A cocktail probe approach to evaluate the effect of hormones on the expression and activity of CYP enzymes in human hepatocytes with conditions simulating late stage of pregnancy.

PURPOSE: Pregnancy-mediated physiological and biochemical changes contribute to alterations in the pharmacokinetics of certain drugs. There is a paucity of data on the systematic evaluation of the underlying mechanisms. The objective of the current study was to examine the impact of changes in circulating and tissue hormonal concentration during the late stage of pregnancy on the activity and expression of hepatic cytochrome P450 (CYP) enzymes using a cocktail probe approach. METHODS: Freshly isolated primary human hepatocytes were incubated with third trimester physiologic (plasma) and projected liver (ten-fold higher) concentrations of female hormones: progesterone (2 µM), estradiol (0.3 µM), estriol (0.8 µM), estrone (0.2 µM), 17α-hydroxyprogesterone (0.1 µM), and human growth hormone (0.005 µM). The metabolic activity of the hepatocytes was assessed using a cocktail of isozyme-specific P450 probe substrates (CYP1A2 (phenacetin), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4 (testosterone)). A validated LC-MS/MS assay was used to measure the corresponding metabolite concentrations. CYP450 protein and mRNA levels were measured using western blot and qRT-PCR, respectively. RESULTS: Female hormones at projected third-semester hepatic concentrations significantly enhanced mRNA and protein expression and increased the metabolic activity of CYP3A4. The expression and activity of other CYP450 enzymes studied were not affected by mixtures of female hormones at concentrations used. CONCLUSION: The increased activity of CYP3A4 is consistent with the clinically observed increase in clearance of CYP3A4 substrates during pregnancy. Overall expression and activity of CYP450 isozymes are differentially regulated during pregnancy.

Pregnancy Alters CYP- and UGT-Mediated Metabolism of Buprenorphine.

BACKGROUND: In the United States, drug addiction has become a nationwide health crisis. Recently, buprenorphine (BUP), a maintenance therapy approved by the Food and Drug Administration, has been increasingly used in pregnant women for the treatment of opioid use disorder. Pregnancy is associated with various anatomic and physiological changes, which may result in altered drug pharmacokinetics (PKs). Previously, we reported that dose-adjusted plasma concentrations of BUP are lower during pregnancy than after pregnancy. The mechanism(s) responsible for this difference has not yet been defined. Our study aimed to evaluate alterations in cytochromes P450 (CYP)- and uridine diphosphate glucunosyltransferases (UGT)-mediated metabolism of BUP during pregnancy to determine the mechanism(s) responsible for this observation. METHODS: Data from 2 clinical studies were included in the current analysis. Study 1 was a prospective, open-labeled, nonrandomized longitudinal BUP PK study in pregnant women with a singleton gestation, stabilized on twice-daily sublingual BUP opioid substitution therapy. Each subject participated in up to 3 studies during and after pregnancy (the second, third trimester, and postpartum). The design of study 2 was similar to study 1, with patients evaluated at different time points during the pregnancy (first, second-half of pregnancy), as well as during the postpartum period. In addition, the dosing frequency of BUP study 2 participants was not restricted to twice-daily dosing. At each study visit, blood samples were collected before a BUP dose, followed by multiple collection times (10-12) after the dose, for up to 12 hours or till the end of the dosing interval. Plasma concentrations of BUP and 3 metabolites were quantified using validated ultraperformance liquid chromatography-tandem mass spectrometric assays. RESULTS: In total, 19, 18, and 14 subjects completed the PK study during 1/2 trimester, third trimester, and postpartum, respectively. The AUC ratios of norbuprenorphine and norbuprenorphine glucuronide to buprenorphine, a measure of CYP3A mediated N-demethylation, were 1.89, 1.84, and 1.33 during the first and second, third trimesters, and postpartum, respectively. The AUC ratios of buprenorphine glucuronide to BUP, indicative of UGT activity, were 0.71, 2.07, and 0.3 at first/second trimesters, third trimester, and postpartum, respectively. Linear mixed-effect modeling analysis indicated that the AUC ratios of CYP- and UGT-mediated metabolism of BUP were significantly higher during pregnancy compared with postpartum. CONCLUSIONS: The CYP and UGT activities were significantly increased as determined by the metabolic ratios of BUP during pregnancy compared with the postpartum period. The increased UGT activity appeared to account for a substantial part of the observed change in metabolic activity during pregnancy. This is in agreement with the need for BUP dose increment in pregnant women to reach similar BUP exposure and therapeutic effect as in nonpregnant subjects.

