Computed-tomography-guided transforaminal intrathecal nusinersen injection in adults with spinal muscular atrophy type 2 and severe spinal deformity. Feasibility, safety and radiation exposure considerations.

BACKGROUND AND PURPOSE: The purpose was to investigate our centre's experience on computed-tomography-guided (CT-guided), transforaminal, intrathecal administration of nusinersen in adult subjects with spinal muscular atrophy (SMA) type 2 and severe spinal deformity. METHOD: This is a retrospective, single-centre study investigating the feasibility and safety of CT-guided, transforaminal, lumbar puncture for the intrathecal administration of nusinersen (Spinranza®; Biogen; Cambridge, MA, USA) in a cohort of adult subjects with SMA type 2, severe neuromuscular scoliosis and previous spinal surgery. Between January 2019 and October 2019, five male, adult, SMA type 2 subjects were eligible to be treated in our centre with nusinersen. The mean age of the patients was 31 ± 9 years (range 19-43 years). The study's outcome measures were technical success, adverse events and radiation exposure. RESULTS: In total, four patients completed the four loading doses, whilst the fifth patient received only one loading dose; two patients also received their first maintenance doses. Overall, 20 consecutive transforaminal, intrathecal treatments were analysed. Technical success was 100% (20/20 intrathecal infusions). No adverse events were documented following the procedures. Mean dose-length product (DLP) value per injection was 665.4 ± 715.5 mGy*cm. Estimated mean effective dose per injection was 12.7 ± 12.9 mSv. Subgroup analysis between the chronologically first 10 versus subsequent 10 procedures demonstrated a clear trend towards less radiation exposure in the latter, although this difference did not reach statistical significance (DLP: 984.7 ± 903.3 vs. 436.7 ± 321.5 mGy*cm, P = 0.165; respectively). CONCLUSIONS: In this retrospective series, CT-guided transforaminal access for intrathecal injection of nusinersen was proven feasible and safe. A decrease in radiation dose over time was noted. Protocols to minimize radiation exposure are essential.

Effect of previous anesthesia experience on patients' knowledge and desire for information about anesthesia and the anesthesiologist: a 500 patients' survey from Greece.

BACKGROUND: This study aimed at assessing the effect of previous anesthesia experience on patients' knowledge of anesthesia and the role of anesthesiologists, on what they would want to know about anesthesia and the way they would like to be informed. METHODS: Questionnaires with fixed questions were distributed to consenting, consecutive surgical patients before the pre-anesthetic visit. Patients were divided into two groups: patients with previous anesthesia experience (Group A) and patients without previous anesthesia experience (Group B). The questionnaires included patients' demographics, questions related to their knowledge about the anesthesiologists' role and about their desire for information. RESULTS: 500 questionnaires were analyzed. The majority of patients (94.2%) know that the anesthesiologist is a specialized doctor and 89.2% believe that the anesthesiologist watches over the patient throughout surgery. These results were similar in both groups. The majority of patients (98.2%) also want to meet the anesthesiologist before surgery and 78% want even more information. Only 65.6% want to be aware of all possible complications, in both groups, while 17.6% do not want to know anything about complications. In general, answers to specific questions regarding what the patients want to know about anesthesia did not differ between groups. The vast majority of patients wish to talk with the anesthesiologist before surgery. CONCLUSION: Previous anesthesia experience did not seem to influence patients' desire for meeting the anesthesiologist and seeking information. A strong desire to personally meet the anesthesiologist is expressed and patients' desire for even more information is noted.

The hypoxia-selective cytotoxin NLCQ-1 (NSC 709257) controls metastatic disease when used as an adjuvant to radiotherapy.

