Lipophilic 1-β-d-arabinofuranosyl cytosine derivatives in liposomal formulations for oral and parenteral antileukemic therapy in the murine L1210 leukemia model

TheN ⁴-alkylcytosine arabinoside derivativeN ⁴-octadecyl-AraC (AraC-Ocd, NOAC) and the (1-octadecylglycero-3-phospho)-AraC (Ocd-GroP-AraC, OPA) conjugate are new lipophilic derivatives of the cytostatic drug 1--d-arabinofuranosylcytosine (AraC) that produce high antileukemic effects in the L1210 murine leukemia model when administered orally or parenterally as liposomal formulations. Between 83% and 100% of the treated animals were cured after five consecutive daily oral drug applications with a total dose of 1 mmol/kg AraC-Ocd or Ocd-GroP-AraC. Corresponding results were obtained after parenteral therapy on days 2 and 6 after tumor inoculation with five- to ten-fold lower concentrations of these two compounds. A comparable cytotoxic activity was found with the orally active AraC-5-(n-stearyl phosphate). However, because of its strong hemolytic toxicity this derivative cannot be used for parenteral therapy. Another AraC conjugate, which was modified with two long-chain hydrocarbons, the (1-octadecylglycero-3-phospho)-N ⁴-hexadecyl-AraC was, probably because of poor oral bioavailability, only active when applied parenterally. The new lipophilic AraC derivatives AraC-Ocd and Ocd-GroP-AraC are compounds with a high potential for the oral treatment of leukemias and possibly also of solid tumors.Abbreviations AraC 1--d-arabinofuranosylcytosine - AraC-Ocd N ⁴-octadecyl-1--d-arabinofuranosylcytosine - Ocd-GroP-AraC 5-O-(1-octadecyl-rac-glycero-3-phospho)-1--d-arabinofuranosylcytosine - OcdP-AraC 1--d-arabinofuranosylcytosine-5-(n-stearylphosphate) - Ocd-GroP-AraC-Hxd 5-O-(1-octadecyl-rac-glycero-3-phospho)-N ⁴-hexadecyl-1--d-arabinofuranosylcytosine     

Oral antitumour activity in murine L1210 leukaemia and pharmacological properties of liposome formulations ofN 4-alkyl derivatives of 1-β-d-arabinofuranosylcytosine

The oral cytostatic activity in L1210 mouse leukaemia of the two newN ⁴-alkyl derivatives of 1--d-arabinofuranosylcytosine (AraC),N ⁴-hexadecyl- andN ⁴-octadecyl-1--d-arabinofuranosylcytosine (NH- AraC, NO-AraC) was investigated. In contrast to AraC, both derivatives were highly cytostatic after oral application as liposome formulations. With treatment schedules of five consecutive dosages or with two applications on days 1 and 4 after intravenous tumour cell inoculation with a total dose of 470–1000 mg/kg NH- AraC or NO-AraC, 70%–100% of the treated animals were cured. The lethal dose in healthy ICR mice after a single intraperitoneal application, corresponding to the LD₅₀, was 524 mg/kg for NO-AraC, whereas NH- AraC proved to be less toxic. The haematological toxicity. remained moderate for both drugs with a mild leucopenia and a drop in platelet counts, which recovered 4–6 days after treatment. The erythrocytes were not affected and haemolytic toxicities were absent. As non-haematological toxicities, at high drug concentrations, a pronounced atrophy of the rapidly dividing epithelia of the small intestines and of the white pulp of the spleen were observed. The blood levels of NH-AraC given orally reached values comparable to those after parenteral application of a four-times lower dose of NH-AraC, suggesting a moderate bioavailability. Thus, these two lipophilic derivatives of AraC are compounds with a potential for the oral treatment of malignant diseases.Abbreviations NH-AraC N ⁴-hexadecyl-1--d-arabino-furanosylcytosine - NO-AraC N ⁴-octadecyl-1--d-arabinofuranosylcytosine - AraC 1--d-arabinofuranosylcytosine     