Impaired adenylate cyclase signaling in acute myocardial ischemia: Impact on effectiveness of P2Y12 receptor antagonists.

INTRODUCTION: P2Y12 receptor antagonists reduce risk of thrombotic complications after stent implantation but increase bleeding risk. Activation of P2Y12 receptors by ADP causes Gi-protein-mediated inhibition of adenylate cyclase (AC), thus limiting platelet response to anti-aggregatory effect of prostacyclin (PGI2). However, P2Y12 blockade reverses this ADP-induced suppression of the platelet PGI2/AC signaling pathway. We previously demonstrated that impairment of this pathway predicts poor response to clopidogrel. OBJECTIVES: To identify clinical correlates of variability in PGI2/AC signaling, and to assess the impact of such variability on individual responses to the direct P2Y12 receptor antagonists ticagrelor (in vivo) and 2-methyl-thioadenosine-monophosphate (2MeSAMP) (in vitro). PATIENTS/METHODS: We compared the inhibitory effects of prostaglandin E1 (PGE1) and the PGI2 analog Iloprost (Ilt) on platelet aggregation in whole blood samples from healthy control subjects (n = 17), and patients with stable angina pectoris (SAP; n = 35) or acute coronary syndromes (ACS; n = 23), with or without associated diabetes/hyperglycemia. RESULTS: Compared to control subjects, patients with ACS and - to a lesser extent - those with SAP, exhibited impaired responses to PGE1, accentuated in the presence of hyperglycemia. Efficacy of ticagrelor treatment, measured as change in platelet reactivity index, was directly related to pre-treatment PGE1 response, both at univariate and multivariate analysis. There was a strong correlation between extent of inhibition of platelet aggregation, whether by PGE1 or Ilt, and the anti-aggregatory effect of 2MeSAMP in vitro. CONCLUSIONS: The integrity of PGI2/AC signaling, which is impaired in the presence of ACS and hyperglycemia, predetermines the anti-aggregatory efficiency of P2Y12 receptor antagonists.

The outcome of a pregnant woman with a minor head injury: an ossified subdural hematoma (OSDH).

We experienced an ossified subdural hematoma (OSDH), which is an extremely rare form of chronic subdural hematoma (SDH), in the dominant hemisphere of a 35-year-old woman. She presented to our outpatient clinic with a complaint of a headache; she had previously experienced a head injury while she was pregnant. We performed surgery with extreme caution because the lesion was attached to the surrounding tissue. Since an OSDH is an extremely rare form of chronic SDH, neurosurgeons might not experienced them during their daily practice. Additionally, head injuries received during pregnancy should be taken seriously, and after delivery, the patient should undergo cranial computed tomography, even if she is asymptomatic.

Silicone Breast Implants: A Rare Cause of Pleural Effusion.

Pleural effusions are one of the rarest complications reported in patients with silicone gel filled breast implants. The silicone implants have potential to provoke chronic inflammation of pleura and subsequent pulmonary complications such as pleural effusion. Herein, we report a 44-year-old female who presented with left sided pleural effusion, six weeks after a silicone breast implantation surgery. The most common infectious, inflammatory, and malignant causes of pleural effusion were excluded with pleural fluid cytology and cultures. With recurrent effusion in the setting of recent surgery, the chemical reaction to silicone breast implants was sought and exploration was performed which revealed foreign body reaction (FBR) to silicone material. The symptoms dramatically improved after the explantation.

Effects of Pringle maneuver and partial hepatectomy on the pharmacokinetics and blood-brain barrier permeability of sodium fluorescein in rats.

