle of the rest span (7 HALO) and most toxic in the middle of the activity phase (19 HALO). Docetaxel or doxorubicin as a single drug were also most effective at 7 HALO, irrespective of dose. Treatment at 7 HALO produced highest rates of complete tumor inhibition (81% versus 11% at 3 HALO for docetaxel, p from chi2 <0.001, and 69% versus 44% at 11 HALO for doxorubicin, not significant) and highest day 60 survival rate (100% versus 28% at 3 HALO for docetaxel, p from chi2 <0.001 and 89% versus 69% at 15 HALO for doxorubicin, not significant). Docetaxel-doxorubicin combinations were most effective following dosing in the beginning of the rest span or short after the onset of the activity span, with regard to the rates of both complete tumor inhibitions (45% at 3 HALO versus 15% at 19 HALO) and day 70 survival rates (85% and 80% at 3 and 7 HALO respectively, versus 20% at 19 HALO). The efficacy of single agent docetaxel or doxorubicin and that of their combination varied largely as a function of circadian dosing time. Single agent docetaxel at 7 HALO was the best treatment option in this model with regard to both tolerability and efficacy. This timing may correspond to the middle of the night in cancer patients.     

Combined treatment of IL-1α and TNF-α potentiates the antitumour effect of hyperthermia

Vasoactive cytokines, such as IL-1α and TNF-α, modulate the homeostatic state at the endothelial surface and cause various types of pathological damage in vascular systems. We investigated the potential therapeutic effects of IL-1α and TNF-α in combination with hyperthermia on SCK tumours grown in the legs of A/J mice. We first determined the effect of cytokines on tumour blood perfusion with the ⁸⁶Rb uptake method. When the host mice were given an i.p. injection of 25 μg/kg IL-1α or 50 μg/kg TNF-α, the tumour blood perfusion markedly declined to 46 and 82% of control, respectively. The combination of IL-1α and TNF-α reduced the ⁸⁶Rb uptake to 41% of control. Hyperthermia at 42·5°C for 1 h reduced the tumour blood flow to 71% of control. The tumour blood perfusion decreased further to 20% of control when the tumours were heated for 1 h at 42.5°C starting 4h after the injection of both IL-1α and TNF-α. The changes in clonogenic cell numbers in SCK tumours, as determined by the in vivo-in vitro assay, following various treatments was also investigated. At 4h after an i.p. injection of 25 μg/kg IL-1α or 50 μg/kg TNF-α, the clonogenicity of SCK tumours significantly decreased to 29 or 37% of control, respectively. Heating at 42.5°C for 1h caused a decline in the clonogenic cell number to 30% of control. When both IL-1α and TNF-α were given and tumours were heated 4h later at 42·5°C for 1h, the clonogenic cell number markedly declined to 0.4% of control. The time needed for control tumours to reach 4x their initial volume was about 3 days, and treatment with IL-1α or hyperthermia alone induced a tumour delay growth by about 1 day. The combined injection of IL-1α and TNF-α followed by a heating at 42·5°C for 1h delayed the tumour growth by 6 days. The results in this study suggest that prior impairment of blood circulation by the combined treatment of IL-1α and TNF-α potentiates hyperthermic damage in tumours.     

The covalent binding of polycyclic hydrocarbons to DNA in the skin of mice of different strains.