Antineoplastic activity of esters and amides of N-[N′-(2-chloroethyl)-N′-nitrosocarbamoyl]-aminoacids

Summary The antineoplastic activity of 7 ester derivatives and 15 amide derivatives of N-[N-(2-chloroethyl)-N-nitrosocarbamoyl (CNC)]-aminoacids was examined in transplanted rat leukemia L 5222. All esters except the ethylester of CNC-l-isoleucine showed only a moderate antitumor activity. CNC-l-isoleucine ethylester effected some cures and showed the lowest toxicity of this series of compounds.The amide derivatives on the other hand were highly active in L 5222; all compounds effected cures in the dose range investigated.     

Tumor-related elevation of serum (α1→3)-l-Fucosyltransferase activity in gastric cancer

Summary (13)-l-Fucosyltransferase activity was measured in serum samples from 90 gastric cancer patients, 10 patients with benign diseases and 100 healthy controls. The enzyme activity was significantly elevated in the serum samples of patients with cancer compared to those from patients with benign diseases (P<0.01) and healthy controls (P<0.001). The elevation of the enzyme activity was found to correlate strongly with the clinical stage of disease. The sensitivity of (13)-l-fucosyltransferase was also demonstrated to be high in comparison with the tumor-associated antigens, such as carcinoembryonic antigen and sialylated Lewis X-i. Follow-up studies of (13)-l-fucosyltransferase in 11 cancer patients with disease at different stages showed that the enzyme activity could be useful for monitoring the postsurgical course of the disease. These results suggest that (13)-l-fucosyltransferase activity has a clinically important potential as a tumor marker in gastric cancer.Abbreviations Gal d-galactose - Fuc l-fucose - GlcNAc N-acetyl-d-glucosamine - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid     

Nephrotoxicity of acyclovir andcis-diamminedichloroplatinum(II)-effect of co-administration in rats

Summary The effect of co-administration of acyclovir andcis-diamminedichloroplatinum(II) (cisplatin) on nephrotoxicity in male Wistar rats was investigated. Animals received acyclovir (15 mg/kg body weight, s.c., three times per day for 5 days) or cisplatin (5 mg/kg body weight, i.p., one single injection) or a combination of both drugs. Acyclovir plasma levels were determined after one single acyclovir s.c. injection. Urines were monitored for volume, pH, osmolality and excretion ofN-acetyl--d-glucosaminidase (NAG), lysozyme and total protein. Concentrations of blood urea nitrogen and plasma creatinine were determined on day 6. Renal cortical slices were monitored to assess the accumulation of weak organic bases (tetraethylammonium) and acids (p-aminohippurate). Cisplatin induced a marked increase in the excretion of NAG, lysozyme and total protein and an increase in urine volume, plasma creatinine and blood urea nitrogen. Urine osmolality and accumulation ofp-aminohippurate were depressed by cisplatin. Acyclovir treatment alone caused no significant symptoms of nephrotoxicity. Co-administration did not impair renal function more than cisplatin treatment alone, excepting a slight rise in lysozyme excretion on day 6. Short-term antiviral therapy with acyclovir, concomitant to cisplatin treatment, may bring, if at all, a slightly increased nephrotoxic risk.Abbreviations Cisplatin cis-diamminedichloroplatinum(II) - NAG N-acetyl--d-glucosaminidase - BUN blood urea nitrogen - TEA tetraethylammonium - PAH p-aminohippurate     

6-Methylguanine and 6-methylguanosine inhibit colony-forming ability in a malignant xeroderma pigmentosum cell line but not in other xeroderma pigmentosum and normal human fibroblast strains after treatment with 1-(2-chloroethyl)-1-nitroso-3-(2-hydroxyethyl)-urea