Liver diseases are known to affect the function of remote organs. The aim of the present study was to investigate the effects of Pringle maneuver, which results in hepatic ischemia-reperfusion (IR) injury, and partial hepatectomy (Hx) on the pharmacokinetics and brain distribution of sodium fluorescein (FL), which is a widely used marker of blood-brain barrier (BBB) permeability. Rats were subjected to Pringle maneuver (total hepatic ischemia) for 20 min with (HxIR) or without (IR) 70% hepatectomy. Sham-operated animals underwent laparotomy only. After 15 min or 8h of reperfusion, a single 25-mg/kg dose of FL was injected intravenously and serial (0-30 min) blood and bile and terminal brain samples were collected. Total and free (ultrafiltration) plasma, total brain homogenate, and bile concentrations of FL and/or its glucuronidated metabolite (FL-Glu) were determined by HPLC. Both IR and HxIR caused significant reductions in the biliary excretions of FL and FL-Glu, resulting in significant increases in the plasma AUC of the marker. Additionally, the free fraction of FL in plasma was significantly increased by HxIR. Although the brain concentrations of FL were increased by almost twofold in both IR and HxIR animals, the brain concentrations corrected by the free FL AUC (and not the total AUC) were similar in both groups at either time points. It is concluded that Pringle maneuver and/or partial hepatectomy substantially alters the hepatobiliary disposition, plasma AUC, plasma free fraction, and brain accumulation of FL without altering the BBB permeability to the marker.

Effects of hepatic ischemia-reperfusion injury on the P-glycoprotein activity at the liver canalicular membrane and blood-brain barrier determined by in vivo administration of rhodamine 123 in rats.

PURPOSE: To investigate the effects of normothermic hepatic ischemia-reperfusion (IR) injury on the activity of P-glycoprotein (P-gp) in the liver and at the blood-brain barrier (BBB) of rats using rhodamine 123 (RH-123) as an in vivo marker. METHODS: Rats were subjected to 90 min of partial ischemia or sham surgery, followed by 12 or 24 h of reperfusion. Following intravenous injection, the concentrations of RH-123 in blood, bile, brain, and liver were used for pharmacokinetic calculations. The protein levels of P-gp and some other transporters in the liver and brain were also determined by Western blot analysis. RESULTS: P-gp protein levels at the liver canalicular membrane were increased by twofold after 24 h of reperfusion. However, the biliary excretion of RH-123 was reduced in these rats by 26%, presumably due to IR-induced reductions in the liver uptake of the marker and hepatic ATP concentrations. At the BBB, a 24% overexpression of P-gp in the 24-h IR animals was associated with a 30% decrease in the apparent brain uptake clearance of RH-123. The pharmacokinetics or brain distribution of RH-123 was not affected by the 12-h IR injury. CONCLUSIONS: Hepatic IR injury may alter the peripheral pharmacokinetics and brain distribution of drugs that are transported by P-gp and possibly other transporters.

Hepatic immunosuppressive effects of systemically administered novel dextran-methylprednisolone prodrugs with peptide linkers in rats.

The hepatic immunosuppressive activities of two novel dextran prodrugs of methylprednisolone (MP) containing one (DMP1) or five (DMP5) amino acids as linkers were studied in rats. At various times (0-2 weeks) after intravenous administration of single 5 mg/kg (MP equivalent) doses of each prodrug or MP succinate (MPS), livers were isolated and immunologically stimulated ex vivo with lipopolysaccharide. The concentrations of tumor necrosis factor (TNF)-α in the outlet perfusate were then quantitated to assess immune response. Additionally, the concentrations of DMP1, DMP5, and/or MP were measured in the liver. MPS, DMP5, or DMP1 injections caused a maximum of 48.9%, 63.5%, or 85.7% decrease in the TNF-α secretion into the perfusate, with the time above the 50% inhibitory effect being <5, <24, or 120 h, respectively. Additionally, the area under the effect-time curve for DMP1 was 11-fold or fourfold higher than that after the administration of MPS or DMP5, respectively. Relatively high concentrations of DMP1 were present in the liver even at the last sampling time of 2 weeks. These data suggest that a single intravenous dose of DMP1 produces an intense and sustained immunosuppression in the liver for a relatively long time, which may be useful in liver transplantation.

Cytochrome P450 induction by phenobarbital exacerbates warm hepatic ischemia-reperfusion injury in rat livers.

Recent studies have shown that cytochrome P450 inhibitors reduce oxidative stress and injury to the liver following warm ischemia-reperfusion (IR). The aim here was to test the effect of P450 induction by phenobarbital on the IR injury in rat livers. Rats were pre-treated with saline or phenobarbital and subjected to IR or sham operation. IR significantly increased the plasma alanine aminotransferase concentrations. Phenobarbital further exacerbated the injury by an additional 50% increase in the alanine aminotransferase levels. Phenobarbital also caused an approximately 40% increase in the total P450 content of the liver, which was also associated with a 75% increase in the reactive oxygen species (ROS) generation in the IR group. There was a strong correlation between the microsomal ROS generation and total P450 content, CYP3A2 activity or CYP2B1 activity. It is concluded that the induction of P450 by phenobarbital significantly increases hepatic production of ROS, leading to significantly higher hepatic IR injury.