The binding of three tritium-labelled carcinogenic polycyclic hydrocarbons, 7,12-dimethylbenz (a) anthracene (DMBA), benzo (a) pyrene (BP) and 3-methylcholanthrene (MCA) to DNA in mouse skin has been studied in C57BL, DBA/2 and Swiss mice following topical application of the hydrocarbons. DNA isolated from the treated areas was hydrolysed to deoxyribonucleosides and chromatographed on Sephadex LH20 columns. The levels of binding of hydrocarbon to DNA were determined from the amount of radioactivity eluted from Sephadex LH20 columns in those fractions containing hydrocarbon-DNA adducts and the radioactivity shown, by rechromatography on AG5OWX4 columns, to be due to tritium incorporation into normal deoxyribonucleosides was not included. C57BL mice were treated with doses of DMBA ranging from 0.025 μmol to 1 μmol/mouse and the levels of hydrocarbon bound to DNA 19 h after treatment were determined; there was no evidence for a threshold dose below which no binding to DNA occurs, and the same hydrocarbon-DNA product peaks were obtained at all doses. The levels of binding of DMBA (1 μmol/mouse) to DNA in skin were compared in C57BL, DBA/2 and Swiss mice at times varying from 6 h to 8 days after treatment. DMBA became bound to similar extents in Swiss and C57BL mice and to a slightly greater extent in DBA/2 mice; the rate of disappearance of bound DMBA from DNA was similar in all three strains. DMBA (0.1 μmol/mouse) was bound to DNA in C57BL and DBA/2 mice to similar extents 19 h after treatment and to a slightly lesser extent in Swiss mice. The ratios of the sizes of the hydrocarbon-DNA product peaks varied with the time after treatment, but were similar at any given time for the three strains. Both BP (1 μmol/mouse) and MCA (1 μmol/mouse) were bound to DNA to similar extents 19 h and 48 h after treatment in all three strains. BP (0.1 μmol/mouse) was bound to DNA in the order DBA/2>C57BL> Swiss 19 h after treatment. The levels of binding for all three hydrocarbons in the different strains do not show a correlation with the reported susceptibilities of the three strains to polycylic hydrocarbon carcinogenesis.     

Prolonged retention of high concentrations of 5-fluorouracil in human and murine tumors as compared with plasma

Summary Concentrations of 5-fluorouracil (5-FU) and its active metabolite 5-fluoro-2-deoxy-5-monophosphate (FdUMP) were measured in biopsy specimens of tumor tissue, normal mucosa, metastatic liver nodules, and normal liver tissue obtained from 39 patients and in two murine colon tumors (colon 26 and colon 38) after a single injection of 5FU at a therapeutic dose (500 mg/m² and 100 mg/kg, respectively). These data were compared with plasma concentrations. Peak plasma concentrations (300–500 m) of 5FU were comparable in human and murine plasma. The half-life of plasma elimination (during the period from 15 to 120 min) in both mouse and man ranged from 10 to 20 min, whereas at between 2 and 8 h, plasma concentrations varied from 0.1 to 1 m, the half-life being about 100 min. In both species, 5FU could be measured in plasma at concentrations ranging from 0.01 to 1 m for several days after 5FU treatment. 5FU concentrations in tissue samples obtained from 14 patients were measured during the time range of 1–6 h, those in samples taken from 7 patients, during the interval of 19–27 h; and those in samples obtained from 18 patients, within the interval of 40–48 h after injection. 5FU tumor concentrations varied between 0.78–21.6, 0.44–6.1, and 0.17–10.8 mol/kg wet wt., respectively. Some of the 48-h samples were obtained from patients who had received leucovorin plus 5FU; coadministration of leucovorin did not alter 5FU tissue concentrations. At between 4 and 48 h, the tissue concentration/plasma concentration ratio was at least 10. 5FU concentrations in murine tumors were measured for up to 10 days after 5FU administration, with plateau 5FU tumor concentrations being about 50 mol/kg wet wt. in colon 38 and about 200 mol/kg wet wt. in colon 26 at 2 h after treatment; after 4 days, values of 0.5 and 4.8 mol/kg, respectively, were obtained and after 10 days, respective concentrations of 0.1 and 0.07 mol/kg were detected. The FdUMP concentrations measured in colon 26 and colon 38 tumors were 214 and 46 pmol/g, respectively, at 2 h after 5FU administration, and these values subsequently decreased to about 15 pmol/g in both tumors. In human tumors the initial FdUMP concentration ranged from 10 to 1000 pmol/g; at later time points the level of FdUMP was just above the detection limit of the assay. In liver metastases, high 5FU concentrations seemed to be related to high levels of FdUMP, which was likely of importance for the antitumor effect. The prolonged retention of 5FU should be taken into consideration in the design of biochemical modulation studies.     

Synergistic effects of intratumor administration of cis-diamminedichloroplatinum(II) combined with local hyperthermia in melanoma bearing mice.