Summary The XP cell strain XP29MA, its malignant counterpart XP29MAmal and a normal human fibroblast strain were tested for colony-forming ability after treatment with HECNU in the presence of m⁶G, m⁶Gua, and he⁷G.In XP29MAmal, inhibition of post-HECNU colony-forming ability was 35% when 0.25 mM of either m⁶G or m⁶Gua were present, whereas in XP29MA and the normal fibroblast strain no inhibition was detected. The he⁷G caused a similar but smaller inhibitory effect in XP29MAmal, but failed to do so in XP29MA.HECNU predominantly exerts its killing effect by alkylating O-6 of DNA-bound guanine and causing DNA interstrand crosslinks. Alkylation of O-6 of guanine can be repaired by 6-methylguanine-DNA methyltransferase. From our experiments we conclude that in XP29MAmal this methyltransferase was inhibited in the presence of the 6-alkylguanines, thus leaving more 2-chloroethylated sites in DNA unrepaired. This results in sensitization in terms of decreased colony-forming ability observed only in the malignant cell line.Abbreviations XP xeroderma pigmentosum - HECNU 1-(2-chloroethyl)-1-nitroso-3-(2-hydroxyethyl)-urea - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - m⁶G 6-methylguanosine - m⁶Gua 6-methylguanine - he⁷G 7-(2-hydroxyethyl)-guanosine This work was supported by the Deutsche Forschungsgemeinschaft, SFB 136The publication is dedicated to Professor E. Hecker on the occasion of his 60th birthday     

A fluorimetric enzyme assay for the diagnosis of Sanfilippo disease C (MPS III C)

Summary Both the - and -anomers of 4-methylumbelliferyl-D-glucosaminide were synthesized and shown to be substrates for the lysosomal acetyl-CoA: glucosaminideN-acetyltransferase. Using the -anomer, fibroblasts and leukocytes from 11 different Sanfilippo C patients showed <1% of mean normalN-acetyltransferase activity. Heterozygotes showed intermediate activities. The enzymatic liberation of the fluorochrome from 4-methylumbelliferyl--d-glucosaminide requires the sequential action of theN-acetyltransferase and -hexosaminidase. Normal -hexosaminidase activity caused complete hydrolysis of the reaction intermediate 4-methylumbelliferyl--d-N-acetylglucosaminide formed by theN-acetyltransferase. In cell extracts with a -hexosaminidase deficiency, however, a second incubation in the presence of excess -hexosaminidase is needed to avoid underestimation of theN-acetyltransferase activity.     

Excessive glucose production,rather than insulin resistance,accounts for hyperglycaemia in recent-onset streptozotocin-diabetic rats

Summary Glucose production and utilization and activities of key enzymes involved in liver and muscle glucose metabolism were studied in post-absorptive streptozotocin-diabetic rats after 12 h of severe hyperglycaemia (17.5±0.5 mmol/l) and insulinopenia (5±1 U/ml). Basal glucose production was increased: 36.6±3.0 mg·kg·min^–1, vs 24.4±2.5 in controls (p<0.05); liver glycogen concentration was decreased by 40% (p<0.05); liver phosphoenolpyruvate carboxykinase and glucose-6-phosphatase activities were increased by 375 and 156%, respectively (p<0.001 and <0.01). During a euglycaemic clamp at a plasma insulin level of 200 U/ml, glucose production was totally suppressed in controls, but persisted at 20% of basal in diabetic rats. In these rats, glucose production was suppressed at a plasma insulin level of 2500 U/ml. Basal whole body glucose utilization rate, 2-deoxy-1-[³H]-d-glucose ([³H]-2dG) uptake by muscles and muscle glycogen concentrations were similar in both groups, as well as total and active forms of pyruvate dehydrogenase and glycogen synthase activities. During the euglycaemic clamp, the total body glucose utilization rates and [³H]-2dG uptake by muscles were similar in control and diabetic rats at a plasma insulin level of 200 U/ ml, but lower in diabetic rats at a plasma insulin level of 2500 U/ml. We conclude 1) in recent-onset severely insulinopenic rats, an excessive glucose production via gluconeogenesis prevailed, mainly accounting for the concomitant hyperglycaemia. This excess glucose output cannot be attributed to liver insulin resistance: the gluconeogenic pathway is physiologically less sensitive than glycogenolysis to the inhibition by insulin. 2) Glucose utilization was apparently normal under hyperglycaemic conditions and at a lower insulin plateau of the euglycaemic clamp but suboptimal in the presence of maximal insulin concentrations, suggesting an early appearance of peripheral insulin resistance.Abbreviations STZ streptozotocin - IDDM insulin-dependent diabetes mellitus - PEPCK phosphoenolpyruvate carboxykinase - PDH pyruvate dehydrogenase - G6Pase glucose 6-phosphatase - NEFA non-esterified fatty acids - DCA sodium dichloroacetate     

