Metformin shows anti-neoplastic properties by inhibition of oxidative phosphorylation and glycolysis in epidermolysis bullosa-associated aggressive cutaneous squamous cell carcinoma.

BACKGROUND: While most cutaneous squamous cell carcinomas (cSCCs) are treatable, certain high-risk cSCCs, such as those in recessive dystrophic epidermolysis bullosa (RDEB) patients, are particularly aggressive. Owing to repeated wounding, inflammation and unproductive healing, RDEB patients have a 68% cumulative risk of developing life-threatening cSCCs by the age of 35, and a 70% risk of death by the age of 45. Despite aggressive treatment, cSCC represents the leading cause of premature mortality in these patients, highlighting an unmet clinical need. Increasing evidence points to a role of altered metabolism in the initiation and maintenance of cSCC, making metabolism a potential therapeutic target. OBJECTIVES: We sought to determine the feasibility of targeting tumour cell energetics as a strategy to selectively hinder the growth advantage of aggressive cSCC. METHODS: We evaluated the cell energetics profiles of RDEB-SCC cells by analysing available gene expression data against multiple gene signatures and single-gene targets linked to metabolic reprogramming. Additionally, we employed real-time metabolic profiling to measure glycolysis and respiration in these cells. Furthermore, we investigated the anti-neoplastic properties of the metformin against human and murine high-risk cSCCs in vitro and in vivo. RESULTS: Gene expression analyses highlighted a divergence in cell energetics profiles between RDEB-SCC and non-malignant RDEB keratinocytes, with tumour cells demonstrating enhanced respiration and glycolysis scores. Real-time metabolic profiling supported these data and additionally highlighted a metabolic plasticity of RDEB-SCC cells. Against this background, metformin exerted an anti-neoplastic potential by hampering both respiration and glycolysis, and by inhibiting proliferation in vitro. Metformin treatment in an analogous model of fast-growing murine cSCC resulted in delayed tumour onset and slower tumour growth, translating to a 29% increase in median overall survival. CONCLUSIONS: Our data indicate that metformin exerts anti-neoplastic properties in aggressive cSCCs that exhibit high-risk features by interfering with respiration and glycolytic processes.

Low rate of new-onset primary biliary cholangitis in a cohort of anti-mitochondrial antibody-positive subjects over six years of follow-up.

BACKGROUND AND AIMS: Anti-mitochondrial antibodies (AMA) are closely linked to primary biliary cholangitis (PBC). The prevalence of AMA in the general population is low, and AMA positivity may precede PBC. We aimed to determine the natural history of subjects with positive AMA. METHODS: In total, 302 patients were tested AMA-positive over a ten-year period. Of these, immunoblotting confirmed specific AMA in 184 (29 male, 155 female, age 59.6 ± 14.1 years). These subjects were invited to our liver outpatient clinic for clinical and biochemical re-evaluation. Detailed clinical history data were additionally collected from the hospital computer system and by telephone. The subsequent course with regard to mortality, liver-related morbidity, extrahepatic co-morbidities and effectiveness of PBC treatment was determined in 150 subjects (81.5%). RESULTS: After 5.8 ± 5.6 years of follow-up (FU), of 184 AMA-positive subjects, 28 subjects (15.2%; liver-related mortality n = 5) were deceased, and 122 subjects (66.3%) completed FU while 34 subjects (18.5%) were not available for FU. The 122 patients who completed FU were 63 patients with established PBC, six de novo cases of PBC (10.2% of 59 initially at risk), 42 (34.4%) subjects were still AMA-positive without PBC, and 11 (9.0%) subjects were AMA-negative at FU. CONCLUSIONS: Anti-mitochondrial antibodies-positive patients without PBC at baseline infrequently developed PBC over six years of FU. AMA positivity represented a transient serological autoimmune phenomenon in a significant proportion of subjects.

Clinical and metabolic characterization of obese subjects without non-alcoholic fatty liver: A targeted metabolomics approach.