The synergistic effect of local hyperthermia (LHT) with intratumor injection (i.t.) of cis-diamminedichloroplatinum (II) (DDP) was studied using a rodent model with implanted B16 melanoma tumors. The hindfoot of the C57BL/6 mouse bearing the tumor was placed in a water bath at 42.5 +/- 0.2 degrees C (intratumor temperature was at 42.3 +/- 0.1 degrees C) for 30 minutes just after local (i.t.) or systemic (intraperitoneal;i.p.) administration of DDP (1-3 mg/kg once in experiment I and 1-3 mg/kg three times in experiment II). The tumor growth ratio (TGR) at 7 days after treatment in the group given DDP 3 mg/kg (i.t.) with LHT was 1.1 in experiment I and 0.5 in experiment II, and there was a statistically significant difference in both experiments compared to findings in other groups (P < 0.01). The mean survival time was 42.1 days in experiment I and 50.2 days in experiment II, with a significant difference in the latter (P < 0.001). Thus regional injection chemotherapy given concomitantly with local hyperthermia promotes the anticancer effects and improves the prognosis without either severe renal injury or the promotion of hematogenic metastasis.     

Cure of malignant ascites and generation of protective immunity by monoclonal antibody–targeted activation of a glucuronide prodrug in rats

We examined the in vivo efficacy of targeting β-glucuronidase (βG) to activate a glucuronide prodrug (BHAMG) of p-hydroxyaniline mustard (pHAM) at hepatoma ascites in Sprague-Dawley rats. Injection i.p. of 500 μg RH1-βG, a conjugate formed between recombinant βG and monoclonal antibody RH1 with specificity for an antigen expressed on AS-30D rat hepatoma cells, into rats bearing AS-30D ascites resulted in the accumulation of 54 μg conjugate per 10⁹ tumor cells after 2 hr. Ascites fluid and serum contained 0.53 and 0 μg/ml, respectively, RH1-βG 2 hr after injection of the conjugate. Conjugate binding to AS-30D cells was heterogeneous and non-saturated, as determined by flow cytometry. BHAMG was less toxic than pHAM to SD rats based on measures of animal mortality, weight loss and hematological toxicity. Treatment of rats bearing established hepatoma ascites with 500 μg RH1-βG followed 2 hr later with a single i.p. injection of 30 mg/kg BHAMG or 3 i.p. injections of 10 mg/kg BHAMG 2, 3 and 4 hr later resulted in the cure of 6/8 and 8/8 animals, respectively. Treatment with BHAMG or pHAM alone did not produce cures, whereas treatment with a control antibody–βG conjugate and BHAMG produced significantly greater hematological toxicity compared to treatment with RH1-βG and BHAMG. All cured rats were completely protected from rechallenge with 2 × 10⁷ AS-30D cells, indicating that successful treatment of animals induced protective immunity. Int. J. Cancer 73:392–402, 1997. © 1997 Wiley-Liss, Inc.     

Endothelin B receptor agonist,IRL 1620, enhances the anti-tumor efficacy of paclitaxel in breast tumor rats

Summary Pharmacological agents that increase tumor blood flow could be utilized to promote the delivery of anti-cancer drugs. We have demonstrated that administration of endothelin-1 (ET-1) to breast tumor bearing rats transiently increased tumor blood flow by stimulating endothelin B (ET_B) receptors. The present study evaluated the effect of ET_B receptor agonist, IRL 1620, on breast tumor perfusion, concentration of [³H]paclitaxel in tumor and tissues, and efficacy of paclitaxel in N-methyl nitrosourea induced breast tumor bearing rats. Administration of IRL 1620 (3 and 9 nmol/kg) significantly increased (203 and 140%, respectively) breast tumor perfusion. BQ 788, an ET_B receptor antagonist, pretreatment completely abolished IRL 1620 induced increase in tumor perfusion. Tumor [³H]paclitaxel concentration was increased by 308% when [³H]paclitaxel was administered 15 min after IRL 1620 (3 nmol/kg) compared to vehicle treated rats. However, IRL 1620 did not increase [³H]paclitaxel concentrations in other organs. Efficacy study showed that paclitaxel (5 mg/kg) administration on every third day for a total of five doses produced 60.0, 4.5 and 0% reduction in tumor volume, tumor progression and complete tumor remission, respectively, compared to saline treated rats. However, paclitaxel (5 mg/kg) when administered 15 min after IRL 1620 (3 nmol/kg) produced 268.9, 210.3 and 20% reduction in tumor volume, tumor progression and complete remission of tumors, respectively, compared to saline treated rats. In conclusion, IRL 1620 significantly enhanced delivery and effectiveness of paclitaxel in an animal model of breast cancer.     