Nitric oxide in ischaemic acute renal failure of streptozotocin diabetic rats

Summary Changes in nitric oxide (NO) levels were determined in ischaemic acute renal failure in streptozotocin-induced diabetes mellitus rats. Two weeks after streptozotocin administration and immediately after right nephrectomy, the left renal artery was occluded for 60 min. Similar procedures were carried out in non-diabetic rats. The nitrite (NO₂) + nitrate (NO₃) levels were measured in plasma and urine. The effects of chronic oral supplementation with l-arginine and an NO synthase inhibitor (N-omega-nitro-l-arginine) were also studied in both diabetic and non-diabetic rats before and after renal artery clamping. The rats with diabetic acute renal failure had a much lower creatinine clearance (90±22 l · min^–1 · 100 g body weight^–1, p<0.005), and higher fractional excretion of sodium (FENa)% (10.90±4.2, p<0.001) and protein excretion (2078±69 g/ml creatinine clearance, p<0.001) compared with the respective values in the non-diabetic groups (163±30; 1.46±86; 453.3±31). The plasma and urine NO₂ + NO₃ levels were significantly higher in the untreated diabetic rats compared with the untreated normal rats before ischaemia (p<0.001). The ischaemic acute renal failure in non-diabetic rats increased the plasma and urinary NO₂ + NO₃ excretion after ischaemia. The urinary excretion of these metabolites decreased significantly and their plasma levels remained unchanged in the ischaemic diabetic rats. The l-arginine administration resulted in a small but significantly higher creatinine clearance after clamping in the non-diabetic rats. The NO synthase inhibitor caused deterioration in renal function in all ischaemic and non-ischaemic groups. In summary, the greater vulnerability to ischaemia of the diabetic kidney seems to be associated with both impaired response to and impaired production of NO.Abbreviations IARF Ischaemic acute renal failure - STZ streptozotocin - NOSi nitric oxide synthase inhibitor - ARF acute renal failure - Ccr creatinine clearance - FENa% fractional excretion of sodium     

Intestinal absorption of bile acid glucuronides in rats

While the intestinal absorption of taurine, glycine, and sulfate conjugates of bile acids has been studied extensively, nothing is known about the absorption of bile acid glucuronides. In the present study, the intestinal phase of the enterohepatic circulation of two bile acid glucuronides was examined. [3-³ H]cholic acid 3-O--d-glucuronide or [3^–3 H]lithocholic acid 3-O--d-glucuronide was perfused through isolated segments of ileum or jejunum with intact blood supply in rats prepared with a biliary fistula. [¹⁴C]Taurocholic acid was perfused simultaneously with each glucuronide to compare glucuronide absorption with that of an actively transported bile acid. Intestinal absorption was determined by measuring the rate of secretion of labeled bile acid in bile. The absorption of [³H]cholic acid glucuronide by the ileum and jejunum was one fortieth and one eighth, respectively, that of [¹⁴C]taurocholic acid. Comparison of the two glucuronides show that [³H]lithocholic acid glucuronide absorption was 18 and 10 times greater than [³H]cholic acid glucuronide absorption from the jejunum and ileum, respectively. Collectively, the above observations suggest that glucuronidation of bile acids markedly reduces absorption from the small intestine.This work was supported in part by the National Institutes of Health, grant HD-14198, and the March of Dimes Foundation, grant G-305.     

