Radiation Sensitivity of Human Malignant Lymphocytes

A simple and rapid in vitro technique to assess the sensitivity of human malignant lymphocytes to roentgen irradiation is described. A variety of established malignant lymphocyte cell lines were cloned in microwells and clone survival was used as the end-point. The survival of the clonogenic malignant lymphocyte down to a fraction of approximately 0.001 could be measured accurately. Except for a T-cell line, the radiation sensitivities of the cell lines were similar to that of normal T-lymphocytes.     

Discovery of novel 2,8-diazaspiro[4.5]decanes as orally active glycoprotein IIb-IIIa antagonists

In our efforts to develop orally active GPIIb-IIIa antagonists with improved pharmaceutical properties, we have utilized a novel 2,8-diazaspiro[4.5]decane scaffold as a template. We describe here our investigation of a variety of templates including spiropiperidinyl-gamma-lactams, spiropiperidinylimide, spiropiperidinylureas, and spiropiperidinylhydantoins. With the appropriate acidic and basic pharmacophores in place, each template yielded analogues with potent GPIIb-IIIa inhibitory activity. One of the compounds, 59 (CT50787), was also used to demonstrate for the first time the use of a pharmacological agent which is alphaIIbbeta3 specific to display biological activity in a lower species such as mouse and to extend bleeding times. Evaluation of the pharmacokinetic properties of selected compounds from each series in rat, dog, and cynomolgus monkey has led to the identification of 22 (CT51464), a double prodrug, with excellent pharmacokinetic properties. It exhibited good pharmacokinetic profile across species (F% = 33 (Cyno), 73 (dog), 22 (rat); t(1/2)(beta)() = 14.2 h (Cyno), 8.97 h (dog), 1.81 h (rat)). The biologically active form, 23 (CT50728), displayed inhibition of platelet aggregation in platelet rich plasma (PRP) with an IC(50) value of 53 nM in citrate buffer, 110 nM in PPACK anticoagulated PRP, and 4 nM in solid-phase GPIIb-IIIa competition binding assay (ELISA). Both 23 and 22 were stable in human liver microsomes, did not inhibit the P450 3A4 isozyme, and had low protein binding (18.22% for 23) and a desirable log P (0.45 +/- 0.06 for 22, and -0.91 +/- 0.32 for 23). It is predicted that the high oral bioavailability for these compounds in multiple species should translate into lower intra- and intersubject variability in man. The long plasma half-life of the lead is consistent with once or twice daily administration for chronic therapy. Analogue 22 (CT51464) thus appears to be a promising oral GPIIb-IIIa inhibitor with significantly improved pharmacokinetic properties over the previously described clinical candidates and may be found useful in the treatment of arterial occlusive disorders.     

Bradycardia following temporomandibular joint ankylosis release

Bradycardia following release of temporomandibular (TM) joint ankylosis is uncommon. We are reporting a case of longstanding TM joint ankylosis, which after release, resulted in significant bradycardia on jaw opening. The possible etiology of this bradycardia is discussed. There is hardly any literature available on this topic.     

Prospective randomised open-label comparison of danaparoid with dextran 70 in the treatment of heparin-induced thrombocytopaenia with thrombosis: a clinical outcome study