BACKGROUND: Metastases cause most cancer-related deaths. We investigated the use of hypoxia-selective cytotoxins as adjuvants to radiotherapy in the control of metastatic tumour growth. METHODS: The NLCQ-1, RB6145 and tirapazamine were assessed against the spontaneously metastasising KHT model. Subcutaneous KHT tumours (250 mm(3)) were irradiated with 25 Gy (single fraction) to control primary growth. Equitoxic drug treatments (NLCQ-1 (10 mg kg(-1)) once daily; RB6145 (75 mg kg(-1)) and tirapazamine (13 mg kg(-1)) twice daily) were administered 3-6 days post-radiotherapy when hypoxic cells were evident in lung micrometastases. Mice were culled when 50% of controls exhibited detrimental signs of lung metastases. RESULTS: In total, 95% of control mice presented with lung disease. This was significantly reduced by NLCQ-1 (33%; P=0.0002) and RB6145 (60%; P=0.02). Semi-quantitative grading of lung disease revealed a significant improvement with all treatments, with NLCQ-1 proving most efficacious (median grades: control, 4; NLCQ, 0 (P<0.0001); RB6145, 1 (P<0.001), tirapazamine, 3 (P=0.007)). Positron emission tomography (PET) was evaluated as a non-invasive means of assessing metastatic development. Primary and metastatic KHT tumours showed robust uptake of [(18)F]fluorodeoxyglucose ([(18)F]FDG). Metastatic burden discernable by [(18)F]FDG PET correlated well with macroscopic and histological lung analysis. CONCLUSION: The hypoxia-selective cytotoxin NLCQ-1 controls metastatic disease and may be a successful adjuvant to radiotherapy in the clinical setting.

Milrinone and theophylline act as lower oesophageal sphincter relaxing agents: a comparative pharmacodynamic study in the rabbit.

This study demonstrates that the inotropic agent milrinone and the bronchodilator drug theophylline exert a relaxing effect on the rabbit lower oesophageal sphincter in vitro. The relaxing effect of milrinone and theophylline, which is concentration-dependent, involves a second messenger 3',5'-cyclic adenosine monophosphate pathway and most probably it is accomplished through inhibition of phosphodiesterase (PDE) type III, as according to the obtained results it is not significantly modified either by nicotinic acid, an inhibitor of adenylate cyclase, or by the inhibitor of nitric oxide-synthetase N(omega)-nitro-L-arginine methylester and the purinergic antagonist suramin; moreover, it persists under non-adrenergic non-cholinergic conditions and it is both hexamethonium- and tetrodotoxin-insensitive. Both milrinone and theophylline display equal efficacy, comparable to that of the calcium blocker verapamil and the non-selective PDE inhibitor papaverine, but milrinone appears 50 times more potent than theophylline and three times less potent than verapamil, as, according to the pIC(50) values the potency rank of order is found to be verapamil (5.56) > milrinone (5.12) > theophylline (3.42). The here obtained pharmacodynamic profiles of the drugs suggest that both milrinone and theophylline may be considered as potent relaxing agents of the lower oesophageal sphincter.

Gene therapy approaches to enhance bioreductive drug treatment.

Hypoxia, or a lack of oxygen, occurs in 50-60% of solid human tumours. Clinical studies have shown that the presence and extent of hypoxia in a tumour cannot be predicted by size or histopathological stage but it is predictive of a poor outcome following radiotherapy, chemotherapy and surgery. However, as a physiological feature of tumours, it can be exploited and researchers have developed many hypoxia-selective chemotherapies or bioreductive drugs that are in varying stages of clinical development. These agents are prodrugs that have two key requirements for their biological activation: they require the reductive environment of a hypoxic tumour cell and the appropriate complement of cellular reductase enzymes. To overcome tumour heterogeneity in reductase enzyme levels and enhance bioreductive drug metabolism a gene therapy strategy can be employed. We have reviewed this field and also present our own pre-clinical research using gene therapy to enhance bioreductive drug treatment for the treatment of cancer. We have specifically focused on studies enhancing lead clinical bioreductive drugs. We consider the metabolic requirements for their activation and we highlight the key in vivo studies supporting the future clinical development of hypoxia-targeted gene-directed enzyme prodrug therapy.

Theophylline and its metabolites produce a stimulating cholinergic effect on the small intestine and a nonadrenergic noncholinergic relaxing effect on the colon: a comparative study in the rabbit intestine.