INTRODUCTION: As a small proportion of obese individuals do not develop metabolic complications and non-alcoholic fatty liver disease (NAFLD), this study aimed to provide a comprehensive clinical, metabolic and genetic description of obese subjects with healthy livers. METHODS: A total of 183 subjects were stratified, according to BMI, presence of metabolic syndrome, biochemical liver tests and hepatic steatosis on ultrasound, into: (i) lean controls (n = 69); (ii) obese healthy (n = 50); and (iii)obese NAFLD (n = 62) groups. Detailed clinical, genetic and metabolic evaluations were then performed. RESULTS: Obese healthy subjects did not differ in glucose parameters from lean controls, and had a lower rate of minor TM6SF2 gene variants compared with obese NAFLD (2/49 vs. 11/60, respectively; P = 0.035) and lean controls (13/64; P = 0.035), but significantly higher leptin concentrations than lean controls (P < 0.001); they also higher adiponectin concentrations (P < 0.001), and lower TNF-α and IL-6 concentrations (P = 0.01 and P < 0.001, respectively), than obese NAFLD subjects. Also, metabolomic studies identified ether- and ester-containing phospholipids [PC ae C44:6, PC ae C42:5, PC aa C40:4; P < 0.001, corrected by the false discovery rate (FDR) method] and found that the amino-acids lysine, glycine and isoleucine (FDR < 0.001) differed between the two obese groups, but not between lean controls and obese healthy subjects. CONCLUSION: Obese people with healthy livers are characterized by intact glucose homoeostasis, lower pro-inflammatory cytokine levels, and higher adiponectin and leptin concentrations compared with obese people with NAFLD. In addition, the major allele of TM6SF2, a set of phosphatidylcholines and several amino acids are associated with healthy livers in obesity.

Aberrant hepatic TRIB3 gene expression in insulin-resistant obese humans.

AIMS/HYPOTHESIS: The pseudokinase tribbles homologue 3 (Drosophila) (TRIB3) negatively interferes with insulin-mediated phosphorylation and activation of v-akt murine thymoma viral oncogene homologue 1 (AKT1, also known as protein kinase B). Animal studies have shown that Trib3 expression was higher in the fasting state and in animal models of diabetes, promoting hyperglycaemia presumably by increasing glucose production in the liver. Less is known about the role of TRIB3 in insulin resistance in humans, although a gain-of-function mutation associated with abnormalities related to insulin resistance has been described in TRIB3. METHODS: We determined hepatic mRNA expression of TRIB3 and selected genes encoding enzymes, transcription factors and coactivators involved in glucose homeostasis. We also determined biochemical variables of intermediary metabolism in obese patients with varying degrees of insulin resistance. RESULTS: In our study population hepatic TRIB3 mRNA expression was associated with surrogate markers of insulin resistance. TRIB3 expression was significantly increased in a subgroup with high HOMA of insulin resistance (HOMA-IR) compared with a low HOMA-IR group (p = 0.0033). TRIB3 transcript levels were correlated with PEPCK (also known as PCK2) mRNA expression (p = 0.0014) and mRNA expression of PPARGC1A (p = 0.0020), PPARGC1B (p < 0.0001), USF1 (p = 0.0017), FOXO1 (p = 0.0003) and SREBP-1c (also known as SREBF1; p = 0.0360). Furthermore ligands of peroxisome proliferator-activated receptor alpha/retinoid X receptor and overexpression of its coactivator PPARGC1A as well as overexpression of SREBP-1c and its coactivator PPARGC1B increased TRIB3 promoter activity in HepG2 cells. CONCLUSIONS/INTERPRETATION: We have found evidence for a role of aberrant hepatic TRIB3 transcript levels in insulin resistance in obese humans and identified potential transcriptional pathways involved in regulation of TRIB3 gene expression in the liver.

Hepatic adiponectin receptors (ADIPOR) 1 and 2 mRNA and their relation to insulin resistance in obese humans.