Comparison of DNA-incising capacities in fibroblast strains from the Mannheim XP collection after treatment with N-acetoxy-2-acetylaminofluorene and UV light

Summary The DNA-incising capacity was determined in 8 normal and 23 XP fibroblast strains of the Mannheim XP collection using the alkaline elution technique after treatment with both UV light and the UV-like carcinogen (Ac)₂ONFln. Experimental conditions were chosen to allow for selective monitoring of repair-specific enzyme-catalyzed breaks. In order to compare DNA-incising capacities of the various cell strains after UV irradiation with those after treatment with (Ac)₂ONFln, dose-response experiments including up to 8 dose levels were performed. The elution curves were analyzed by linear regression analysis. Elution velocities (in terms of DNA singlestrand breaks per 10⁶ nucleotides) were plotted against the square root of the doses. The slope of the resulting regression line yielded a characteristic term, designated E ₀, for the DNA-incising capacity of each cell strain. In contrast to normal fibroblasts, E ₀ was found to be reduced in all XP cell strains belonging to the complementation groups A, C, D, E, F (or G) and I investigated, after treatment with both UV light or (Ac)₂ONFln. Surprisingly, XP variant strains also exhibited lower E ₀ values. A comparison of post-UV with post-(Ac)₂ONFln DNA-incising capacities revealed that reduction in the E ₀ values was very similar in all XP cell strains tested. These data suggest that the sensitivity of XP cells towards UV light or (Ac)₂ONFln is due to the same enzymatic defect, namely impaired incision of DNA containing pyrimidine dimers or (Ac)₂ONFln-DNA adducts.Abbreviations XP xeroderma pigmentosum - (Ac)₂ONFln N-acetoxy-2-acetylaminofluorene - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - ara-C 1--d-arabinofuranosyl cytosine Dedicated to Prof. W. Kunz on the occasion of his 65th birthdayThis work was supported by the Deutsche Forschungsgemeinschaft, SFB 136     

Investigations on the acute and chronic nephrotoxicity of the new platinum analogue carboplatin

Summary In order to detect even subclinical hints of nephrotoxicity after application of carboplatin, sensitive non-invasive methods, e.g. determination of urinary enzyme (lactate dehydrogenase, leucine aminopeptidase,-glutamyltransferase,N-acetyl--glucosaminidase), glomerular and tubular protein excretion (albumin,-1-microglobulin) and determination of the creatinine clearance, were used. Eighteen patients with small-cell lung cancer entered the study. All patients were treated with the three-drug combination chemotherapy: vincristine (1.5 mg i.v. on days 1, 8, 15, 22), etopside (escalating doses: 100–160 mg/m² on days 1–3) and carboplatin (300 mg/m² day 1). Investigations were made during the first, third and fifth treatment cycles. Deterioration of renal function occurred in 4 out of 18 patients in all three observed treatment courses. Abnormal amounts in the excretion of at least one of four urinary enzymes were found in 6 out of 18 patients during the first cycle and in 4 out of 8 patients during the third and fifth cycles. All patients with pathological enzymuria during the first treatment course also developed an increased enzymuria during cycles 3 and 5. Four patients developed pathological proteinuria during the first and 2 patients during the third and fifth cycles. These findings demonstrate that the new platinum analogue, carboplatin, is capable of inducing renal damage. In comparison with cisplatinum, the nephrotoxicity of this new analogue is reduced but not completely eliminated.     

Early glomerular hyperfiltration and the development of late nephropathy in Type 1 (insulin-dependent) diabetes mellitus

Summary We performed a follow-up study of the glomerular function in a series of 29 Type 1 (insulin-dependent) diabetic patients who had been studied 18 years previously. Initial median duration of diabetes was 2 years (range 0–9) and at follow-up 21 (17–27) years. At follow-up, 8 diabetic patients exhibited increased urinary albumin excretion rate 515 (32-3234) g/min with glomerular filtration rates significantly lower than 21 diabetic patients with normal urinary albumin excretion (85 vs 126ml/min/1.73 m²; p<0.01). The patients with increased urinary albumin excretion rate also had higher arterial blood pressure (145/90 vs 120/80) mm Hg; p<0.02) and increased frequency of proliferative retinopathy (7 out of 8 vs 2 out of 21; p = 0.0001) as compared to the group with normal urinary albumin excretion. However, we found no association of increased urinary albumin excretion rate (incipient or overt nephropathy) to early glomerular hyperfiltration as median initial glomerular filtration rate was 142 ml/min/1.73 m² in the diabetic patients with increased urinary albumin excretion and 147 ml/min/1.73 m² in the patients with normal excretion rate (p>0.05)     