Local disposition kinetics of floxuridine after intratumoral and subcutaneous injection as monitored by [19F]-nuclear magnetic resonance spectroscopy in vivo

Purpose: To test the utility of [¹⁹F]-nuclear magnetic resonance (NMR) spectroscopy for studying the kinetics of local drug disposition after interstitial application in vivo. Methods: Floxuridine at 30 μmol (2.5% of the reported i.p. 50% lethal dose, LD₅₀) was injected into rats either intratumorally (Morris hepatoma M3924A) or s.c. [¹⁹F]-NMR spectra were obtained at the site of administration for up to 5 h after injection using a 2-cm diameter surface coil at 2.0 T. Signal-time data obtained for floxuridine and the metabolite 5-fluorouracil were analyzed using linear compartment models. Results: The lower limit for the quantitation of drug remaining at the site of administration was 1 μmol for tumors and 0.2 μmol for the s.c. injection site. Local drug disposition was biexponential in four of six tumors where the half-lives of the fast and slow components of disposition ranged from 4 to 26 and from 33 to 289 min, respectively. It was monoexponential in the remaining two tumors (half-lives 49 and 128 min) and in the s.c. injection experiments (n = 4, half-life 6–9 min). 5-Fluorouracil could be quantitated in three of six tumors; the estimated fraction of floxuridine converted intratumorally into 5-fluorouracil was 11–23%. α-Fluoro-β-alanine was detected in the sum spectra of three of the six tumours. Conclusions: Local drug-disposition kinetics after interstitial application can be monitored noninvasively by in vivo [¹⁹F]-NMR spectroscopy. Disposition kinetics after local injection is highly variable and has a slow component in this tumor, whereas it is much less variable and relatively fast in subcutaneous tissue. The results suggest that NMR spectroscopy may be useful for in vivo studies of drug release from depot preparations designed for interstitial application. Received: 7 August 1998 / Accepted: 17 December 1998     

A new pulsed electric field therapy for melanoma disrupts the tumor's blood supply and causes complete remission without recurrence

We have discovered a new, ultrafast therapy for treating skin cancer that is extremely effective with a total electric field exposure time of only 180 μsec. The application of 300 high‐voltage (40 kV/cm), ultrashort (300 nsec) electrical pulses to murine melanomas in vivo triggers both necrosis and apoptosis, resulting in complete tumor remission within an average of 47 days in the 17 animals treated. None of these melanomas recurred during a 4‐month period after the initial melanoma had disappeared. These pulses generate small, long‐lasting, rectifying nanopores in the plasma membrane of exposed cells, resulting in increased membrane permeability to small molecules and ions, as well as an increase in intracellular Ca²⁺, DNA fragmentation, disruption of the tumor's blood supply and the initiation of apoptosis. Apoptosis was indicated by a 3‐fold increase in Bad labeling and a 72% decrease in Bcl‐2 labeling. In addition, microvessel density within the treated tumors fell by 93%. This new therapy utilizing nanosecond pulsed electric fields has the advantages of highly localized targeting of tumor cells and a total exposure time of only 180 μsec. These pulses penetrate into the interior of every tumor cell and initiate DNA fragmentation and apoptosis while at the same time reducing blood flow to the tumor. This new physical tumor therapy is drug free, highly localized, uses low energy, has no significant side effects and results in very little scarring. © 2009 UICC     

Immunological studies on mouse mammary tumors. I. Induction of resistance to tumor isograft in C3H/He mice