Effects of cytochrome p450 inhibition by cimetidine on the warm hepatic ischemia-reperfusion injury in rats.

BACKGROUND: Cimetidine is an H(2)-antagonist with cytochrome P450 (P450) inhibitory activity. Recent studies showed that cimetidine improves warm ischemia-reperfusion (IR) injury in isolated rat heart and rabbit lung and in primary cultures of rat proximal tubule epithelial cells by inhibiting P450-mediated reactive oxygen species generation. Here, we studied the effects of cimetidine on the warm IR injury in the liver. METHODS: Three groups of rats were treated with a single i.p. dose (0.6 mmol/kg) of cimetidine or ranitidine (an H(2) antagonist without a significant P450 inhibitory activity) or with saline 1.5 h before surgery. Livers were then subjected to 1 h of in vivo ischemia, followed by 1 h of ex vivo reperfusion using a physiologic buffer in a recirculating manner. A fourth group of animals, receiving saline pretreatment underwent sham operation instead of ischemia. Perfusate and bile samples were collected during the reperfusion, and the liver tissue was collected at the end of reperfusion period for measurement of various biochemical markers. RESULTS: Warm IR resulted in a significant increase in the perfusate concentrations of liver enzymes (3- to 4.5-fold) and hepatic concentrations of lipid hydroperoxides (2-fold). Whereas the glutathione concentrations in the liver tissue were not affected by IR injury, the injury caused a significant decrease ( approximately 40%) in the biliary glutathione excretion. Cimetidine treatment completely or partially reversed all the IR-mediated changes, while ranitidine was ineffective. The protective effects of cimetidine were associated with a 60% decline in the microsomal CYP2C11 activity. CONCLUSIONS: Whereas cimetidine, an H(2) blocker with substantial P450 inhibitory activity, is protective in warm IR injury, ranitidine, a similar drug with no significant P450 inhibitory activity, is devoid of any protective effects. Therefore, P450 inhibition appears to be the underlying mechanism in the protective effects of cimetidine in this model of IR injury.

Protective effects of diallyl sulfide, a garlic constituent, on the warm hepatic ischemia-reperfusion injury in a rat model.

PURPOSE: The aim of this study is to evaluate the effects of diallyl sulfide (DAS) on the warm hepatic ischemia-reperfusion (IR) injury in a rat model. METHODS: Rats (n = 8-10/group) were subjected to sham operation or warm ischemia (1 h)-reperfusion (3 h) preceded by a single intraperitoneal dose (1.75 mmol/kg) of DAS or vehicle, and relevant biochemical parameters were monitored. RESULTS: Warm IR injury caused a significant increase in the plasma markers of liver injury, which was attenuated by DAS. The hepatoprotective effects of DAS were associated with significant reductions in lipid peroxidation markers and in situ generation of superoxide in the liver and increases in the glutathione levels of the liver and bile, suggestive of an antioxidant effect for DAS. Additionally, DAS caused an almost twofold increase in the protein expression of the liver heme oxygenase-1, an enzyme that confers cytoprotection against oxidative stress. Whereas the total cytochrome P450 remained unchanged, the protein levels and activity of CYP2E1, which plays an important role in the generation of reactive oxygen species, significantly decreased by DAS pretreatment. CONCLUSIONS: DAS protects the liver from warm IR injury by reducing oxidative stress through, at least in part, induction of heme oxygenase-1 and inhibition of CYP2E1.

Metabolic bioactivation and toxicity of ethyl 4-hydroxybenzoate in human SK-MEL-28 melanoma cells.