Tumor cell recruitment in the mouse adenocarcinoma EO 771 directly demonstrated by double labeling with [3H]- and [14C] thymidine and flow cytometry

Summary Tumor cell recruitment in the mouse adenocarcinoma EO 771 after application of 1--d-arabinofuranosylcytosine (AraCyt) has been directly demonstrated by double labeling with [³H]- and [¹⁴C]-thymidine. This method enables a quantitative study of the extent and time course of tumor cell recruitment in this solid mouse tumor. The results show that tumor cell recruitment is a continuous process that starts early after AraCyt application (about 4–8 h) and lasts about 1 day with a maximum at about 12 h after AraCyt application. Up to 40% of all cells are recruited at that time. The recruited tumor cells re-enter the resting state after having passed through one cycle.Abbreviations used AraCyt 1--d-arabinofuranosylcytosine - d'Thd thymidine - LI labeling index, MI, mitotic index This work was supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Mechanism of bradykinin-induced cyclic GMP accumulation in bovine tracheal smooth muscle

Bradykinin (10^–8-10^–5M) caused a concentration-dependent increase in cyclic GMP (cGMP) production in bovine tracheal smooth muscle in the absence of epithelium. The effect was calcium-dependent and was inhibited by pyrogallol (10 M) and methylene blue (10 M). The inhibition of pyrogallol was reversed by superoxide dismutase (100 Usnowml). Nitric oxide (NO) synthase inhibitors, N ^G-methyl-l-arginine (10–100 M) and N ^G-nitro-l-arginine (10–100 M) reduced cGMP accumulation induced by bradykinin in a concentration-dependent fashion, and the inhibition was reversed by l-arginine. Immunohistochemistry with a specific antibody against neuronal NO synthase from rat cerebellum showed positive staining localized in some nerve fibers. Bradykinin-induced cGMP accumulation appears to be related to the release of NO, part of which is probably synthesized in nonadrenergic noncholinergic nerve in bovine trachea.Offprint requests to: Dr. Hong Sheng     

Streptozotocin: Its excretion and metabolism in the rat

Summary The excretion of radioisotope following the administration of three specifically¹⁴C-labelled forms of streptozotocin was investigated in the rat using ureter and bile duct cannulation techniques. The urine collected during the first hour following the administration of the drug contained the highest proportion of injected radioactivity (approximately 34% with (3-methyl-¹⁴C)-streptozotocin and approximately 40% each with (1-¹⁴C)- and (2-¹⁴C)-streptozotocin. Over the entire experimental period (6 hours), approximately 70% of the injected radioactivity of (1-¹⁴C)- and (2-¹⁴C)-streptozotocin appeared in the urine. With (3-methyl-¹⁴C)-streptozotocin, only 53% of the injected radioactivity appeared in the urine over the same period. In contrast to the high urinary excretion, less than 3 % of the injected radioactivity from all three radiolabelled streptozotocin samples appeared in the bile. The in vivo and in vitro metabolism of streptozotocin was also investigated. In addition to substantial amounts of unchanged drug, three radiolabelled metabolites (two major and one minor) were detected in the urine during the 6 hour collection period following the administration of (1-¹⁴C)- and (2-¹⁴C)-streptozotocin. In contrast, only unchanged (3-methyl-¹⁴C)-streptozotocin was detected in the urine collected over the same period following the administration of the methyl labelled drug. The two major metabolites were also produced when (1-¹⁴C)- and (2-¹⁴C)-streptozotocin were incubated with a rat liver supernatant fraction (100,000 X g). The liver was further demonstrated to be the major site of metabolism in isolated liver perfusion studies in which both (1-¹⁴C)- and (2-¹⁴C)-streptozotocin were quantitatively converted to the two major metabolites. The two major metabolites of (1-¹⁴C)-streptozotocin, whether produced in vivo or in vitro, were chromatographically homogeneous with the two major metabolites formed from (2-¹⁴C)-streptozotocin. Nicotinamide pretreatment had no apparent effect on the urinary excretion of streptozotocin and its metabolites.     