AIM: To compare clinical outcomes in a randomised comparison of treatment with danaparoid sodium (a heparinoid), or dextran 70, for heparin-induced thrombocytopaenia (HIT) plus thrombosis. METHODS: Forty-two patients with recent thrombosis and a clinical diagnosis of probable HIT who presented at ten Australian hospitals during a study period of six and one half years were randomly assigned to open-label treatment with intravenous danaparoid or dextran 70, each combined with oral warfarin. Thirty-four patients (83%) had a positive platelet aggregation or 14C-serotonin release test for HIT antibody. Twenty-five received danaparoid as a bolus injection of 2400 anti-Xa units followed by 400 units per hour for 2 h, 300 units per hour for 2 h, and then 200 units per hour for five days. Seventeen received 1000 mL dextran 70 on day one and then 500 mL on days 2-5. Patients were reviewed daily for clinical evidence of thrombus progression or resolution, fresh thrombosis or embolism, bleeding or other complications. The primary trial endpoint was the proportion of thromboembolic events with complete clinical resolution by the time of discharge from hospital. RESULTS: With danaparoid, there was complete clinical recovery from 56% of thromboembolic events compared to 14% after dextran 70 (Odds Ratio 10.53, 95% Confidence Interval 1.6-71.4; p = 0.02). Clinical recovery with danaparoid was complete or partial in 86% of thromboembolic events compared with 53% after dextran 70 (Odds Ratio 4.55, 95% Confidence Interval 1.2-16.7; p = 0.03). Overall clinical effectiveness of danaparoid was rated as high or moderate in 88% of patients compared with 47% for dextran 70 (p = 0.01). One patient given danaparoid died of thrombosis compared with three patients given dextran 70. The platelet count returned to normal after a mean of 6.7 days with danaparoid and 7.3 days with dextran 70. There was no major bleeding with either treatment. CONCLUSION: danaparoid plus warfarin treatment for HIT with thrombosis is effective, safe, and superior to dextran 70 plus warfarin.     

Preclinical evaluation for noninvasive reversal following long-term vas occlusion with styrene maleic anhydride in langur monkeys

A preclinical evaluation for reversal through a noninvasive approach following long-term vas occlusion with styrene maleic anhydride (SMA) has been attempted in langur monkeys at the level of semen parameters, sperm functional tests, semen biochemistry, histology and ultrastructure of reproductive organs, hematology and serum clinical biochemistry including antisperm antibodies (ASA), prostate-specific antigen (PSA) and testosterone. Noninvasive reversal through palpation, percutaneous squeezing and electrical stimulation, forced vibratory movements and suprapubic percussion in the inguinal segments and per-rectal digital massage was attempted in seven langur monkeys after 540 days following vas occlusion. The results revealed instant azoospermia reversal on the same day of reversal with impaired sperm quality, which showed gradual improvement and normospermia with normal motility and viability after 60-90 days of reversal. Sperm functional tests, including ultrastructure of spermatozoa, indicative of sterility in the initial ejaculations, reached normalcy after 90-120 days of reversal. The seminal plasma biochemistry indicative of obstructive azoospermia regained a normal pattern after 90-120 days of reversal. The morphology of testes that showed focal degeneration during 540 days of vas occlusion and that of vasa deferentia that showed exfoliation of epithelial cells resumed to normal morphology comparable with control animals after 150 days of reversal. The morphology of the epididymis, seminal vesicle and prostate did not show appreciable changes following vas occlusion and after noninvasive reversal compared with those of control animals. Hematology, serum clinical chemistry, ASA, PSA and testosterone fluctuated within control limits, indicating safety of the procedure at the level of accessory reproductive organs. The results suggest that noninvasive reversal is feasible even after long-term vas occlusion with SMA and is safe without adverse side effects.     