OBJECTIVE: Adiponectin signalling attenuates insulin resistance (IR) and steatosis hepatis in animal models. As adiponectin receptor (ADIPOR)1 and ADIPOR2 are critical components in the adiponectin signalling cascade, we studied hepatic ADIPOR1/2 mRNA levels in humans and their relation to IR. DESIGN: We determined metabolic risk factors and levels of hepatic mRNA transcribed from ADIPOR1, ADIPOR2 and FOXO1, a putative up-stream regulator, in 43 and 34 obese subjects with low and high homeostasis model assessment-IR, respectively. RESULTS: Plasma adiponectin and metabolic risk factors showed associations with IR as expected. Both hepatic ADIPOR1 and ADIPOR2 mRNA expression levels were higher in insulin-resistant subjects (P<0.0035). ADIPOR1 mRNA correlated with FOXO1 mRNA in obese insulin resistant (P=0.0034), but not insulin-sensitive subjects, while no correlations of ADIPOR2 with FOXO1 mRNA were noted. FOXO1 enhanced transcription from the ADIPOR1, but not the ADIPOR2 promoter in HepG2 cells. CONCLUSION: Increased hepatic ADIPOR1 and ADIPOR2 mRNA in insulin-resistant obese subjects may, at least in part, reflect a compensatory mechanism for reduced plasma adiponectin. FOXO1 may contribute to enhanced ADIPOR1, but not ADIPOR2 transcription in IR.

The SREBF-1 locus is associated with type 2 diabetes and plasma adiponectin levels in a middle-aged Austrian population.

CONTEXT: The sterol regulatory element-binding protein-1c (SREBP-1c) is a transcription factor involved in the regulation of lipid and glucose metabolism and has been implicated in the pathophysiology of type 2 diabetes mellitus (T2DM). OBJECTIVE: We aimed to confirm associations of the SREBF-1 gene with T2DM in an Austrian population and to study possible associations with diabetes-related quantitative traits. DESIGN, SETTINGS AND PARTICIPANTS: We genotyped a diabetic cohort (n=446) along with a control group (n=1524) for a common C/G variation that is located in exon 18c (rs2297508) and has been associated with obesity and T2DM in French populations. MAIN OUTCOME MEASURES: Body mass index (BMI), indices of insulin sensitivity and beta-cell function, plasma adiponectin, T2DM and single-nucleotide polymorphism rs2297508. RESULTS: Genotype distributions associated with rs2297508 differed by T2DM status (P=0.0045), but not by BMI. The variant G allele was associated with a modest, but significant, increase in the prevalence of T2DM after adjustment for age, sex and BMI (G/G: odds ratios (OR) (95% confidence intervals)=1.45 (0.99-2.11) and G/C: OR=1.37 (1.04-1.81)). In a cross-sectional population of non-diabetic subjects, associations of rs2297508 genotypes with plasma adiponectin levels adjusted for age, sex and BMI (P=0.0017) were observed in that the risk G/G genotype displayed the lowest adiponectin levels. CONCLUSIONS: We observed associations of rs2297508 with T2DM prevalence and plasma adiponectin. SREBP-1c has been implicated in the regulation of adiponectin gene expression. Our results therefore raise the possibility that sequence variations at the SREBF-1 gene locus might contribute to T2DM risk, at least in part, by altering circulating adiponectin levels.

Synthesis and antiviral/antitumor evaluation of 2-amino- and 2-carboxamido-3-arylsulfonylthiophenes and related compounds as a new class of diarylsulfones.

