AMG 151 (ARRY-403), a novel glucokinase activator, decreases fasting and postprandial glycaemia in patients with type 2 diabetes.

Phase I studies have shown that AMG 151 activates glucokinase, a key enzyme in glucose homeostasis. The present randomized, placebo-controlled phase IIa study evaluated the dose-effect relationship of the glucokinase activator AMG 151 relative to placebo on fasting plasma glucose (FPG) in 236 patients (33-35 patients per arm) with type 2 diabetes treated with metformin. Patients received oral AMG 151 at 50, 100 or 200 mg twice daily, AMG 151 at 100, 200 or 400 mg once daily or matching placebo for 28 days. A significant linear dose-effect trend was observed with the twice-daily regimen (p = 0.004) for change in FPG to day 28. No trend was observed with the once-daily regimen. A higher incidence of hypoglycaemia and hypertriglyceridaemia was observed with AMG 151 administration. AMG 151 significantly reduced FPG when administered twice daily but not when administered once daily in patients with type 2 diabetes treated with metformin.

Tramadol hydrochloride 75 mg/dexketoprofen 25 mg oral fixed-dose combination in moderate-to-severe acute pain: sustained analgesic effect over a 56-h period in the postoperative setting.

Multimodal analgesia constitutes a common strategy in pain management. A tramadol hydrochloride 75 mg/dexketoprofen 25 mg oral fixed combination (TRAM/DKP 75 mg/25 mg) has been recently registered and released in Europe for the treatment of moderate-to-severe acute pain. This paper provides additional analyses on the results of two phase III clinical trials (DEX-TRA-04 and DEX-TRA-05) on postoperative pain to document its sustained effect. The analysis was applied to a modified intention-to-treat population (mITT, n = 933) of patients undergoing active treatment from the first dose, to assess the sustained effect of TRAM/DKP 75 mg/25 mg on pain intensity (PI-VAS 0-100) over 56 h from first drug intake. The superior analgesic effect of TRAM/DKP 75 mg/25 mg over 56 h in terms of difference in PI-VAS (mean [SE]) was shown for DEX-TRA-04 (-11.0 [0.55] over dexketoprofen 25 mg and -9.1 [0.55] over tramadol 100 mg, P ≤ 0.0001) and for DEX-TRA-05 (-10.4 [0.51] over dexketoprofen 25 mg and -8.3 [0.51] over tramadol 100 mg, P ≤ 0.0001). The statistical analysis performed on data coming from both studies confirms the superior sustained analgesia of TRAM/DKP 75 mg/25 mg over tramadol 100 mg and dexketoprofen 25 mg. These results are consistent with the previously published data obtained on the ITT population and strongly support the role of this oral fixed-dose combination in the treatment of moderate-to-severe acute pain.

Exacerbation of chronic renovascular hypertension and acute renal failure in heme oxygenase-1-deficient mice.

Heme oxygenase (HO) is a cytoprotective enzyme that degrades heme (a potent oxidant) to generate carbon monoxide (a vasodilatory gas that has anti-inflammatory properties), bilirubin (an antioxidant derived from biliverdin), and iron (sequestered by ferritin). Because of properties of HO and its products, we hypothesized that HO would be important for the regulation of blood pressure and ischemic injury. We studied chronic renovascular hypertension in mice deficient in the inducible isoform of HO (HO-1) using a one kidney-one clip (1K1C) model of disease. Systolic blood pressure was not different between wild-type (HO-1(+/+)), heterozygous (HO-1(+/-)), and homozygous null (HO-1(-/-)) mice at baseline. After 1K1C surgery, HO-1(+/+) mice developed hypertension (140+/-2 mm Hg) and cardiac hypertrophy (cardiac weight index of 5.0+/-0.2 mg/g) compared with sham-operated HO-1(+/+) mice (108+/-5 mm Hg and 4.1+/-0.1 mg/g, respectively). However, 1K1C produced more severe hypertension (164+/-2 mm Hg) and cardiac hypertrophy (6.9+/-0.6 mg/g) in HO-1(-/-) mice. HO-1(-/-) mice also experienced a high rate of death (56%) within 72 hours after 1K1C surgery compared with HO-1(+/+) (25%) and HO-1(+/-) (28%) mice. Assessment of renal function showed a significantly higher plasma creatinine in HO-1(-/-) mice compared with HO-1(+/-) mice. Histological analysis of kidneys from 1K1C HO-1(-/-) mice revealed extensive ischemic injury at the corticomedullary junction, whereas kidneys from sham HO-1(-/-) and 1K1C HO-1(+/-) mice appeared normal. Taken together, these data suggest that chronic deficiency of HO-1 does not alter basal blood pressure; however, in the 1K1C model an absence of HO-1 leads to more severe renovascular hypertension and cardiac hypertrophy. Moreover, renal artery clipping leads to an acute increase in ischemic damage and death in the absence of HO-1.