An isografted tumor MM2, originating from a spontaneous mammary tumor in a C3H/He/mouse, killed 4- to 5-week-old mice within 30 days through intraperitoneal injection of 2 × 10⁴ cells per mouse. It was shown that resistance to the isograft was induced by injection of 1.5 × 10⁵ or 2.0 × 10⁵ tumor cells sensitized with serum (5 μg antibody N) of rabbits immunized with the tumor. Four weeks after injection of the sensitized MM2 cells, 173 C3H/He mice were challenged intraperitoneally with fresh unsensitized MM2 in doses of 5 × 10⁵ to 1 × 10⁶ cells per mouse. Of these, 119 mice survived without any sign of tumor growth while all untreated mice died. The mean survival time for untreated mice was 18.1 days (± 1.7) when inoculated with 5 × 10⁵ cells and 16.2 days (± 1.8) when inoculated with 1 × 10⁶ cells. After the second challenge with the same number of fresh unsensitized cells, 117 out of 119 mice survived without any sign of tumor growth. All of 15 mice randomly selected from these resistant mice were tested for acceptance of skin grafts from normal C3H/He mice. The grafts showed no rejection even 150 days after the second graft. After hyperimmunization of these resistant mice, the serum of the spleen extract taken from the mice inhibited growth of tumors when the tumors were premixed in vitro and injected intraperitoneally into C3H/He mice.     

Targeted therapy of Schwannoma cells in immunocompetent rats with an erbB2-specific antibody-toxin

Over-expression of the erbB2-receptor tyrosine kinase is frequently observed in many human tumors of epithelial origin. Due to its causal involvement in malignant transformation and its presence on the tumor cell surface erbB2 is an attractive target for directed tumor therapy. We earlier described the potent anti-tumoral activity of the recombinant single-chain antibody toxin scFv(FRP5)-ETA in vitro and in nude mouse tumor models in vivo. This molecule consists of the variable domains of the heavy and light chains of an erbB2-specific antibody genetically fused to a truncated Pseudomonas exotoxin A. Here we have investigated the in vivo effects of this immunotoxin on erbB2 expressing NV2Cd schwannoma cells growing as s.c. tumors in syngeneic BDIX rats. Established tumors were treated either locally by intratumoral injection of scFv(FRP5)-ETA or systemically by injection into the tail vein. Both routes of application resulted in pronounced inhibition of tumor growth with local treatment being more effective. Treatment with 25 μg/day of scFv(FRP5)-ETA for 10 days suppressed tumor growth almost completely. Antibodies directed mainly against the toxin domain of the fusion protein developed in all animals treated. Int. J. Cancer 73:117–124, 1997. © 1997 Wiley-Liss, Inc.     

Development of an early induction model of medulloblastoma in Ptch1 heterozygous mice initiated with N‐ethyl‐N‐nitrosourea

Mice heterozygous for the ptch1 gene (ptch1 mice) are known as a valuable model of medulloblastoma, a common brain tumor in children. To increase the incidence and reduce the time required for tumor development, allowing for evaluation of modifier effects on medulloblastoma in a short time, we attempted to develop an early induction model of medulloblastoma in ptch1 mice initiated with N‐ethyl‐N‐nitrosourea (ENU). Ptch1 mice and their wild‐type littermates received a single intraperitoneal injection of ENU (10, 50 or 100 mg/kg) on postnatal day 1 (d1) or 4 (d4), and histopathological assessment of brains was conducted at 12 weeks of age. The width of the external granular layer (EGL), a possible origin of medulloblastoma, after injection of 100 mg ENU on d1 or d4 was measured in up to 21‐day‐old mice. Cerebellar size was apparently reduced at the 50 mg dose and higher regardless of genotype. Microscopically, early lesions of medulloblastomas occurred with a high incidence only in ptch1 mice receiving 10 mg on d1 or d4, but a significant increase was not observed in other groups. Persistent EGL cells and misalignment of Purkinje cells were increased dose‐dependently. Although EGL was strikingly decreased after ENU injection, strong recovery was observed in mice of the d1‐treated group. In summary, neonatal treatment with ENU is available for the induction of medulloblastoma in ptch1 mice, and 10 mg of ENU administered on d1 appeared to be an appropriate dose to induce medulloblastoma.     