The present study examines comparatively the effects of theophylline and its metabolites, 1-methylxanthine (1-MX), 3-methylxanthine (3-MX), 1,3-dimethyluric acid (1,3-DMU) and 1-methyluric acid (1-MU) along the rabbit intestine, and explores the underlying mechanism(s). In the small intestine, theophylline produces atropine- and hexamethonium-sensitive increases in both the amplitude of phasic contractions and the basal tone. All metabolites mimic the theophylline's stimulating effect. In particular, concerning the phasic contractions, all metabolites are more potent than theophylline in the duodenum and jejunum, while in the ileum, only 1-MU is more potent. Regarding the basal tone, the metabolites show, in most cases, higher efficacy in all small intestinal regions, the maximum effects of 3-MX and 1-MU on the duodenum and ileum being double or triple the one of theophylline. In the ascending colon, while lower concentrations of theophylline produce an atropine- and hexamethonium-sensitive increase in the basal tone, higher ones produce a postsynaptic, nonadrenergic noncholinergic (NANC) relaxing effect. 1-MU mimics, in a weaker manner, theophylline's effect, while the other metabolites produce only relaxation, the potency rank of order being 3-MX>1-MX=1,3-DMU>theophylline. It is suggested that the theophylline and its metabolites stimulatory effect involves a cholinergic pathway, while the relaxing one is due to 3('),5(')-cyclic adenosine monophosphate (cAMP) elevation mediated by the theophylline and its metabolites inhibitory action on phosphodiesterases (PDEs).

Interaction of strong DNA-intercalating bioreductive compounds with topoisomerases I and II.

Nitro(imidazole/triazole)-linked acridines (NLAs) have been previously developed in our laboratory as DNA-intercalating bioreductive drugs. Such compounds demonstrate toxicity through the formation of bulky monoadducts with cellular macromolecules upon activation and reductive metabolism under hypoxic conditions. However, NLAs also demonstrate considerable aerobic toxicity. Based on the ability of NLAs to bind strongly to DNA through intercalation, we investigated whether their relatively high aerobic cytotoxicity and their relatively low hypoxic selectivity in vitro are associated with topoisomerases I and II (Topo I and II) inhibition. DNA Topo I or II-mediated activity studies have been performed using supercoiled or kinetoplast DNA plasmids. Calf thymus or human Topo I and human Topo II purified enzymes were used. All NLA derivatives strongly inhibited relaxation of supercoiled DNA catalyzed by either Topo I or II, in a concentration-dependent manner, without stabilization of a cleavable complex. Aerobic toxicity correlated well with the inhibition of Topo II-mediated decatenation of kinetoplast DNA, whereas the intracellular concentrations of NLAs were 27-152-fold greater than those needed for 50% inhibition of Topo-II mediated decatenation of DNA. These results suggest that topoisomerase inhibition accounts for NLAs aerobic toxicity.

Synergistic interaction between cyclophosphamide or paclitaxel and the bioreductive compound NLCPQ-1, in vivo.

The antitumor effect of cyclophosphamide (CPM) and paclitaxel was investigated in BALB/c mice bearing EMT6 tumors, in combination with the bioreductive compound NLCPQ-1 by using the in vivo/in vitro assay as the endpoint. An optimum administration schedule for a synergistic interaction between NLCPQ-1 and CPM/paclitaxel was determined and dose modification factors (DMF) were calculated for antitumor effect and bone marrow toxicity. All drugs were given by IP injection; NLCPQ-1 at 15 mg/kg, which is much less than its maximally tolerated dose (MTD greater than 50 mg/kg), paclitaxel up to 25 mg/kg, and CPM up to 200 mg/kg. Bone marrow toxicity studies were performed in parallel by using a modified CFU-GM assay. A schedule-dependent synergistic interaction was observed for both chemotherapeutic agents combined with NLCPQ-1 but with entirely different patterns, as has been previously seen with the analog NLCQ-1. The optimal degree of potentiation, P (percentage of tumor cells that were killed due to clear potentiation), was 31 and 33 when NLCPQ-1 was administered 2 h before CPM and 3-3.5 h after paclitaxel, respectively. At the above time schedules, NLCPQ-1 modified the dose of CPM and paclitaxel, for 60% tumor cell killing, by a factor of 1.8 and 2.1, respectively. Bone marrow toxicity was not enhanced by combining either chemotherapeutic agent with NLCPQ-1. Comparison with results from previous similar studies with NLCQ-1 revealed that, on a molar basis, NLCPQ-1 was a less potent chemosensitizer than NLCQ-1. However, the results still suggest a potential clinical use of NLCPQ-1 as an adjuvant to CPM or paclitaxel therapy against solid tumors.