The metabolism and toxicity of ethyl 4-hydroxybenzoate (4-HEB) were investigated in vitro using tyrosinase enzyme, a melanoma molecular target, and CYP2E1 induced rat liver microsomes, and in human SK-MEL-28 melanoma cells. The results were compared to 4-hydroxyanisole (4-HA). At 90 min, 4-HEB was metabolized 48% by tyrosinase and 26% by liver microsomes while the extent of 4-HA metabolism was 196% and 88%, respectively. The IC50 (day 2) of 4-HEB and 4-HA towards SK-MEL-28 cells were 75 and 50 microM, respectively. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased 4-HEB toxicity towards SK-MEL-28 cells indicating o-quinone formation played an important role in 4-HEB induced cell toxicity. Addition of ascorbic acid and GSH to the media was effective in preventing 4-HEB cell toxicity. Cyclosporin A and trifluoperazine, inhibitors of permeability transition pore in mitochondria, were significantly potent in inhibiting 4-HEB cell toxicity. 4-HEB caused time-dependent decline in intracellular GSH concentration which preceded cell death. 4-HEB also led to reactive oxygen species (ROS) formation in melanoma cells which exacerbated by dicoumarol and 1-bromoheptane whereas cyclosporin A and trifluoperazine prevented it. Our findings suggest that the mechanisms of 4-HEB toxicity in SK-MEL-28 were o-quinone formation, intracellular GSH depletion, ROS formation and mitochondrial toxicity.

Attenuation of acute rejection in a rat liver transplantation model by a liver-targeted dextran prodrug of methylprednisolone.

BACKGROUND: The use of methylprednisolone (MP) and other corticosteroids for the treatment of acute liver allograft rejection is associated with severe toxicities in nontarget tissues. Therefore, selective delivery of MP to the liver may improve its efficacy and alleviate its side effects. We investigated the effects of a novel liver-targeted dextran prodrug of MP (DMP) in an orthotopic rat liver transplantation (OLT) model. METHODS: The model consisted of a high responder rejection strain combination (Dark Agouti donors and Lewis recipients). Liver recipients were intravenously administered saline or a single subtherapeutic dose of MP (5 mg/kg) as the parent drug (MP) or its prodrug (DMP). Different groups were then monitored for graft survival or euthanized 5 or 9 days posttransplantation. Plasma chemistry, including alkaline phosphatase and bilirubin, allograft histology, and survival duration were determined. RESULTS: Untreated recipients exhibited elevated plasma levels of liver injury markers, progressive portal and venous inflammation and cellular infiltration in liver allografts, and a mean graft survival time (MST) of 10.5 days. MP treatment did not alter any of these parameters. In contrast, a single dose of DMP resulted in a decrease in plasma levels of liver injury markers, a decrease in histological grade of rejection on day 5, and a substantial increase in MST (27.5 days). CONCLUSIONS: These results demonstrate attenuation of acute rejection following local (allograft) immunosuppression with a single subtherapeutic dose of MP delivered as a liver-targeted prodrug. Dextran prodrugs may be useful for selective delivery of immunosuppressants to the liver following liver transplantation.

Rapid determination of reduced and oxidized glutathione levels using a new thiol-masking reagent and the enzymatic recycling method: application to the rat liver and bile samples.

A microtiter plate assay for quantitation of reduced (GSH) and oxidized (GSSG) glutathione in the rat liver tissue and bile is described. The assay is based on the established enzymatic recycling method and a new thiol-masking reagent, 1-methyl-4-vinyl-pyridinium trifluoromethane sulfonate (M4VP). Samples were first processed by homogenization with (liver) or addition of (bile) sulfosalicylic acid. The total glutathione and GSSG were then determined before and after rapid (< or = 2 min) and efficient (100%) masking of the GSH content of the samples with M4VP followed by the enzymatic recycling assay. The percentages of error and coefficient of variation of the assay were within the accepted guidelines, indicating the accuracy and precision of the assay in the range of 6.25-100 pmol GSH per microplate well and 2.17-140 pmol GSSG per well, with lower limit of quantitation of 6.25 and 2.17 pmol per well for GSH and GSSG, respectively. Furthermore, the recoveries of added GSH or GSSG from the liver and bile samples were accurate and precise. The assay was applied to measurement of GSH, GSSG, and GSH:GSSG ratio in the liver and serially collected bile samples in sham-operated and ischemic rat livers, demonstrating a depletion of glutathione and a decrease in the GSH:GSSG ratio as a result of ischemia. The developed assay is rapid, sensitive, accurate, and precise and is suitable for studies of the redox status of liver under physiologic and pathophysiologic conditions.

Streptozocin and o,p'DDD in the treatment of adrenocortical cancer patients: long-term survival in its adjuvant use.