Endotoxin-induced mortality is related to increased oxidative stress and end-organ dysfunction, not refractory hypotension, in heme oxygenase-1-deficient mice.

BACKGROUND: Heme oxygenase (HO)-1 is an enzyme that degrades heme to generate CO (a vasodilatory gas), iron, and the potent antioxidant bilirubin. A disease process characterized by decreases in vascular tone and increases in oxidative stress is endotoxic shock. Moreover, HO-1 is markedly induced in multiple organs after the administration of endotoxin (lipopolysaccharide [LPS]) to mice. METHODS AND RESULTS: To determine the role of HO-1 in endotoxemia, we administered LPS to mice that were wild-type (+/+), heterozygous (+/-), or homozygous null (-/-) for targeted disruption of HO-1. LPS produced a similar induction of HO-1 mRNA and protein in HO-1(+/+) and HO-1(+/-) mice, whereas HO-1(-/-) mice showed no HO-1 expression. Four hours after LPS, systolic blood pressure (SBP) decreased in all the groups. However, SBP was significantly higher in HO-1(-/-) mice (121+/-5 mm Hg) after 24 hours, compared with HO-1(+/+) (96+/-7 mm Hg) and HO-1(+/-) (89+/-13 mm Hg) mice. A sustained increase in endothelin-1 contributed to this SBP response. Even though SBP was higher, mortality was increased in HO-1(-/-) mice, and they exhibited hepatic and renal dysfunction that was not present in HO-1(+/+) and HO-1(+/-) mice. The end-organ damage and death in HO-1(-/-) mice was related to increased oxidative stress. CONCLUSIONS: These data suggest that the increased mortality during endotoxemia in HO-1(-/-) mice is related to increased oxidative stress and end-organ (renal and hepatic) damage, not to refractory hypotension.

Role of macrophage-expressed adipocyte fatty acid binding protein in the development of accelerated atherosclerosis in hypercholesterolemic mice.

Atherosclerosis is an inflammatory disease process associated with elevated levels of plasma cholesterol, especially low-density lipoproteins. The latter become trapped within the arterial wall and are oxidized and taken up by macrophages to form foam cells. This process is an initiating event for atherosclerosis. Fatty acid binding proteins (FABP) are involved in fatty acid metabolism and cellular lipid transport, and adipocyte FABP (aP2) is also expressed in macrophages. We recently generated mice lacking both apolipoprotein (Apo)E and aP2 (ApoE-/-aP2-/-) and found that these mice, compared with ApoE-/- mice, developed markedly smaller atherosclerotic lesions that contained fewer macrophages. Here we investigated the mechanism(s) responsible for this prevention of atherosclerotic lesion formation. Bone marrow transplantations were performed in ApoE-/- mice, receiving cells from either ApoE-/- or ApoE-/-aP2-/- mice. The lack of aP2 in donor marrow cells led to the development of smaller (5.5-fold) atherosclerotic lesions in the recipient mice. No differences were found in plasma cholesterol, glucose, or insulin levels between recipients of bone marrow cells from ApoE-/- or ApoE-/-aP2-/- mice. However, the expression of chemoattractant and inflammatory cytokines was decreased in macrophages from ApoE-/-aP2-/- mice compared with ApoE-/- mice, which may contribute to the decrease in atherosclerotic lesion formation. Taken together, we demonstrate the importance of macrophage aP2 in the development of atherosclerotic lesions.

The influence of alumina and ultra-high molecular weight polyethylene particles on osteoblast-osteoclast cooperation.