Antitumour effect of a gonadotropin-releasing-hormone antagonist (MI-1544) and its conjugate on human breast cancer cells and their xenografts

Our gonadotropin-releasing hormone (GnRH) antagonist analogue MI-1544 ([Ac-d-Trp^1,3,d-Cpa²,d-Lys⁶,d-Ala¹⁰]GnRH) was developed as a potential contraceptive material, because it decreased the luteinizing hormone level without unfavourable side-effects. The antagonist was covalently bound to poly[Lys-(Ac-Glu_0.96-dl-Ala_3.1)] (AcEAK) —a branched polypeptide having a polylysine backbone — resulting in a MI-1544-AcEAK conjugate. According to our in vitro experiments the MI-1544 induced a 33%–35% decrease in cell numbers of MCF-7 and MDA-MB-231 human breast cancer cell lines at a dose of 30 M. The biodegradable polymeric carrier, AcEAK, alone inhibited cell proliferation by only 13%–15%, while the MI-1544-AcEAK conjugate, applied at the same dose, was capable of producing 45%–50% inhibition of cell proliferation. Our in vivo experiments using immunosuppressed mice showed that MI-1544, applied twice daily s.c., inhibited the growth of oestrogensensitive and-insensitive xenografts by 65% and 30% respectively. This effect was potentiated (70%) in both types of xenografts by the presence of the polymeric carrier in the conjugate; however, the carrier by itself did not cause tumour growth inhibition. The polymeric polypeptide carrier is supposed to increase the stability of the GnRH antagonist and to prevent the rapid excretion of the covalently bound peptide molecule. The antagonist and its conjugate may have various direct and indirect effects on breast cancer cells and, as a consequence, the new GnRH antagonist conjugates are suitable for treating an extended range of breast cancers.Abbreviations GnRH gonadotropin-releasing hormone - FSH folliclestimulating hormone - LH luteinizing hormone - ER oestradiol receptor - EGF epidermal growth factor - IGF insulin-like growth factor - MI-1544 [Ac-d-Trp^1.3,d-Cpa²,d-Lys⁶,d-Ala¹⁰]GnRH antagonist - EAK poly[Lys-(Glu_i-dl-Ala_m)] - AcEAK poly[Lys-(Ac-Glu_0.96-dl-Ala_3.1)] Supported by National Science Research Fund (OTKA 325 and T4217)     

Hexose metabolism in pancreatic islets

Islets from fed and 3-day-starved rats were incubated for 60 min in the presence of either 2.8 or 16.7 mM d-glucose, mixed with tracer amounts ofd-[5-³H]glucose,d-[3,4-¹⁴C]glucose,d-[6-¹⁴C]glucose andd-[2-¹⁴C]glucose. Starvation decreased the generation of³HOH fromd-[5-³H]glucose and the production of¹⁴CO₂ from¹⁴C-labelledd-glucose, both at low and high hexose concentrations. In islets from starved and fed rats, a rise ind-glucose concentration preferentially stimulated oxidative glycolysis, pyruvate decarboxylation and acetyl residue oxidation, relative tod-glucose utilization. At both low and high hexose concentrations and in both fed and starved rats, the decarboxylation of pyruvate exceeded the oxidation of glucose-derived acetyl residues, the C₁ of such residues being more efficiently converted to¹⁴CO₂ than their C₂. Starvation decreased oxidative glycolysis more severely than non-oxidative glycolysis, impaired the preferential stimulation ofd-[3,4-¹⁴C]glucose oxidation relative tod-[5-³H]glucose utilization as observed in response to a rise in hexose concentration, and lowered the ratio betweend-[6-¹⁴C]glucose oxidation and hexose utilization. It is proposed, therefore, that the short-term regulation of mitochondrial oxidative events byd-glucose is itself modulated in islet cells by the nutritional status.     