Therapeutic advantage from combining 5-fluorouracil with the hypoxia-selective cytotoxin NLCQ-1 in vivo; comparison with tirapazamine.

PURPOSE: The antitumor effect and bone marrow toxicity of 5-fluorouracil (5FU) in combination with the hypoxia-selective cytotoxins NLCQ-1 or tirapazamine (TPZ) were investigated in vivo. METHODS: Using appropriate intraperitoneal administration schedules for optimal synergistic interactions, the antitumor effect and the bone marrow toxicity of combinations of NLCQ-1 or TPZ and 5FU were determined in EMT6/BALB/c and SCCVII/C3H models in terms of dose modification factors (DMF) using the in vivo-in vitro clonogenic assay as endpoint. Bone marrow toxicity studies were performed in parallel using a modified CFU-GM assay. The antitumor efficacies of each combination treatment under optimal administration conditions were evaluated in the SCCVII/C3H model using also the tumor regrowth assay as endpoint. RESULTS: A schedule-dependent and tumor-specific synergistic interaction was observed for NLCQ-1 plus 5FU and DMFs of 2.0-2.3 and 1.0 were obtained for the antitumor effect and bone marrow toxicity, respectively, in both tumor models. The antitumor effect of 5FU was slightly potentiated (DMF 1.2) by TPZ in the EMT6/BALB/c model but not in the SCCVII/C3H model when the in vivo-in vitro assay was used as the endpoint. Significant additional tumor regrowth delays (about 11 and 6 days for NLCQ-1 and TPZ, respectively) were observed, compared to the effect of 5FU alone, when an equitoxic dose of NLCQ-1 (10 mg/kg) or TPZ (23 mg/kg) was administered 1 h before 5FU (50 mg/kg) twice a day at 4-h intervals on days 0 and 9. CONCLUSIONS: These results corroborate the therapeutic advantage of combining hypoxia-selective cytotoxins such as NLCQ-1 and TPZ with chemotherapy.

Optimizing the use of combined radioimmunotherapy and hypoxic cytotoxin therapy as a function of tumor hypoxia.