Based on general SARs previously described for anti-HIV-1 diarylsulfone derivatives, a series of 2-amino- and 2-carboxamido-3-arylsulfonylthiophenes has been prepared and evaluated as potential antiviral and antitumor agents. In cell culture, some of the 2-aminothiophenes exhibited moderate and selective activity against HIV-1, with 2-amino-3-(2-nitrophenylsulfonyl)thiophene (7e) being most attractive (EC(50)=3.8 microg/mL, CC(50)=>100 microg/mL). In broad-spectrum antiviral assays, the 3-arylsulfonyl-2-(trifluoroacetamido)thiophenes (8c-g) and 2-acetamido-3-arylsulfonyl-5-nitrothiophenes (9f-g) proved considerably active (IC(50)=0.1-10 microg/mL) against human cytomegalovirus (CMV) and/or varicella zoster virus (VZV). Based on the activity of the trifluoroacetamides, ring-modified furan, N-(substituted)pyrrole, phenyl, and 3,4-thiophene analogues were prepared, and these compounds were also active against CMV and/or VZV, with the notable exception of the 3,4-thiophene derivative. In contrast to other amines, the 2-aminopyrrole precursors (13a-d) also exhibited potent activity against CMV. Unfortunately, most of these compounds displayed significant cytotoxicity against human fibroblasts, the cells supporting CMV and VZV replication, and thus selectivity indices were low. The most notable exception to this was the naphthyl-substituted aminopyrrole 13d, which exhibited both potent (IC(50)=0.3 microg/mL) and selective (CC(50)=>50 microg/mL) activity against CMV. Finally, thiophene aryl amides 8i-k displayed moderate in vitro activity against certain leukemia, breast, and colon cancer cell lines.

Lymphogranuloma venereum presenting as a rectovaginal fistula.

Lymphogranuloma venereum (LGV) is a rare form of the sexually transmitted disease caused by Chlamydia trachomatis. In the United States, there are fewer than 350 cases per year. In a review of the world's literature, there has not been a case reported in the last thirty years of a case of LGV presenting as a rectovaginal fistula. We present a case of an otherwise healthy American woman who presented with a rectovaginal fistula. Although uncommon, LGV does occur in developed countries and may have devastating tissue destruction if not recognized and treated before the tertiary stage.

Effects of ethanol on rat somatosensory cortical neurons.

In this study, we characterized the local effects of ethanol (EtOH) on postsynaptic potentials (PSPs) and membrane properties of layer II-III (L2-3) and layer V (L5) somatosensory cortical neurons. Intracellular recordings were done using the in vitro slice preparation of rat somatosensory cortex. Our results show that EtOH exerts local effects on cortical cell membrane at physiologically relevant concentrations. A predominant effect of EtOH was to reduce excitability of L2-3 and L5 neurons by increasing the rheobase, decreasing input resistance and repetitive firing, reducing PSPs amplitude and the probability of evoking action potentials. Early (6 ms) and late (18 ms) PSP components were affected differentially by EtOH, the late components being more suppressed. Overall, EtOH-mediated suppression of PSPs was stronger in L5 neurons. Cortical neurons were divided into three subtypes: regular spiking adapting (RS-A), regular spiking non-adapting (RS-NA) and bursting (D-IB) neurons. PSPs evoked in RS-A neurons were more sensitive to EtOH suppressant effects. EtOH effects on input resistance were distributed differentially among the three groups of neurons. These results support the notion that EtOH disrupts higher processing of somatosensory information via a differential alteration of cortical neuron's membrane properties and synaptic transmission.

Effect of omeprazole on the bioavailability of unmodified and phospholipid-complexed aspirin in rats.

BACKGROUND: Treatment and prevention of non-steroidal anti-inflammatory drug-induced gastropathy involve the concurrent use of antisecretory drugs. Recently, we have shown that the ability of these drugs to increase the intragastric pH to values > > pKa of NSAIDs compromises their therapeutic activity. In the present study, we evaluated the potential of omeprazole to interfere with the bioavailability of aspirin administered to rats either alone or complexed with the zwitterionic phospholipid, dipalmitoylphosphatidylcholine (DPPC). METHODS: Aspirin or aspirin/DPPC was administered intragastrically to rats pre-dosed with either saline or omeprazole. Concentrations of aspirin and salicylic acid in the blood and the gastric mucosa were assessed by HPLC and the 6-keto-PGF1 alpha gastric mucosal concentration by radioimmunoassay. RESULTS: Gastric absorption of aspirin and its relative bioavailability were reduced by an antisecretory dose of omeprazole; its inhibitory effect on gastric prostaglandin synthesis was consequently attenuated. However, these effects could be partly overcome if aspirin was administered as a complex with DPPC. CONCLUSIONS: These observations suggest that: (i) DPPC increases the lipid solubility and gastric permeability of NSAIDs; and (ii) neutralization of the gastric pH results in a shift of aspirin absorption toward the intestine where it could be degraded to salicylic acid.