BACKGROUND: To evaluate the efficacy of streptozocin and o.p'DDD (SO) in adrenocortical cancer (ACC) patients since other chemotherapeutic regimens have limited effects. PATIENTS AND METHODS: We performed a phase II study with SO therapy in 40 ACC patients (median age 44 years). Oral o,p'DDD administration (1-4 g/d, every day) was given together with intravenous streptozocin (1 g/d for five days, thereafter 2 g once every three weeks). 5HT3-receptor blocker was used as standard premedication for streptozocin. RESULTS: The SO therapy was found to have significant effects on disease-free interval (P = 0.02) as well as on survival (P = 0.01) in adjuvantly treated cases (n = 17) in comparison to the patients who did not get any therapy after complete resection (n = 11). Complete or partial response was obtained in 36.4% of patients with measurable disease (n = 22). The overall two-year and five-year survival rates were 70% and 32.5%, respectively. The presence of metastases at diagnosis was identified as a poor prognostic factor (P = 0.02). CONCLUSIONS: The present study necessitates further randomized clinical study of SO therapy in the treatment of ACC, mainly as adjuvant treatment immediately after curative intended surgery, and could be developed into a regular treatment regimen.

Expression of CD44 variant isoforms and association to the benign form of endocrine pancreatic tumours.

BACKGROUND: The expression of CD44 and its isoforms have been shown in many neoplastic tissues to serve as prognostic indicators, therefore, the feasibility of using these as prognostic markers in endocrine pancreatic tumour patients was examined. PATIENTS AND METHODS: Immunohistochemistry (IHC) was performed on 26 tumour samples (5 gastrinomas, 3 glucagonomas, 10 non-functioning tumours, 6 insulinomas, 2 mixed insulinoma and glucagonomas) with monoclonal antibodies against CD44s (standard form) and variant isoforms (v4, v5, v6, v7, v7-8, v9, v10). Staining was correlated to the tumour proliferation, malignancy, metastasis and patients survival. RESULTS: There was variable expression of CD44s. All tumours showed complex expression of many isoforms. CD44v6 and CD44v9 were down regulated in malignant tumours. There was statistical significance of CD44v6 expression in benign tumours (P < 0.05) compared to malignant tumours and near significance in CD44v9 expression (P = 0.0574). Survival of the patients with CD44v6 positive staining was higher than those who were negative (P = 0.0822). Moreover, the expression was well correlated to the patients without any distant metastases (CD44v6, p < 0.001; CD44v9, P < 0.01). Tumour proliferation (Ki67 index) correlated directly to the malignancy (P < 0.05) and there was inverse correlation between Ki67 index and CD44v6 (P < 0.05) as well as v9 (P < 0.05). CONCLUSIONS: Endocrine pancreatic tumours express CD44s and isoforms differentially. Expression of the two isoforms of CD44, namely v6 and v9 seem to be related more to benign form of the tumour and could serve as a predictor of good prognosis.

High-dose treatment with lanreotide of patients with advanced neuroendocrine gastrointestinal tumors: clinical and biological effects.

BACKGROUND: Neuroendocrine tumors usually present with inoperable metastatic disease and severe hormonal symptoms. Specific chemotherapy, alpha-interferon and the somatostatin analog octreotide are established therapies in these patients but all of them eventually fail. Other somatostatin analogs, e.g., RC-160 and lanreotide, are currently being studied in different doses and modes of administration. PATIENTS AND METHODS: Nineteen patients with advanced neuroendocrine gastrointestinal tumors [13 carcinoids and six endocrine pancreatic tumors (EPT)], liver metastases being present in 18, most of them heavily pretreated, were included. Seventeen out of 18 patients had somatostatin receptors demonstrated by octreotide scintigraphy. Lanreotide was given as four daily subcutaneous injections, starting with 750 microg/d, then increasing every week up to 12,000 microg/d after six weeks, a dose which was maintained, if tolerated, for 12 months, or until progression. RESULTS: There was a significant tumor size response (>50%) in one patient (5%), whereas 12 patients (70%) had tumor stabilization for 12 months. Bichemical tumor markers were significantly reduced at six months (urinary 5-hydroxyindoleacetic acid and plasma chromogranin) and 12 months (chromogranin) and the overall biochemical response rate was 58% with this high dose of lanreotide. Adverse events were observed and four patients stopped the treatment due to adverse events. Studies of tumor biopsies before and during treatment indicated induction of apoptosis in patients with tumor stabilization and biochemical response. CONCLUSION: High-dose treatment with lanreotide (12,000 microg/d) produced tumor size response in 5%, stabilization in 70% and a biochemical response in 58% of patients. These results should be related to the advanced stage of the disease as indicated by the mean duration of disease of more than four years, but they do not appear to be better than those achieved with standard doses of somatostatin analogs. However, in responding patients we observed induction of apoptosis in the tumors, a phenomenon not seen with regular doses of somatostatin analogs, but often produced by chemotherapeutic agents.