Pharmacology of N-benzyladriamycin-14-valerate in the rat

Purpose: N-Benzyladriamycin-14-valerate (AD 198) is a semisynthetic anthracycline analogue superior to doxorubicin (DOX) both in vitro and in experimental rodent tumor models, and with differing mechanistic properties from those of the parental antibiotic agent. In the present study, we examined the metabolic fate and hematotoxicity of AD 198 in rats, with a view to determining whether some of the therapeutic properties observed for this drug might be due to a DOX prodrug effect. Methods: Samples of plasma, bile and urine were obtained at various times following intravenous (i.v.) [¹⁴C]–AD 198 administration to rats and were analyzed by reversed-phase HPLC with flow–fluorescence detection and complementary liquid scintillography. In other animals, red blood cell and white blood cell (WBC) counts were determined for blood obtained by retrobulbar sampling on selected days from groups of animals receiving either AD 198 or DOX at several dose levels, as well as from vehicle controls. Results: Following a single iv dose of [¹⁴C]-AD 198 (5 mg/kg; equivalent to the optimal murine antitumor dose) in anesthetized rats, a triphasic plasma decay pattern for parental drug was evident with extremely rapid α and β phases followed by a very long terminal elimination phase. Principal plasma products included N–benzyladriamycin (AD 288) and N–benzyladriamycinol (AD 298) together with very low levels of DOX and doxorubicinol (DOXOL). Analysis of bile from anesthetized and conscious animals receiving AD 198 revealed DOX to be the principal biliary fluorescent species together with low levels of AD 288, AD 298 and DOXOL; no parental drug was seen. By contrast, AD 288 was the principal urinary product, together with low levels of AD 298 and DOX; again, no parental drug was evident. Dose recovery (8 h) in the respective bile and urine of anesthetized rats was 12.4% and 13.2% based upon total fluorescence versus 1% and 15.3% of the administered radiolabel. In conscious animals, 13.4% of drug fluorescence was recovered in the bile (48 h), while in urine 16.6% and 77.1% of drug fluorescence and radiolabel, respectively, were eliminated over 72 h. The discrepancy between recovery of drug fluorescence and ¹⁴C was due to the production of nonfluorescent hippuric acid (benzoylglycine) and N–benzyl daunosamine as a consequence of hepatic and renal drug metabolism. In the separate hematotoxicity studies, AD 198 (24.6 mg/kg i.v.; equivalent to the murine LD₅₀ dose), produced a 45% reduction (nadir day 3–5) in WBC count, with recovery by day 10. By contrast, DOX (10 mg/kg i.v.; equivalent to the mouse highest nonlethal dose) produced an 80% decline in WBC with only partial recovery by day 17. Conclusions: By virtue of the low systemic DOX levels and low hematotoxicity observed in rats receiving AD 198, the in vivo therapeutic superiority of AD 198 cannot be attributed to substantial intracellular DOX generation. The conclusion that the therapeutic superiority of AD 198 compared to DOX results from the mechanistic differences between these two drugs is further supported by recent observations on their biochemical differences with regard to protein kinase C and topoisomerase II inhibition. Received: 26 January 1998 / Accepted: 5 August 1998     

Comparative uptake and retention of adriamycin andN-benzyladriamycin-14-valerate in human CEM leukemic lymphocyte cell cultures