Bleomycin induces apoptosis in human alveolar macrophages.

Bleomycin (BLM) is an effective antineoplastic drug; however, cumulative dosage is often associated with inflammation that can progress to pulmonary fibrosis. The mechanisms by which this occurs are not understood, but they have been proposed to involve the alveolar macrophage (AM). In this study, we examined the in vitro effects of BLM on human AM cytotoxicity and the role of heat shock proteins (HSP or stress proteins) in this process. Although BLM did not cause marked necrosis, it caused significant DNA fragmentation detected by in situ DNA labeling and confirmed by BLM-induced DNA ladder formation after 24 h. The DNA fragmentation was significantly blocked by 10 and 50 microM ZnCl2, suggesting that BLM was inducing apoptosis. BLM did not alter intracellular protooncogene bcl-2 or glutathione levels. However, BLM significantly (50%) blocked HSP-72 expression by 4 h during a mild heat stress (39.8 degrees C). This inhibition occurs without affecting mRNA levels (in situ hybridization) for HSP-72 or overall protein synthesis ([35S]methionine incorporation), suggesting that BLM is blocking the stress response relatively specifically and post-transcriptionally. In summary, these results suggest that BLM causes apoptosis in human AM in vitro that is preceded by the inhibition of HSP-72 induction that appears to be caused by a posttranscriptional mechanism.

Antibiotic prophylaxis in cerebrospinal fluid shunting: a prospective randomized trial in 129 patients.

The efficacy of a single dose of cefotiam, a cephalosporin of the second generation, as prophylaxis for postoperative infection was analyzed in a prospective randomized study of 129 patients undergoing cerebrospinal fluid shunting. The main focus of interest was the rate of shunt infection requiring operative shunt removal. Data were evaluated in the total group and subgroups formed for normal and high risk patients, respectively. The overall rate of shunt infection was 7.5% in the cefotiam group and 12.9% in the control group. In the high risk subgroup infection rate was 14.3% with and 26.3% without cefotiam as opposed to 4.3% and 6.9%, respectively, in the normal risk subgroup. Although our results do not reach statistical significance, there is a noticeable difference of infection rate between those patients who receive the antibiotic and those who do not. Therefore, we favor single dose antibiotic prophylaxis in shunting procedures.

Phase I clinical investigation of benzisoquinolinedione (amonafide) in adults with refractory or relapsed acute leukemia.

After Phase I studies of benzisoquinolinedione (amonafide) in solid tumors identified myelosuppression as the dose-limiting toxicity, we conducted a Phase I study in patients with relapsed or refractory acute leukemia to define the optimal dose. Amonafide was given i.v. over 2-4 h daily for 5 days. The starting dose was 600 mg/m2/day with subsequent escalation to 750, 900, 1100, 1400, and 1800 mg/m2/day. Thirty-eight courses were administered to 24 patients, of whom 12 participated in concomitant pharmacological studies. Nausea and vomiting, transient orange discoloration of the skin, and tinnitus occurred at all dose levels. The latter symptom, along with lightheadedness and flushing, was related to infusion duration; this was increased to 4 h with doses greater than or equal to 900 mg/m2. The dose-limiting toxicities were mucositis and painful skin erythema which occurred in all 4 patients treated with 1800 mg/m2. No remissions occurred. Clearing of peripheral blood blasts occurred in 67% of patients treated with 1100 mg/m2 and in all patients treated with greater than or equal to 1100 mg/m2/day. A decrease in marrow leukemic infiltrate (% blasts x % cellularity) to less than 10% occurred in 15 and 50% of patients treated at these levels, respectively. There were 10 deaths (42%), which were unrelated to dosage. The harmonic mean terminal plasma half-life was 4.6 h (range, 2.5-35.5 h). Three patients had long drug half-lives of 9.7, 16.4, and 35.5 h and each had initial bilirubin levels greater than 1.0 mg/dl. The average urinary excretion of amonafide over 5 days was 3.5% of the total dose. This establishes 1100-1400 mg/m2/day for 5 days as the maximally tolerated dose of amonafide for studies in acute leukemia.