Combined radioimmunotherapy (RAIT) and hypoxic cytotoxin therapy (SR4233 or NLCQ-1) have been evaluated with both modalities administered on the same day with only moderate improvement compared with the effects of RAIT alone. In a series of studies using oxygen electrodes, immunohistochemistry and radiotracers, we have demonstrated that RAIT induces a prolonged state of hypoxia in most tumors, without affecting the pO(2) levels in normal tissues. Using serial microelectrode measurements through subcutaneous (s.c.) GW-39 human colonic xenografts, we established that the median pO(2) was unrelated to the initial size of the tumor, over a range of sizes from 1.0 to 4.0 cm. Fourteen days after mice were given a 240-microCi dose of (131)I-MN-14 anti-carcinoembryonic antigen immunoglobulin G, their median pO(2) declined from 26.1 +/- 9.6 mmHg to 9.8 +/- 3.9 mmHg (p < 0.001). Using the radiotracer (3)H-MISO that accumulates in hypoxic regions, uptake in GW-39, LoVo and LS174T s.c. human colonic tumors increased 3.0- to 4.2-fold from day 14 through day 28 post-RAIT, but uptake of (3)H-MISO in CALU-3 tumors remained unchanged after RAIT. Normal tissue (liver, kidney, lung) uptake of (3)H-MISO did not exhibit significant changes. The increase in tumor hypoxia was also demonstrated visually using anti-PIMO staining of tumor sections. We postulated that sequential delivery of the 2 therapeutic agents, with the hypoxic cytotoxin given 2 weeks after RAIT when tumor pO(2) levels were at their nadir, would improve the therapeutic response above either modality alone or above the 2 agents delivered on the same day. Tumor growth was compared in mice given either RAIT or cytotoxin alone, the combined treatment on the same day or with the cytotoxin delivered 14 days after RAIT. Tumor size on day 35 for RAIT-treated and SR4233-treated GW-39 were 3.56 +/- 0.40 and 7.98 +/- 2.50 cm(3). When RAIT + SR4233 were delivered on the same day, tumor size dropped to 2.78 +/- 0.80 cm(3). If RAIT was given on day 0 and SR4233 on day 14, size further declined further to 1.74 +/- 0.32 cm(3) (p < 0.05 compared with same day delivery). For LS174T, tumor size on day 28 for RAIT-treated and SR4233-treated tumors were 1.14 +/- 0.36 cm(3) and 3.65 +/- 0.78 cm(3), respectively. When RAIT + SR4233 were delivered on the same day, size was 0.51 +/- 0.174 cm(3). If RAIT was dosed on day 0 and SR4233 was given on day 14, tumor size was 0.13 +/- 0.07 cm(3) (p < 0.05). Similar results were obtained for LoVo, but not for CALU-3 tumors. Another hypoxic cytotoxin, NLCQ-1, was also more efficacious 2 weeks after RAIT, compared with same-day dosing. Thus, information on tumor hypoxia after radioantibody therapy could be important for ascertaining a window of opportunity when the surviving tumor regions are most responsive to hypoxic cytotoxins.

In vitro evaluation of 4-[3-(2-nitro-1-imidazolyl)-propylamino]-7-trifluoromethylquinoline hydrochloride (NLTQ-1), a new bioreductive agent as a hypoxia marker by 19F-magnetic resonance spectroscopy (19F-MRS).

19F-labeled bioreductive drugs bound to hypoxic cells in tumors could be detected by nuclear magnetic resonance, provided that they do not lose 19F during their metabolism. NLTQ-1, a 2-nitroimidazole-linked 7-trifluoromethylquinoline, has been synthesized to furnish this aim. NLTQ-1 demonstrated hypoxic selectivities of 7-10 in various cell-lines, in vitro. Uptake studies in V79 cells showed a 5 to 6 fold greater intracellular than extracellular concentration at a range of 100-300 microM input concentrations. A strong sharp peak, which was identified as the parent compound, was observed in the 19F-NMR spectrum of 90% MeCN extracts of V79 cells aerobically exposed to NLTQ-1, indicating that NLTQ-1 was not metabolized under aerobic conditions. Similarly, 19F NMR efflux studies in intact cells showed that the NLTQ-1 was bound to the cells predominantly under hypoxic conditions. 19F-NMR spectra of intact cells, exposed under hypoxic conditions to NLTQ-1, and of their lysates, after precipitation of various cellular components, indicated that possible covalent binding of NLTQ-1 had occurred with macromolecules such as proteins and nucleic acids. Therefore, NLTQ-1 might be suitable as a 19F-MRS/MRI hypoxia probe, although further in vivo work is necessary to verify this matter.

4-[3-(2-Nitro-1-imidazolyl)propylamino]-7-chloroquinoline hydrochloride (NLCQ-1), a novel bioreductive agent as radiosensitizer in vitro and in vivo: comparison with tirapazamine.