Salivary acetaldehyde increase due to alcohol‐containing mouthwash use: A risk factor for oral cancer

Increasing evidence suggests that acetaldehyde, the first and genotoxic metabolite of ethanol, mediates the carcinogenicity of alcoholic beverages. Ethanol is also contained in a number of ready‐to‐use mouthwashes typically between 5 and 27% vol. An increased risk of oral cancer has been discussed for users of such mouthwashes; however, epidemiological evidence had remained inconclusive. This study is the first to investigate acetaldehyde levels in saliva after use of alcohol‐containing mouthwashes. Ready‐to‐use mouthwashes and mouthrinses (n = 13) were rinsed in the mouth by healthy, nonsmoking volunteers (n = 4) as intended by the manufacturers (20 ml for 30 sec). Saliva was collected at 0.5, 2, 5 and 10 min after mouthwash use and analyzed using headspace gas chromatography. The acetaldehyde content in the saliva was 41 ± 15 μM, range 9–85 μM (0.5 min), 52 ± 14 μM, range 11–105 μM (2 min), 32 ± 7 μM, range 9–67 μM (5 min) and 15 ± 7 μM, range 0–37 μM (10 min). The contents were significantly above endogenous levels and corresponding to concentrations normally found after alcoholic beverage consumption. A twice‐daily use of alcohol‐containing mouthwashes leads to a systemic acetaldehyde exposure of 0.26 μg/kg bodyweight/day on average, which corresponds to a lifetime cancer risk of 3E?6. The margin of exposure was calculated to be 217,604, which would be seen as a low public health concern. However, the local acetaldehyde contents in the saliva are reaching concentrations associated with DNA adduct formation and sister chromatid exchange in vitro, so that concerns for local carcinogenic effects in the oral cavity remain. © 2009 UICC     

Phase I clinical study of the recombinant antibody toxin scFv(FRP5)-ETA specific for the ErbB2/HER2 receptor in patients with advanced solid malignomas

Introduction

ScFv(FRP5)-ETA is a recombinant antibody toxin with binding specificity for ErbB2 (HER2). It consists of an N-terminal single-chain antibody fragment (scFv), genetically linked to truncated Pseudomonas exotoxin A (ETA). Potent antitumoral activity of scFv(FRP5)-ETA against ErbB2-overexpressing tumor cells was previously demonstrated in vitro and in animal models. Here we report the first systemic application of scFv(FRP5)-ETA in human cancer patients.

Methods

We have performed a phase I dose-finding study, with the objective to assess the maximum tolerated dose and the dose-limiting toxicity of intravenously injected scFv(FRP5)-ETA. Eighteen patients suffering from ErbB2-expressing metastatic breast cancers, prostate cancers, head and neck cancer, non small cell lung cancer, or transitional cell carcinoma were treated. Dose levels of 2, 4, 10, 12.5, and 20 μg/kg scFv(FRP5)-ETA were administered as five daily infusions each for two consecutive weeks.

Results

No hematologic, renal, and/or cardiovascular toxicities were noted in any of the patients treated. However, transient elevation of liver enzymes was observed, and considered dose limiting, in one of six patients at the maximum tolerated dose of 12.5 μg/kg, and in two of three patients at 20 μg/kg. Fifteen minutes after injection, peak concentrations of more than 100 ng/ml scFv(FRP5)-ETA were obtained at a dose of 10 μg/kg, indicating that predicted therapeutic levels of the recombinant protein can be applied without inducing toxic side effects. Induction of antibodies against scFv(FRP5)-ETA was observed 8 days after initiation of therapy in 13 patients investigated, but only in five of these patients could neutralizing activity be detected. Two patients showed stable disease and in three patients clinical signs of activity in terms of signs and symptoms were observed (all treated at doses ≥ 10 μg/kg). Disease progression occurred in 11 of the patients.

Conclusion

Our results demonstrate that systemic therapy with scFv(FRP5)-ETA can be safely administered up to a maximum tolerated dose of 12.5 μg/kg in patients with ErbB2-expressing tumors, justifying further clinical development.     