Particle-induced macrophage activation, mainly by UHMWPE wear, has been recognized as the biological mechanism leading to periprosthetic bone resorption, which is responsible for the loosening of the total hip replacements (THR). Ceramic-on-ceramic implants have been advocated as a means of reducing wear products. Many studies investigated the effect of alumina (Al(2)O(3)) particles on monocytes/macrophages, but only limited information are available on their participation to bone turnover. An in vitro model was performed to investigate how Al(2)O(3) and UHMWPE particles may influence the osteoblast-osteoclast interaction: human osteoblasts (HOB) were obtained from trabecular bone, while osteoclasts were derived from peripheral blood mononuclear cells (PBMC) of healthy donors. The amount of IL6, TNF alpha, GM-CSF, and other factors acting on the bone turnover, i.e. the 'receptor activator of NF kappa B' ligand (RANKL) and osteoprotegerin (OPG), was detected in culture medium of particle-challenged HOB (HOB-CM). The Al(2)O(3) and UHMWPE particles did not affect either cell viability or TNF and GM-CSF release, while the increase in IL6 release seemed to be dependent on the particle concentration. UHMWPE increased the release of RANKL from HOB, while OPG and OPG-to-RANKL ratio were significantly inhibited. The ability of HOB-CM to promote osteoclastogenesis was tested via osteoblast/monocyte cooperation: after seven days of culture UHMWPE HOB-CM induced a large amount of multinucleated TRAP-positive giant cells, as well as significantly reduced the amount of IL6, GM-CSF and RANKL in the supernatant. With regard to the inductive effect on the osteoclastogenesis, our results show that the Al(2)O(3) wear debris are less active.

Loxoribine affects fludarabine activity on freshly isolated B-chronic lymphocytic leukemia cells.

Purine analogues like fludarabine have been shown to be superior to conventional therapy for B-cell chronic lymphocytic leukemia (B-CLL). In order to improve the activity of fludarabine, we tested its combination with loxoribine, a guanine ribonucleotide derivative, known to enhance the sensitivity of B-CLL cells to cytotoxic drugs. B-CLL cells from 6 patients were studied; co-incubation with loxoribine 100 microM increased the activity of fludarabine by 12% to 48%, as demonstrated by XTT colorimetric assay; while 1000 microM loxoribine exerted a protective effect. Accordingly, fludarabine-induced apoptosis was enhanced by the addition of loxoribine 1000 microM (39% increase). These results indicate that the combination of loxoribine and fludarabine could be of interest in B-CLL.

Cytotoxic combination of loxoribine with fludarabine and mafosfamide on freshly isolated B-chronic lymphocytic leukemia cells.

Fludarabine has shown a definite clinical activity in B-cell chronic lymphocytic leukemia (B-CLL). Recently it has been demonstrated that loxoribine, a guanine ribonucleotide derivative, is able to increase the cytotoxicity of fludarabine in B-CLL cells, in vitro. We have here extended these findings by testing the activity of loxoribine in combination with fludarabine and mafosfamide. As we have previously demonstrated, loxoribine enhances the activity of fludarabine at all concentrations, while only lower doses of mafosfamide seem to be positively affected by loxoribine. The combination of fludarabine and mafosfamide is synergistic on CLL cells, and the cytotoxic activity is increased by the addition of loxoribine. We have also evaluated the pro-apoptotic activity of each drug, both alone and in combination; these results are concordant with the cytotoxicity data, thus demonstrating that, even though loxoribine is more active in combination with fludarabine than with mafosfamide, the efficacy of the triple combination is higher than that obtained with any other agent alone or in double combination.

Diagnosis of infection after total hip replacement.