Pattern of sodium handling and its consequences in patients with preascitic cirrhosis

The initial abnormalities in the renal sodium handling in patients with cirrhosis before developing ascites remain unknown. The aim of this study is to further characterize sodium metabolism and the effects of sodium loading in preascitic cirrhosis. Eight male, preascitic patients with cirrhosis and 6 volunteers had their daily urinary sodium excretion level measured while on a strictly metabolically controlled diet, first consisting of 20 mmol then of 200 mmol sodium per day each for 7 days. Central blood volume, plasma norepinephrine, and atrial natriuretic factor levels were measured during each diet. Preascitic patients with cirrhosis had significantly less daily urinary sodium excretion on both diets. Volume expansion in the patients with cirrhosis was indicated by significantly greater weight gain and higher atrial natriuretic factor levels for each diet. Patients with cirrhosis had central blood volume expansion (1725 ± 54 mL/m²) compared with controls (1495 ± 81 mL/m²; P = 0.03) on a low-sodium diet. This increased significantly in the controls (1864 ± 164 mL/m²; P = 0.04) on a high-sodium diet, associated with suppression of plasma norepinephrine, but not in the patients with cirrhosis (1679 ± 107 mL/m²; P > 0.05). Failure of further central blood volume expansion in the patients with cirrhosis on high-sodium diet in the presence of significant weight gain suggests maldistribution away from the effective arterial blood volume. This study provides further reasons why preascitic patients with cirrhosis might benefit from sodium restriction.     

Modulation of sensitivity to mitomycin C and a dithiol analogue by tempol in non-small-cell lung cancer cell lines under hypoxia

We examined the mechanisms involved in the bioactivation of mitomycin C (MMC) and a newly developed MMC analogue: 7-N-(2-{[2-(-l-glutamylamino)ethyl]dithio}ethyl)mitomycin C, KW-2149, in non-small-cell lung cancer (NSCLC) cell lines under aerobic and hypoxic conditions. To investigate these mechanisms, we used MMC-resistant non-small-cell lung cancer cell lines (PC-9/MC4) that had been established in our laboratory from the parent PC-9 cell line by continuous exposure to MMC. We previously reported that the MMC-resistant cell line (PC-9/MC4) was poor in NAD(P)H dehydrogenase (quinone) activity and approximately 6-fold more resistant than the parent cells (PC-9) to MMC on 2-h exposure under aerobic conditions. In this study, the subline PC-9/MC4 was 6.7-fold more resistant to MMC than PC-9, the parent cell line, under aerobic conditions, and 5.2-fold more resistant under hypoxic conditions after 2h exposure to MMC. However, on co-incubation with tempol, an inhibitor of the one-electron reduction pathway, the sensitivity of PC-9/MC4 to MMC was impaired under hypoxic conditions, but the impairment was not evident under aerobic conditions. KW-2149, the newly developed MMC analogue, was cytotoxic for both PC-9/MC4 and PC-9 cells, and the sensitivity of both cell lines to KW-2149 was not changed by exposure to hypoxic conditions or by coincubation with tempol. There were no significant differences in the intracellular uptake of MMC and the activities of cytosolic detoxification enzymes between the PC-9 and PC-9/MC4 cell lines. These results support the hypothesis that the one-electron reduction pathway plays a partial role in the bioactivation of MMC, but not of KW-2149, and that KW-2149 is excellent at circumventing resistance to MMC in NSCLC.Abbreviations KW-2149 7-N-(2-{[2-(-l-glutamylamino)ethyl]dithio}ethyl)mitomycin C - MMC mitomycin C - NSCLC non-small-cell lung cancer - PBS calcium and magnesium-free Dulbecco's phosphate-buffered saline - P-450 reductase NADPH-cytochromeP-450 reductase