Allogeneic and autologous stem-cell transplantation in advanced Ewing tumors

Background:An update of results from the High Risk Protocol ofthe Meta-EICESS Study, conducted at the Pediatric Stem-Cell Transplant Centersof Düsseldorf and Vienna. In order to evaluate a possible therapeuticbenefit after allogeneic SCT in patients with advanced Ewing tumors (AET), wecompared outcome after autologous and allogeneic stem-cell transplantation(SCT). Patients and methods:We analyzed 36 patients treated with themyeloablative Hyper-ME protocol (hyperfractionated total body irradiation,melphalan, etoposide ± carboplatin) between November 1986 and December1994. Minimal follow-up for all patients was five years. All patientsunderwent remission induction chemotherapy and local treatment beforemyeloablative therapy. Seventeen of thirty-six patients had multifocal primaryEwing's tumor, eighteen of thirty-six had early, multiple or multifocalrelapse, one of thirty-six patients had unifocal late relapse. Twenty-six ofthirty-six were treated with autologous and ten of thirty-six with allogeneichematopoetic stem cells. We analyzed the following risk factors, that couldpossibly influence the event-free survival (EFS): number of involved bones,degree of remission at time of SCT, type of graft, indication for SCT, bonemarrow infiltration, bone with concomitant lung disease, age at time ofdiagnosis, pelvic involvement, involved compartment radiation,histopathological diagnosis. Results:EFS for the 36 patients was 0.24 (0.21) ± 0.07.Eighteen of thirty-six patients suffered relapse or died of disease, nine ofthirty-six died of treatment related toxicity (DOC). Nine of thirty-sixpatients are alive in CR. Age 17 years at initial diagnosis (P< 0.005) significantly deteriorated outcome. According to the type ofgraft, EFS was 0.25 ± 0.08 after autologous and 0.20 ± 0.13after allogeneic SCT. Incidence of DOC was more than twice as high afterallogeneic (40%) compared to autologous (19%) SCT, even thoughthe difference did not reach significance (P = 0.08, Fisher's exacttest). Conclusions:Because of the rather short observation period,secondary malignant neoplasm (SMN) may complicate the future clinical courseof some of our patients who are currently viewed as event-free survivors. EFSin AET is not improved by allogeneic SCT due to a higher complication rate.The patient group was to small to analyze for a possiblegraft-versus-tumor effect.     

Pharmacokinetics of WR-1065 in mouse tissue following treatment with WR-2721

Levels of reduced glutathione (GSH) and N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065) were measured in tissues of Balb/c mouse bearing EMT₆ tumors at time intervals ranging from 5 min to 48 hr after i.v. injection of S-2-(3-aminopropylamino)ethyl phosphorothioate (WR-2721) at 500 mg per kg. In all tissues examined (liver, kidney, lung, heart, muscle, brain, tumor, spleen, and salivary gland), maximal WR-1065 levels occurred 5–15 min after injection, with levels in liver, kidney, lung, and salivary gland exceeding one μmole per gm. The post-maximum decline in WR-1065 varied markedly with tissue, lung exhibiting a 6-fold drop by 30 min and salivary gland falling only 1596 after 3 hr. In a mouse treated with carbon-14 labeled WR-2721 it was found after 15 min that WR-1065 accounted for over half of the total drug in all tissues except tumor, where it accounted for a third of the total drug. There was no evidence that GSH levels were substantially altered by WR-2721 treatment. The results provide the first direct evidence supporting the widely held view that WR-2721 treatment results in intracellular WR-1065 and they demonstrate that high levels of WR-1065 occur very soon after i.v. injection.     

The potentiation of radiation response in human colon carcinoma cells in vitro and murine lymphoma in vivo by AG337 (thymitaq™), a novel thymidylate synthase inhibitor

Purpose: To determine whether the administration of Thymitaq^TM (AG337), a selective inhibitor of thymidylate synthase (TS), enhances radiation-induced cytotoxicity in vitro and increases tumor control rate in vivo.Methods and Materials: In vitro studies were carried out with HT-29 human colon carcinoma cells. In vivo studies were carried out using L5178Y(TK⁻) murine lymphoma implanted in DBA/2 mice.Results: Pretreatment of HT-29 cells to nontoxic concentration of AG337 (<10 μM) for a short period of time (< 24 h) significantly enhanced the radiation induced cell lethality. The radiosensitizing enhancement ratio was 1.7. In contrast, there was no increased cell killing when the drug was exposed immediately after irradiation. In studies using L5178Y(TK⁻) tumors, the drug alone (50 mg/kg, i.p. × 5) had a minimal tumor growth delay, while a single dose of radiation (17 Gy) resulted in < 10% tumor control at day 30. When radiation and drug (17 Gy + AG337, 50 mg/kg, i.p. × 5) were combined, the tumor control rate reached 90% at Day 30. Using the local tumor control assay (TCD₅₀), the radiation dose modification factor after a single dose of radiation was 2.6.Conclusion: The concentration of drug shown to be of radiosensitizing value in the in vivo studies is achievable in humans. The results of the present study further supports the potential utility of AG337 in the treatment of human tumors by radiotherapy.     