Subclinical infection in patients with pain following total hip replacement (THR) is an underestimated condition that needs consideration because it mimics aseptic loosening, contributes to periprosthetic osteolysis, and necessitates proper treatment. We aimed to define the reliability of diagnostic parameters that are routinely used before revision surgery for the assessment of infection. A continuous series of 26 subjects who underwent THR revision surgery was considered, including 21 cases diagnosed as aseptic loosening (group A) and 5 hip revisions with a clinical diagnosis for infection (group B). Seven subjects at the time of the primary arthroplasty were used as negative controls (group C). Technetium-99m labeled hydroxymethylene diphosphonate [(99m)Tc-HDP]- and technetium-99m hexamethylpropyleneamine oxide [(99m)Tc-HMPAO)]-labeled granulocyte scintigraphy, histology of peri-implant tissues, laboratory tests for inflammation, and microbiology were performed. Scintigraphy was positive for loosening [positive (99m)Tc-HDP scan] but negative for infection [negative (99m)Tc-HMPAO-labeled granulocyte scan] in all group A patients, whereas in 11 cases (52%) a positive culture was unexpectedly obtained. Histology showed conflicting results: Polymorphonuclear cells (PMNs) were found only in 5 of 11 culture-positive patients, whereas in 2 cases the presence of PMNs did not correspond to a positive culture. In group B patients, both isotope scans and microbiology were found to be positive. All control subjects (group C) had negative cultures. In our opinion, smoldering infection could be present in a significant proportion of cases of failed hip implants currently diagnosed as "nonseptic." The inflammatory response to wear debris and the presence of superimposed, slowly growing bacteria could act synergically, both contributing to the pathogenesis of periprosthetic osteolysis.

Deep peroneal nerve paresis in a runner caused by ganglion at capitulum peronei. Case report and review of the literature.

Although lateral popliteal sciatic nerve damage is not one of the commonest diseases in the general population, it is quite frequent among athletes. Several physiopathologic mechanisms have been thought to bring about this damage in athletes. Soft tissue ganglions with neurological involvement of the lateral popliteal sciatic nerve or its terminal rami are in differential diagnosis with several lesions of this area, as direct or indirect trauma, subcutaneous rupture of anterior tibialis muscle and long peroneal muscle, disc hernia, intraspinal tumor, anterior tarsal tunnel syndrome, cysts, neurofibroma, baker's cyst, vascular claudication, stenosing or inflammatory pathology of 2(nd) motoneuron, antimicrobial agents for urinary tract infection (nitrofurnantoin). The authors report the case of a 34-year-old amateur athlete with a recent paralysis of the hallux extensor, paresis of the toe extensor and hyposthenia of the tibialis anterior. The patient had been suffering from episodes of lumbalgia for a long time. He was sent to us because neurological damage due to disc herniation was suspected. Electromyography, sonography, and CT showed peripheral compression of the deep peroneal nerve caused by a mucous cyst at the capitulum peronei, a ''rare'' condition. The patient underwent surgery to excise the cyst, which led to the rapid resolution of the nerve deficit shown by clinical and electromyographical tests. A meticulous anamnesis and accurate objective examination, followed by specific tests (radiographs, sonography, and possibly CT scan) generally enable a correct diagnosis to be made. If diagnosis and therapy are carried out correctly, and without delay, symptoms quickly resolve and the nerve deficit progressively regresses.

[Familial Mediterranean fever: a report of 2 cases of Italian origin]. / Febbre mediterranea familiare: descrizione di due casi di origine italiana.

Familial Mediterranean fever is an inherited disease, occurring almost exclusively in Arabs, Jews and Turks. Cases are very rarely described in the USA, USSR, France, and patients are all natives to the Mediterranean area. This paper describes two cases of familial Mediterranean fever in brothers native to Campania, Italy. Both had complained of repeated episodes of fever, with acute abdomen, thoracalgia and arthralgia since the age of about 20. One of them had had pleuritis when he was 6 years old. In the period preceding our first observation, both underwent laparotomy to evaluate abdominal symptoms, with negative results. After ruling out other diseases with similar signs and symptoms, we raised the hypothesis of familial Mediterranean fever, despite the fact that the literature has described very few Italian natives affected by this disease. The diagnostic hypothesis was confirmed by the positivity of the metaraminol provocation test. At the same time we evaluated the presence of amyloidosis by rectal biopsy, with negative results. Treatment with colchicine 1 mg/day per os was established. Dramatic improvement of the symptoms was observed in both patients. The present paper stresses the importance of familial Mediterranean fever, its correct diagnosis in Italy and the fundamental role played by the metaraminol provocation test as a determinant diagnostic tool. It allows establishment of appropriate treatment as soon as possible, so that renal amyloidosis, the most severe complication and major prognostic determinant of familial Mediterranean fever, can be prevented. Inappropriate, useless and potentially harmful surgical diagnostic procedures are also avoided.