The novel hypoxia-selective cytotoxin NLCQ-1, which is a weak DNA intercalator, was studied in conjunction with radiation against V79 cultured cells and EMT6 or SCCVII tumors in their syngeneic mice and compared with tirapazamine (TPZ). NLCQ-1 was a very potent and efficient radiosensitizer of hypoxic V79 cells, providing SER values of 2.27-2.56 at 20-80 microM concentration (measured at 10% survival level). Its C1.6 (concentration for an SER of 1.6 to be obtained) was 7.2+/-0.2 microM. Its in vitro therapeutic index (ThI, defined as CT50(Air),/C1.6) varied by the exposure time from 57 (1-h exposure) to 145 (4.5-h exposure). The corresponding C1.6 value for TPZ was 16.9 microM whereas its in vitro therapeutic index was 49 (3-h exposure). A schedule-dependent synergistic interaction was observed between NLCQ-1 or TPZ and 20 Gy of radiation in both tumor models examined, by using the in vivo-in vitro assay as endpoint. Optimal synergism (> 1 log) was observed in EMT6 tumors when each bioreductive drug was given between 45 and 60 min before irradiation. NLCQ-1 alone had no significant antitumor activity at 10 mg/kg (28% of its single LD50), whereas a 0.4 surviving fraction was obtained by TPZ at 30 mg/kg (38% of its single LD50). SER values of 1.52 and 1.25 were obtained with 10 mg/kg NLCQ-1 and 30 mg/kg TPZ, respectively, in EMT6 tumors. An SER value of 1.58 was obtained for both hypoxia-selective cytotoxins, at equitoxic doses, in SCCVII tumors, by using a fractionated regimen. These results suggest a possible use of NLCQ-1 or TPZ as adjuvants to radiotherapy.

Schedule-dependent potentiation of chemotherapeutic drugs by the bioreductive compounds NLCQ-1 and tirapazamine against EMT6 tumors in mice.

PURPOSE: Comparisons of schedule-dependent interactions between the hypoxic cytotoxins NLCQ-1/ tirapazamine (TPZ) and various chemotherapeutic drugs in BALB/c mice bearing EMT6 tumors. METHODS: The antitumor effects of the single or combined drugs were assessed with various administration time intervals using the in vivo-in vitro clonogenic assay as the endpoint. The chemotherapeutic drugs tested were cisplatin (cisDDP), melphalan (L-PAM), cyclophosphamide (CPM), 5-fluorouracil (5-FU), doxorubicin (Doxo), etoposide (VP-16) and Taxol at doses of 8, 5, 100, 150, 12, 35 and 20 mg/kg, respectively. NLCQ-1 was given at 10 mg/kg (28% of its single LD50 value) and TPZ was given at 30 mg/kg (38% of its single LD50 value). All drugs were given by i.p. injection in saline or as commercially available pharmaceutical solutions. RESULTS: Schedule-dependent synergistic interactions with different patterns for each bioreductive drug were observed with almost all of the chemotherapeutic agents examined. Potentiation accounting for more than 25% of the total tumor cell killing was observed with NLCQ-1/TPZ and cisDDP, L-PAM, CPM, 5-FU and Taxol at the optimal administration intervals. Potentiation accounting for 70% of the total tumor cell killing was found with NLCQ-1 and CPM. CONCLUSIONS: These results suggest a potential clinical use of NLCQ-1/TPZ as adjuvants to certain chemotherapeutic agents.

4-[3-(2-Nitro-1-imidazolyl)propylamino]-7-chloroquinoline hydrochloride (NLCQ-1), a novel bioreductive compound as a hypoxia-selective cytotoxin.

A novel weakly DNA-intercalative bioreductive compound. 4-[3-(2-nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1). has been synthesized and studied as a hypoxia-selective cytotoxin in vitro. NLCQ-1, which shares a similar structure with the DNA-intercalative antimalarial drug chloroquine, bound more strongly to DNA than the nonchlorinated analog NLQ-1 (4-[3-(2-nitro-1-imidazolyl)propylamino]-quinaldine hydrochloride). Thus, NLCQ-1 exhibited a C50 [concentration for 50% displacement of the ethidium bromide (EB) from a DNA-EB complex] of 44 microM, whereas a C50 value could not be reached for NLQ-1 up to 225 microM. NLCQ-1 demonstrated significant hypoxic selectivity in several rodent (V79, EMT6, SCCVII) or human (A549, OVCAR-3) tumor cell lines. Its potency as a hypoxic cytotoxin (expressed as the product of exposure time and concentration for 50% survival) ranged between 10 and 136 microM x h, for the cell lines tested, at 30 microM input concentration. Because uptake in all cell lines was similar, the differences in potency may reflect differences in the enzymatic profile or damage repair processes among the cell lines. In addition, however, the most striking feature of NLCQ-1 was that hypoxic selectivity increased with exposure time, a common feature normally found in only bis-bioreductive agents carrying two moieties with different redox potentials. Thus, hypoxic selectivity of NLCQ-1 in V79 cells at 50% survival was increased from fivefold up to 388-fold by increasing exposure time from 1 to 4.5 h, as the result of a concomitant increase and decrease in its hypoxic and aerobic potency, respectively, over time. Because the nonchlorinated analog NLQ-1 did not demonstrate similar behavior, we hypothesized that the C-7 chlorine of NLCQ-1 might play a significant role in this phenomenon.