Induction of apoptosis in neuroendocrine tumors of the digestive system during treatment with somatostatin analogs.

The extent of apoptosis identified by in situ DNA nick end labelling (TUNEL) on tissue samples obtained from patients with neuroendocrine tumors was correlated with the clinical outcome in patients treated with high-dose somatostatin analog (lanreotide 12 mg/day), n = 8, or other biotherapy including interferon-alpha (IFN-alpha), n = 4, low-dose somatostatin analog (octreotide or lanreotide), n = 3, or a combination of both, n = 1. Biopsies were obtained before the start of treatment and/or after 6 months and 12 months. After 6 months of treatment, 5 patients receiving high-dose somatostatin analog showed a biochemical response (decrease in different neuroendocrine tumor markers) and 4 of these showed an increase in apoptotic index (AI: percentage of apoptotic cells) by 1.94 +/- 1.71%. At 12 months, AI was also increased in patients with a biochemical response (4.22 +/- 3.93%). However, none showed a decrease in tumor size on computerized tomography (CT) and none of the patients treated with low-dose somatostatin analog or IFN-alpha showed any significant increase in AI during treatment. In an experimental model, nude mice were xenografted with the neuroendocrine cell line (BON-1). From the 2nd day of tumor implantation, they received treatment with either placebo, high-dose octreotide, IFN-alpha, or a combination of both, for 28 days. In mice receiving treatment with high-dose octreotide (300 microg/kg, t.i.d) there was a threefold increase in apoptotic cells as compared to the placebo group (p = 0.0084), while the combination group had few cells with ultra-structural changes indicating apoptosis and the IFN-alpha treated group showed no significant changes. However, tumor growth inhibition was more pronounced in the combination group (p = 0.0011). This probably denotes that tumor growth inhibition could be achieved more efficiently by blocking the cell cycle than by inducing apoptosis. We concluded that treatment with high-dose somatostatin analogs may induce apoptosis in neuroendocrine tumors, while this is not found during treatment with low-dose somatostatin analogs or IFN-alpha. We also found that an increase in AI during high-dose somatostatin analog treatment was correlated with the biochemical response, but not with the tumor size as detected by CT in patients or with the tumor mass in the experimental model.

Interferon-alpha induces bcl-2 proto-oncogene in patients with neuroendocrine gut tumor responding to its antitumor action.

We have studied the expression of apoptosis regulating genes bcl-2 and bax in neuroendocrine gut tumors. The expression pattern of these genes was compared with the clinical response (changes in the tumor markers and changes of the tumor size determined by radiology) after treatment with interferon-alpha (IFN-alpha, n = 13), somatostatin analog (octreotide, n = 3; lanreotide, n = 2) or a combination of both (n = 5). Immunohistochemistry and in situ RT-PCR were performed and expressions were scored from 0 (no staining) to 6 (strong and wide-spread staining). With regard to clinical outcome, the scores (mean +/- SEM) of immunohistochemical staining of bcl-2 and bax were 1.77 +/- 0.25 and 4 +/- 0.22 for patients with stable disease and, 0.54 +/- 0.28 and 4.68 +/- 0.21 for patients with progressive disease. The scores of bcl-2 and bax staining for IFN-alpha-treated patients were 1.96 +/- 0.35 and 4.12 +/- 0.31 and for untreated patients were 0.5 +/- 0.25 and 4.5 +/- 0.21, respectively. Expression of bcl-2 was observed in all IFN-alpha-treated patients who responded to the drug but not in nonresponsive patients (p = 0.0027). In contrast, bax, a promotor of apoptosis was expressed in all patients with higher degree of expression seen in patients with progressive disease (p = 0.0364). We have also detected bcl-2 expression by western blot analysis in neuroendocrine tumor tissue grown in nude mice, which were treated with IFN-alpha for 28 days. Our results indicate that, IFN-alpha can induce bcl-2. Thus, we, propose that bcl-2 may be used as a prognostic marker for IFN-alpha sensitivity of neuroendocrine tumors.