Association of the Budd-Chiari syndrome with lupus anticoagulant. Case report and critical review.

The Budd-Chiari syndrome (BCS) was diagnosed in a 30-year-old male hospitalized with hepatomegaly, abdominal collateral vessels and hepatic veins and inferior vena cava thrombosis (IVC) in 1988. The presence of circulating lupus anticoagulant (LAC) was suspected and demonstrated on this occasion in view of an earlier diagnosis of systemic lupus erythematosus (SLE) and recurrent vein thrombosis dating from 1981. There are sporadic reports of an association of BCS with SLE and other autoimmune diseases. The recent literature also describes associations with hypercoagulability due to LAC. These are reviewed together with the personal case to provide the rationale for correct diagnosis and therapy.

Antagonizing and measurement: approaches to understanding of hemodynamic effects of kinins.

Hemodynamic effects of the kallikrein-kinin system can be investigated by experimental administration of specific kinin antagonists and by measurement of kinin levels in the circulating blood. In conscious normal rats, the bradykinin analog B4162 blunts the hypotensive effect of exogenous bradykinin. This kinin antagonist has no blood pressure effect in control rats, but it enhances the pressor effect of vasoconstrictor substances such as vasopressin or angiotensin II when they are infused at subpressor doses. Endogenous kinins may therefore participate in blood pressure regulation by antagonizing pressor substances. Plasma levels of endogenous kinins are normally in the low picomolar range. They are rapidly generated and destroyed in biological fluids. Thus, measurement of plasma kinins requires sensitive assays based on high-affinity antibodies and careful sample-handling techniques. Nonpolar solid-phase extraction on phenylsilylsilica provides a rapid, reliable, and easy extraction of kinins from plasma with constant and high recoveries.

Plasma kinins increase after angiotensin-converting enzyme inhibition in human subjects.

1. In patients treated with angiotensin-converting enzyme inhibitors, kinin-related effects have been postulated repeatedly, but information on changes in plasma kinin levels in these patients is sparse. Difficulties in the measurement of plasma kinins account for this, at least in part. 2. The main purpose of the present study was to investigate, in normal human subjects, the effect of the angiotensin-converting enzyme inhibitor quinapril on plasma kinins. 3. High-affinity antisera (Kd < 10(-11) mol/l) of C-terminal specificity were raised in rabbits for radioimmunoassay of immunoreactive kinins activating the bradykinin B2-receptor, and three different liquid- and solid-phase extraction methods for plasma kinins were evaluated. Ethanol and subsequent petroleum ether extraction of 5-40 fmol of bradykinin added to plasma yielded recoveries of 39 +/- 16% (mean +/- SD); normal kinin levels in human plasma were 18.6 +/- 3.3 pmol/l (mean +/- SEM). Solid-phase extraction on urea-equilibrated phenylsilylsilica produced recoveries of 89 +/- 5% and normal values of 36.4 +/- 18 pmol/l. Finally, with an assay based on ethanol extraction alone, recoveries of 100 +/- 16% and normal values of 16.8 +/- 5.8 pmol/l were obtained, with a detection limit of 1.5 fmol/ml of plasma. Blanks were below the detection limit. Serial dilution of plasma extracts (n = 4) provided linear kinin concentrations (r = 0.99). For two different plasma pools, coefficients of variation for within-assay precision were 16.7% and 21.7%, respectively. Between-assay coefficients of variation were 12.8% and 17.4%.(ABSTRACT TRUNCATED AT 250 WORDS)

Osteoid osteoma simulating an osteocartilaginous exostosis.

We describe a case of osteoid osteoma in the tibia of a 3-year-old patient who presented with a clinical and radiographic picture that suggested an exostosis. The formation of osteoid osteoma with a radiographic picture similar to that of osteophytes or exostosis has been previously documented only rarely. The authors hypothesize that the exostosis-like formation observed was actually the calcification of soft tissues that formed after the intense periosteal inflammatory reaction caused by the osteoid osteoma. As a result of its peculiar clinical and radiographic presentation, diagnosis of this lesion was delayed. Being located close to the medial growth plate of the tibia, it caused lengthening of the limb with a pronounced valgus deviation of the knee. An excisional biopsy provided histological evidence, clinical resolution and immediate pain relief, but incomplete resolution of the valgus deformity of the knee.