Phase II study of 4-demethoxydaunorubicin in previously untreated extensive disease non-small cell lung cancer.

Fifteen patients with previously untreated extensive non-small cell lung cancer (E-NSCLC) were treated with oral 4-demethoxydaunorubicin (4-DMDR) at the dose of 10 mg/m2/day x 5 days every 3 weeks with routine administration of antiemetic drugs. They received a median of two courses of treatment with the cumulative dose range from 50-712.5 mg/m2. One patient achieved partial remission with a duration of 14 weeks. Two patients had minor responses with durations of 14 and 24 weeks. Stable disease occurred in three patients (21, 22, and 27 weeks). Median survival was 33 weeks (range 3-73+ weeks). Toxicities were tolerable. Neutropenia (less than 1,000 mm3) occurred in only 16% of all treatment courses. Three patients developed correctable arrhythmias (two with atrial fibrillation and one with accelerated junctional rhythm). The cause of arrhythmia was unclear. No clinical evidence of congestive heart failure or decreased cardiac ejection fraction was observed. Nausea and vomiting were common but tolerable. Alopecia and mucositis were uncommon. Clinical pharmacokinetic studies were done in nine patients. However, plasma 4-DMDR levels were below the limit of detection (3 ng/ml). Because 4-DMDR has shown some activity in previously untreated E-NSCLC and the toxicities at this dose schedule are mild, we suggest that further studies of this drug at a higher dose in this schedule are indicated.

Prospective randomized trial of high-dose cisplatin and fluorouracil infusion with or without sodium diethyldithiocarbamate in recurrent and/or metastatic squamous cell carcinoma of the head and neck.

Previous studies have indicated that sodium diethyldithiocarbamate (DDTC) can reduce cisplatin's (CP) toxic effects without altering the antitumor activity. DDTC has also been shown to have immunostimulative properties. Sixty patients with objectively measurable recurrent and/or metastatic squamous cell carcinoma (SCC) of the head and neck were randomized to receive either (A) CP at 120 mg/m2 over one hour on day 1, plus fluorouracil (5-FU) at 1,000 mg/m2 over 24 hours as a continuous infusion on days 1 through 5, or (B) CP/5-FU as in A, plus DDTC at 600 mg/m2 over 30 minutes administered intravenously (IV) exactly 30 minutes after CP infusion. Group B also received DDTC at 200 mg/m2 administered IV over 30 minutes on days 8 and 15. Each cycle was repeated at 3-week intervals. Objective responses were achieved in 41% of the CP/5-FU group and in 29% of the CP/5-FU with DDTC group (P = .26). Median survival was 9 months in group A and 10 months in group B. CP-related toxicity between the groups was equivalent with respect to nausea and vomiting, renal impairment, neurotoxicity, ototoxicity, and hematologic toxicity. The pharmacokinetics of reactive platinum species in plasma ultrafiltrate and urine samples obtained from both groups were comparable. The immune status of 48 patients was evaluated before and after completion of therapy. There were no significant differences in mean pretreatment and posttreatment values within or between groups A or B, except for absolute pretreatment OKT4 values (P = .02). We conclude that (1) the present dose and infusion schedule of DDTC did not significantly reduce CP-mediated toxic effects, (2) DDTC did not alter the disposition of ultrafilterable platinum species, (3) DDTC did not affect immune responses, and (4) the addition of DDTC improved neither the clinical response nor the survival of patients with recurrent SCC of the head and neck.