Summary N-Benzyladriamycin-14-valerate (AD 198) is a new lipophilic adriamycin (ADR) analogue that shows marked therapeutic superiority to ADR in murine tumor model systems yet differs mechanistically from ADR in a number of ways. Among its other properties, AD 198 produces a delayed but profound effect on cell-cycle progression and a pattern of continuing DNA damage in cultured cells briefly exposed to the drug. Using radiolabeled drug forms and radioassays combined with HPLC separation and fluorimetric detection techniques, aspects of drug accumulation, biotransformation, and retention in cultured human CEM leukemic lymphocytes were studied, in part to determine a possible pharmacologic basis for the latent effects seen with this drug. In addition, the cellular pharmacology of AD 198 and ADR were comparatively examined under identical experimental conditions. When CEM cells were incubated with drug at equi-growth inhibitory/minimally cytotoxic concentrations (AD 198, 1.0 M; ADR, 0.1 M), a number of differences were apparent. Under conditions of continuous 24-h drug exposure, a slow cellular accumulation and equilibration was observed with ADR (cell: medium equilibrium, 1:11 after 4–6 h), whereas the uptake of AD 198 was rapid and extensive (cell: medium equilibrium, 3:1 within 30 min). In drug-retention studies, when cells were pretreated at the same drug concentrations as before (AD 198 for 1 h; ADR for 4 h) and then transferred to drug-free media, both compounds re-equilibrated their intracellular drug content with the fresh media, losing about 50% of their respective anthracycline levels. Liquid chromatographic analysis of ADR-treated cultures under both sets of conditions showed the parent drug to be the only intracellular anthracycline species, whereas analysis of AD 198-treated cultures revealed two fluorescent signals corresponding to the parent drug and its 14-deesterified biotransformation product,N-benzyladriamycin (AD 288). Levels of AD 288 rose from 2% of the total intracellular anthracycline content immediately on drug admixture to 61% following 24 h continuous drug exposure and to 69% at 24 h in cells exposed to drug for 1 h and then continued in drug-free media for 24 h. At all times, the balance of the intracellular anthracycline fluorescence was attributable to the parent drug; no ADR was detectable in AD 198-treated cells by either fluorescence detection or radioassay. Thus, AD 198 is not a prodrug form of ADR, and the in vitro effects of this agent, including the latent effects on cell-cycle inhibition and DNA damage seen in cells following short-term drug exposure, can be explained on the basis of the high levels of active parent drug and biotransformation product that accumulate and persist in the cells.Abbreviations ADR adriamycin (doxorubicin) - AD 198 N-benzyladriamycin-14-valerate - AD 288 N-benzyladriamycin - AD 32 N-trifluoroacetyladriamycin-14-valerate - AD 143 N-trifluoroacetyladriamycin-14-0-hemiadipate - AD 41 N-trifluoroacetyladriamycin - [¹⁴C]-AD 198 [benzyl]--methylene-¹⁴C]-N-benzyladriamycin-14-valerate - [¹⁴C]-ADR [14-¹⁴C]-adriamycin - HPLC high-performance liquid chromatography - TLC thin-layer chromatography - DMSO dimethylsulfoxide - S-MEM Eagle's minimum essential medium for suspension culture - PBS phosphate-buffered saline (pH 7.0)     

Regulation of motility,invasion, and metastatic potential of squamous cell carcinoma by 1α,25‐dihydroxycholecalciferol

BACKGROUND:

The active metabolite of vitamin D 1α,25‐dihydroxycholecalciferol (1,25D₃) has exhibited broad‐spectrum antitumor activity in xenograft animal models. However, its activity against metastatic disease has not been extensively investigated.

METHODS:

Squamous cell carcinoma (SCC) or 1,25D₃‐resistant variant SCC‐DR cells were treated with 1,25D₃. Actin organization was examined by immunofluorescence assay. Cell migration was assessed by “wound” healing and chemotactic migration assays. Cell invasion was assessed by a Matrigel‐based invasion assay and in situ zymography. Matrix metalloproteinase 2 (MMP‐2) and MMP‐9 expression and secretion were examined by immunoblot analysis and an enzyme‐linked immunosorbent assay, respectively. E‐cadherin expression was assessed by flow cytometry, immunoblot analysis, and immunohistochemistry. Knockdown of E‐cadherin was achieved by small interfering RNA. An experimental metastasis mouse model was created by intravenous injection of tumor cells; and lung tumor development in the mice was assessed by magnetic resonance imaging, gross observation, and histology.

RESULTS:

SCC cellular morphology and actin organization were altered by 10 nM 1,25D₃. 1,25D₃ inhibited SCC cell motility and invasion, which were associated with reduced expression and secretion of MMP‐2 and MMP‐9, and 1,25D₃ promoted the expression of E‐cadherin. These findings were not observed in SCC‐DR cells. Knock down of E‐cadherin rescued 1,25D₃‐inhibited cell migration. Intravenous injection of SCC or SCC‐DR cells resulted in the establishment of extensive pulmonary lesions in saline‐treated C3H mice. Treatment with 1,25D₃ resulted in a marked reduction in the formation of lung tumor colonies in mice that were injected with SCC cells, but not in mice that were injected with SCC‐DR cells.

CONCLUSIONS:

1,25D₃ suppressed SCC cell motility, invasion, and metastasis, partially through the promotion of E‐cadherin‐mediated cell‐cell adhesion. Cancer 2013. © 2012 American Cancer Society.