Retinoids increase idarubicin cytotoxicity in human myeloid leukemia cell lines.

All-trans-retinoic acid (ATRA) has proven useful in acute promyelocytic leukemia (APL). In order to reduce the side effects and to improve the efficacy of this compound, conventional chemotherapy, and anthracyclines in particular, are frequently added either during remission induction or in consolidation therapy. In this study we aimed at investigating the rationale of the combination of ATRA plus idarubicin in two human leukemic cell lines, HL-60 and K562, that display a different sensitivity to ATRA treatment. The effects of ATRA were compared with those of two clinically active retinoids, 13-cis-retinoic acid (13-cis-RA) and 9-cis-retinoic acid (9-cis-RA). Both in HL-60 and in K562 cells, the majority of the combinations of ATRA and idarubicin were synergistic, while the combinations with 9-cis-RA and 13-cis-RA were more effective in HL-60 and K562 cells, respectively. A 72 h pre-incubation with retinoids was able to further increase the cytotoxicity of ATRA plus idarubicin in the two cell lines. Intracellular idarubicin accumulation was enhanced by retinoids, as demonstrated by a cytofluorimetric method. Our results could contribute to provide a rationale for ATRA plus idarubicin combinations not only in APL but also in acute leukemia of other cytotypes.

Reduction of heat-shock protein-70 after prolonged treatment with retinoids: biological and clinical implications.

Heat shock proteins (HSPs) are a group of highly conserved polypeptides involved in cellular response to heat or other physical or chemical stresses. It has been recently reported that HSPs could play a role in cellular differentiation. In this study we have evaluated, by a cytofluorimetric method, the presence of HSP-70 in HL-60 cells during treatment with all-trans retinoic acid (ATRA), 9-cis retinoic acid (9-cis RA), and 13-cis retinoic acid (13-cis RA). After 1 and 3 days of incubation at 10(-7) M, HSP-70 did not show any variation compared to control; prolonging the exposure, together with the appearance of cellular differentiation along the granulocytic pathway and apoptosis, a progressive decrease of HSP-70 was observed that, after 8 days of treatment, was reduced by 40% with ATRA and by 28% with 9-cis RA compared to untreated samples, while only minimal changes were evident by incubating the cells with 13-cis RA. Reduction of HSP-70 was not associated with decreased protein synthesis, as demonstrated by [3H] leucine incorporation. Double labeling with propidium iodide showed a decrease in HSP-70 in all the phases of the cell cycle concomitant with a reduced percentage of cycling cells in ATRA-treated samples. Dot blot and Northern blot analysis demonstrated no change in HSP-70 mRNA after retinoid treatment, thus suggesting a post-transcriptional regulation of the phenomenon. This reduced production of HSP-70 caused by ATRA and by 9-cis RA, though to a lesser extent, could render the cells more sensitive to cytotoxic agents and could provide the rationale for the efficacy of ATRA + chemotherapy combinations.

Adenoviral mediated gene transfer can be accomplished in human myeloid cell lines and is inhibited by all-trans retinoic acid-induced differentiation.

BACKGROUND AND OBJECTIVE: Gene transfection could potentially represent a useful therapeutic tool for genetic and neoplastic hematological diseases. After having long been considered poorly able to transfect myeloid cells, adenoviral vectors have recently been demonstrated to be capable of introducing foreign DNA into purified CD34+ cells from human bone marrow or cord blood. In the present study we evaluated the feasibility of adenoviral-mediated gene transfer in two human leukemic cell lines, both at baseline and after differentiation induction by all-trans retinoic acid (ATRA). METHODS: We used a recombinant adenovirus expressing beta-galactosidase (Ad-RSV-beta-gal) to transfect K562 and HL-60 cell lines. The effects of 10(-6)M ATRA were evaluated after 8 days of exposure. The efficacy of transfection was verified by X-gal staining. RESULTS: Ad-RSV-beta-gal was able to transfect both the HL-60 and, to a minor extent, the K562 cell lines. The addition of ATRA had no effect on transfection of K562 cells, while a lower percentage of beta-gal-positive cells was detected in HL-60, which underwent differentiation upon ATRA treatment. INTERPRETATION AND CONCLUSIONS: These data suggest that adenoviral-mediated gene transfer could be feasible in myeloid leukemia cell lines and that it is inhibited by ATRA in differentiation-sensitive cells. The latter effect merits further investigation in order to verify whether this represents an ATRA-related or a differentiation-related phenomenon.