Mechanisms involved in the potentiation of melphalan by the bioreductive compound THNLA-1 in vitro.

9-[3-(2-Nitro-1-imidazolyl)propylamino]-1,2,3,4-tetrahydroacridine hydrochloride (THNLA-1) is a 2-nitroimidazole-based, weakly DNA-intercalating bioreductive agent that significantly potentiates the toxic effects of commonly used antitumor drugs such as melphalan (L-PAM) or cis-DDP in sensitive or resistant cell lines in culture, as well as in solid tumors in mice. Potentiation in vitro was observed when cells were preexposed to THNLA-1 under hypoxic conditions before exposure to L-PAM under aerobic conditions. In this study we investigated possible mechanisms involved in the potentiation of L-PAM by THNLA-1 in V79 Chinese hamster cells. Limited depletion of glutathione with buthionine sulfoximine or THNLA-1 under hypoxic pretreatment conditions accounted for only 8.3% of the potentiation induced by THNLA-1. However, DNA, RNA, and protein synthesis were inhibited in a synergistic way in cells preexposed to THNLA-1 under hypoxic conditions (2 h, 37 degrees C) and then coexposed to various doses of L-PAM under aerobic conditions (1 h, 37 degrees C). Cell cycle analysis by flow cytometry showed a slow traverse through the S phase in the L-PAM-alone-treated cells. However, this phenomenon was more prominent in the THNLA-1 plus L-PAM-treated cells. Under aerobic co-incubation conditions with L-PAM, no difference was observed in the cell cycle of L-PAM-alone-treated cells vs. THNLA-1 plus L-PAM-treated cells. Significantly increased apoptosis was observed in the hypoxia-pretreated cells with THNLA-1, 12 and 24 h posttreatment. Comet and alkaline elution assay analysis showed increased DNA cross-links in the hypoxia-pretreated cells with THNLA-1 compared to the L-PAM-alone-treated cells. Finally, potential lethal damage repair was totally suppressed only in the hypoxia-pretreated cells with THNLA-1. In conclusion, DNA damage and hindrance in its repair are the most important mechanisms in the potentiation of L-PAM by THNLA-1, under hypoxic pretreatment conditions.

NLCQ-1, a novel hypoxic cytotoxin: potentiation of melphalan, cisDDP and cyclophosphamide in vivo.