Phase I clinical investigation of benzisoquinolinedione.

A phase I study of benzisoquinolinedione (amonafide) was conducted in 30 patients with advanced solid tumors refractory to conventional therapy. The starting dose was 10 mg/m2/day X 5 days and the highest tolerated dose was 625 mg/m2/day X 5. The daily dose was mixed in 100 ml of normal saline and infused over 30-60 minutes. The dose-limiting toxicity was myelosuppression with nadirs of blood counts reached on Day 15 and recovery by Day 21-28. Other side effects included mild nausea and vomiting, mild phlebitis, skin rashes, and alopecia in some patients. A majority of the patients experienced dizziness, tinnitus, and hot flushes occurring predominantly at the higher dose levels. These were related to the rate of drug infusion and resolved on prolonging the infusion to 60 minutes. Pharmacokinetic studies of amonafide revealed a monoexponential plasma disappearance curve with a mean half-life of 3.5 +/- 1.9 hours. The recommended dose of amonafide for phase II studies in solid tumors is 400 mg/m2/day X 5 for good-risk and 300-320 mg/m2/day X 5 days for poor-risk patients with courses repeated at 21-28-day intervals.

Pharmacokinetics and metabolism of the antitumor drug amonafide (NSC-308847) in humans.

Pharmacokinetics and urinary excretion of Amonafide (5-amino-2-[2-(dimethylamine)ethyl]-1H-benz[de]isoquinoline-1,3-(2H)- dione) were examined in seven patients who were administered 400 mg/m2 of drug as a 30-min infusion on a daily schedule for 5 consecutive days. Amonafide concentrations in plasma and urine were determined using reversed phase HPLC. Amonafide was eliminated from plasma with a terminal half-life of 3.5 hr. Renal excretion accounted for 23% of the administered dose. Amonafide pharmacokinetic parameters after the initial dose (day 1) were similar to those calculated after the fifth daily dose. Amonafide undergoes a significant amount of metabolism and eight urinary metabolites have been identified using a thermospray liquid chromatography-mass spectrometry (LC/MS) technique. Various N-acetylated species appear to be the major metabolites, although no evidence of N-acetylation was found in urine obtained from two patients. Two of the primary metabolites, the N(N5)-acetyl and N'(N1)-oxide metabolites of Amonafide, were tested in vitro for cytotoxicity against P388 murine leukemia cells. In this test system, the N-acetyl metabolite was observed to be only slightly less cytotoxic than the parent compound. The N'-oxide of Amonafide, however, proved to be inactive. These results are discussed together with the pharmacokinetic and metabolism data of this new investigational antitumor drug.

Pulmonary disposition and pharmacodynamics of verapamil.

Verapamil, a calcium channel blocker, has received recent attention as a potential therapeutic agent in pulmonary hypertension. Accordingly, the pulmonary disposition and pharmacodynamics of verapamil were evaluated in isolated rat lungs perfused at a constant rate with a physiological salt solution. Isolated rat lungs sequestered but did not metabolize verapamil. The efflux of verapamil into drug-free perfusate occurred from two kinetically distinct pools with half-lives of 1.3 and 14.1 min. The theoretical amount of verapamil effluxed at infinite time was less than the amount taken up during the infusion, thereby suggesting that verapamil was also bound in a third "noneffluxable" pool. The time course for decline of verapamil inhibition of pulmonary vasoconstriction was compared with the rate of verapamil efflux. Inhibition of pulmonary vasoconstriction was related to the amount of verapamil in a pool exhibiting mono-exponential efflux of drug with a half-life of 12.6 min. This half-life suggests association of the inhibitory response with the efflux component having a half-life of 14.1 min. These findings indicate that the verapamil persists in the lungs in the form of a noneffluxable pool and that verapamil in the pool with half-life of 14.1 min is responsible for some of the vasoactivity in the pulmonary circulation.