In vitro study of the combination gemcitabine + fludarabine on freshly isolated chronic lymphocytic leukemia cells.

BACKGROUND AND OBJECTIVE: Fludarabine has shown a definite clinical activity in B-cell chronic lymphocytic leukemia (CLL). If the effects of this drug could be potentiated, it could be useful in order to obtain complete remissions. In this study we evaluated the effects of the combination of fludarabine and gemcitabine, a deoxycytidine analog that has shown both in vitro and in vivo activity against a variety of solid tumors. DESIGN AND METHODS: CLL cells from 10 patients were cultured in vitro in the presence of fludarabine (0.5-1,000 microg/mL) and gemcitabine (0.1-5,000 microg/mL), both alone and in different combinations. Cytotoxic activity was tested by the XTT colorimetric assay. Furthermore we evaluated BCL-2 protein expression and, subsequently, the induction of apoptosis at baseline and after exposing cells to different concentrations of fludarabine and gemcitabine. RESULTS: The IC(50) of fludarabine and gemcitabine on CLL cells was 550 and 1,100 microg/mL, respectively, in our series of samples; the cytotoxicity of either drug was not influenced by the percentage of BCL-2 positive cells in the same sample. The addition of gemcitabine increased fludarabine-induced cytotoxicity; however, isobologram analysis of the data showed synergism only when lower doses of gemcitabine were combined to fludarabine. Induction of apoptosis reflected this pattern of activity. INTERPRETATION AND CONCLUSIONS: Gemcitabine was able to increase the activity of fludarabine only when low doses of the former were employed. As both compounds incorporate into DNA blocking chain elongation, our results could be explained by the drugs interfering at that level. The possibility of potentiating the effects of fludarabine with low doses of gemcitabine renders this combination promising in view of an in vivo use.

Plasma bradykinin in angio-oedema.

BACKGROUND: Bradykinin is believed to be the main mediator of symptoms in hereditary (HA) and acquired (AA) angio-oedema due to C1 esterase inhibitor deficiency, as well as in angio-oedema that complicates treatment with inhibitors of angiotensin-converting enzyme (ACE). Difficulties in the measurement of kinin concentrations, however, have so far precluded the demonstration of an incontrovertible change in plasma bradykinin concentrations in these disorders. By developing a reliable assay we have been able to follow bradykinin concentrations during attacks and during remission in HA and in AA, and also in a patient treated with an ACE-inhibitor. METHODS: Liquid-phase extraction, high-performance liquid chromatography, and RIA were used for specific measurement of plasma bradykinin concentrations in 22 patients with HA and in 22 healthy volunteers of similar age and sex distribution. Four patients with AA and one hypertensive patient treated with the ACE inhibitor captopril were also studied. FINDINGS: Among the healthy volunteers plasma bradykinin concentration was inversely proportional to age. The geometric mean plasma bradykinin concentration in the healthy volunteers was 2.2 fmol/mL (SD 2.2), compared with 3.9 fmol/mL (3.7) among patients with HA during remission (p=0.095). Bradykinin was also high in the patients with AA (10.4 fmol/mL [1.6]). During acute attacks of oedema, in both HA and AA, plasma bradykinin rose to two to 12 times the upper limit of normal. Infusion of C1-esterase inhibitor (the deficient factor in both HA and AA) immediately lowered bradykinin concentrations. In the patient receiving the ACE-inhibitor captopril, bradykinin concentration was very high at 47 fmol/mL during an acute attack of angio-oedema, but normal at 3.2 fmol/mL in remission after withdrawal of the drug. INTERPRETATION: A sensitive method for measurement of plasma bradykinin provided the means to show that concentrations of this peptide decrease with age in healthy people. Although the differences between patients in remission and healthy controls did not reach statistical significance, there were substantial rises in bradykinin during acute attacks of hereditary, acquired, or captopril-induced angio-oedema.