PURPOSE: To investigate in vivo interactions between the recently developed bioreductive agent 4-[3-(2-nitroimidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1) and the chemotherapeutic agents melphalan (L-PAM), cis-platin (cisDDP) and cyclophosphamide (CPM). METHODS AND MATERIALS: EMT6 and FSaIIC tumor cells were inoculated (subcutaneously) into the leg(s) of female Balb/c and male C3H mice, respectively. Treatment was initiated at 10 mm (EMT6) and 5 mm (FSaIIC) mean tumor diameter. The in vivo-in vitro and tumor regrowth assays were used, respectively, as endpoints. Bone marrow toxicity studies were also performed when the in vivo-in vitro assay was used. Drugs were given by i.p. injection. Tumors were excised 18-h after chemotherapeutic drug administration (Balb/c mice) or measured daily until three times their original size (C3H mice). The optimum administration schedule for potentiation between NLCQ-1 and each chemotherapeutic drug, as well as dose modification factors (DMF) at the optimum time, were determined with the in vivo-in vitro assay. When the tumor regrowth assay was used, each chemotherapeutic agent was given either as a single dose or as a split dose over two consecutive days at the optimum administration time after a 10 mg/kg NLCQ-1 i.p. injection. RESULTS: NLCQ-1 (at 0.33 times MTD), strongly potentiated the antitumor effect of L-PAM, cisDDP and CPM without concurrent enhancement in bone marrow toxicity. Potentiation was strictly schedule dependent and the optimum effect (1.5 to 2 logs killing beyond additivity) was observed when NLCQ-1 was given 60-, 45-, and 110-min before L-PAM, cisDDP, and CPM, respectively. The DMF values at 30% survival were 2.5, 1.9, and 3.8 for L-PAM, cisDDP, and CPM, respectively. DMF values for bone marrow toxicity at 50% survival were ca. 1 for all chemotherapeutic drugs. Pretreatment with NLCQ-1 resulted in 4-12 days extra delay in the regrowth of FSaIIC tumors. CONCLUSIONS: These results support the clinical investigation of NLCQ-1 as a chemosensitizer.

9-[3-(2-Nitro-1-imidazolyl)propylamino]-cyclopenteno[b]quinoline hydrochloride (NLCPQ-1). A novel DNA-affinic bioreductive agent as chemosensitizer: mechanistic studies. II.

NLCPQ-1 is a novel, weak, DNA-affinic bioreductive compound with enhanced chemosensitizing ability for commonly used chemotherapeutic agents, both in vitro and in vivo. In the present report we investigated possible mechanisms involved in the potentiation of cis-DDP and L-PAM in V79 cells. Potentiation was observed when cells were pretreated under hypoxic conditions with NLCPQ-1 prior to their aerobic exposure to each chemotherapeutic agent studied and in the presence of NLCPQ-1. The dominant mechanisms, under hypoxic pretreatment conditions, participating in the potentiation were: a) extensive DNA damage, as measured by the comet and alkaline elution assays, b) DNA, RNA, and protein synthesis inhibition, c) significant delay in the traverse through the S phase, as observed by flow cytometry, and possibly d) suppression of PLD repair. Apoptosis was also detected 36 h posttreatment in the chemotherapeutic drug-treated as well as the combination drug-treated cells. Glutathione depletion by NLCPQ-1 metabolites under hypoxic conditions was also involved in the potentiation process, but its contribution in potentiation was minimal.

Favorable outcome of ovarian germ cell malignancies treated with cisplatin or carboplatin-based chemotherapy: a Hellenic Cooperative Oncology Group study.

PURPOSE: To evaluate the outcome and the prognosis of patients with ovarian germ cell malignancies who were treated with platinum-based chemotherapy immediately after initial surgery. METHODS: We conducted a retrospective review of patients with ovarian germ cell tumors who were referred for consideration of treatment to the Departments of Medical Oncology participating in the Hellenic Cooperative Oncology Group. RESULTS: Over a 14-year period 53 patients were included in our study. There were 13 patients with dysgerminoma and 40 patients with nondysgerminomatous tumors. Forty percent of patients underwent complete resection of their tumors. Platinum-based chemotherapy consisted primarily of cisplatin, vinblastine, and bleomycin (PVB) in 9 patients; bleomycin, etoposide, and cisplatin (BEP) in 15 patients; and bleomycin, etoposide, and carboplatin (BEC) in 25 patients. With a median follow-up of 39 months, 5 patients developed progressive disease and died of their tumor and 1 patient died of bleomycin-induced lung toxicity with no evidence of active tumor. The 5-year overall survival was 100% for patients with dysgerminoma and 85% for patients with nondysgerminomatous tumors. Eighty percent of patients with advanced nondysgerminomatous tumors and residual disease after surgery remain disease free. CONCLUSION: With this study we confirm that patients with ovarian germ cell malignancies have a favorable outcome when treated with platinum-based chemotherapy.