Semiquantitative assessment of the microdistribution of fluorescence‐labeled monoclonal antibody in small peritoneal disseminations of ovarian cancer

(Cancer Sci 2010; 101: 820–825) Uniform antibody microdistribution throughout tumor nodules is crucial for antibody‐targeted therapy, because non‐uniform microdistribution leads to suboptimal therapeutic effect, a commonly observed limitation of therapeutic antibodies. Herein, we evaluated the microdistribution of different doses of intraperitoneally injected fluorescence‐labeled full‐antibody trastuzumab (15, 50, and 150 μg) and its Fab fragment (trastuzumab‐Fab: 15 and 50 μg) in a mouse model of ovarian cancer with peritoneal disseminated tumor. A semiquantitative approach (central/peripheral accumulation ratio; C/P ratio) was developed using in situ fluorescence microscopy. Furthermore, we compared the microdistribution of intact trastuzumab with a mixed injection of trastuzumab and trastuzumab‐Fab or serial injections of trastuzumab using in situ multicolor fluorescence microscopy. Fluorescence images after the administration of 15 or 50 μg trastuzumab and 15 μg trastuzumab‐Fab demonstrated antibody accumulation in the tumor periphery, whereas administration of 150 μg trastuzumab and 50 μg trastuzumab‐Fab showed relatively uniform accumulation throughout the tumor nodule. Using serial injections (19‐h interval) of trastuzumab‐rhodamine green and carboxytetramethylrhodamine (TAMRA), it was observed that the latterly injected trastuzumab‐TAMRA was distributed more centrally than trastuzumab‐rhodamine green injected first, whereas no difference was observed in the control mixed‐injection group. Moreover, the mixed injection of trastuzumab and trastuzumab‐Fab showed that trastuzumab‐Fab distributed more centrally than the same amount of co‐injected trastuzumab. Our results suggest that the strategies of increasing dose and using Fab fragments can be used to achieve a uniform antibody distribution within peritoneal disseminated nodules after intraperitoneal injection. Furthermore, serial‐injection and mixed‐injection strategies can modify antibody microdistribution within tumors and have the potential for preferential delivery of anticancer drugs to either the tumor periphery or its center.     

Combination of vesicular stomatitis virus matrix protein gene therapy with low‐dose cisplatin improves therapeutic efficacy against murine melonoma

Vesicular stomatitis virus (VSV) matrix protein (MP) can directly induce apoptosis via the mitochondrial pathway due to the inhibition of host gene expression. Our previous studies have demonstrated that MP gene therapy efficiently suppressed the growth of malignant tumor in vitro and in vivo. The present study was designed to determine the possibility that the combination of MP gene therapy with low‐dose cisplatin would improve therapeutic efficacy against murine melanoma. Immunocompetent C57BL/6 mice bearing B16‐F10 melanoma were established. Mice were treated once every 5 days with i.v. administration of 10 μg pVAX‐MP/30 μg liposome complex per mouse for 16 days and i.p. delivery of cisplatin at 4 mg/kg/mouse on days 6 and 12 after the initiation of MP treatment. We found that MP + cisplatin treatment resulted in significant inhibition of tumor growth and improved the survival time of melanoma‐bearing mice. MP successfully inhibited angiogenesis as assessed by CD31. Histological examination revealed that the combination therapy led to significant increased induction of apoptosis, tumor necrosis, and elevated CD8⁺ lymphocyte infiltration. Furthermore, the induction efficacy of the CTL response was dramatically enhanced by the combination therapy. Our findings may prove useful in further explorations of the application of these combinational approaches to the treatment of malignant melanoma. (Cancer Sci 2010; 101: 1219–1225)