Phase 1 dose-escalation study of a siRNA targeting the RTP801 gene in age-related macular degeneration patients.

BACKGROUND: To evaluate the safety, tolerability, pharmacokinetics, and dose-limiting toxicities of a single intravitreal (IVT) injection of PF-04523655, a 19-nucleotide, O-methyl stabilized, double-stranded small interfering ribonucleic acid targeting the RTP801 gene in patients with neovascular age-related macular degeneration (AMD). METHODS: Prospective, phase 1, clinical multicentre trial, enrolled 27 patients with neovascular AMD unresponsive to prior treatment and best corrected visual acuity (BCVA) ≤ 20/200 in the study eye in stratum 1: (dose-escalating, open-label: 50 to 3000 µg of PF-04523655) and 27 patients who had potential to benefit from therapy and BCVA of ≤ 20/100 and ≥ 20/800 in stratum 2 (parallel, masked study of 1000, 1500, 2250, and 3000 µg of PF-04523655). The primary outcome was safety and tolerability assessment as well as pharmacokinetic profiling following a single IVT injection of PF-04523655. RESULTS: Doses of PF-04523655 ≥ 400 µg were generally detectable in the plasma at 1, 4, and 24 h post-injection. And all doses were below the lowest level of quantification by day 14. A single IVT injection of 50 to 3000 µg of PF-045237655 was generally safe and well tolerated over 24 months. There were no dose-limiting toxicities. CONCLUSION: A single IVT injection of PF-0523655 ≤ 3000 µg seems safe and well tolerated in eyes with neovascular AMD.

Suppression of ocular neovascularization with siRNA targeting VEGF receptor 1.

In this study, we used small interfering RNA (siRNA) directed against vascular endothelial growth factor receptor 1 (vegfr1) mRNA to investigate the role of VEGFR1 in ocular neovascularization (NV). After evaluating many siRNAs, Sirna-027 was identified; it cleaved vegfr1 mRNA at the predicted site and reduced its levels in cultured endothelial cells and in mouse models of retinal and choroidal neovascularization (CNV). Compared to injection of an inverted control sequence, quantitative reverse transcriptase-PCR demonstrated statistically significant reductions of 57 and 40% in vegfr1 mRNA after intravitreous or periocular injection of Sirna-027, respectively. Staining showed uptake of 5-bromodeoxyuridine-labeled Sirna-027 in retinal cells that lasted between 3 and 5 days after intravitreous injection and was still present 5 days after periocular injection. In a CNV model, intravitreous or periocular injections of Sirna-027 resulted in significant reductions in the area of NV ranging from 45 to 66%. In mice with ischemic retinopathy, intravitreous injection of 1.0 mug of Sirna-027 reduced retinal NV by 32% compared to fellow eyes treated with 1.0 mug of inverted control siRNA. These data suggest that VEGFR1 plays an important role in the development of retinal and CNV and that targeting vegfr1 mRNA with siRNA has therapeutic potential.

Aleukaemic leukaemia cutis presenting as a benign-appearing eruption.

A 68-year-old Caucasian male presented with a 5-week history of a widespread pruritic papular eruption. Histology from a papule on the left shoulder showed a dense dermal infiltrate of large mononuclear cells which were positive for leucocyte common antigen, KP1 and PGM1, with an MIB-1 proliferating fraction of 40%, diagnostic of acute monocytic (M5) leukaemia cutis. Full blood count revealed pancytopaenia but no blasts. Bone marrow aspirate showed reduced red cell precursors and 10% blasts, consistent with myelodysplastic syndrome (refractory anaemia with excess blasts). The patient was managed with a 3 unit transfusion of packed red cells, after which his skin eruption resolved within 6 weeks and his peripheral blood counts returned to normal. No chemotherapy was administered. In conclusion, leukaemia can present in the skin, the eruption may be nonspecific and it may precede systemic involvement by either myelodysplastic syndrome or acute leukaemia.

A multicenter, double-blind, dose-ranging, randomized, placebo-controlled study of recombinant human interleukin-1 receptor antagonist in patients with rheumatoid arthritis: radiologic progression and correlation of Genant and Larsen scores.

OBJECTIVE: To evaluate radiographic progression and the relationship of radiologic scores obtained by the Genant and Larsen methods in a clinical trial of recombinant human interleukin-1 receptor antagonist (IL-1Ra). METHODS: Patients with rheumatoid arthritis (RA) were randomized into 4 groups: placebo (n = 121) or IL-1Ra at a daily dosage of 30 mg (n = 119), 75 mg (n = 116), or 150 mg (n = 116). Hand radiographs obtained at baseline, 24 weeks, and 48 weeks were scored using both methods. RESULTS: At 24 weeks, by the Genant method, there was significant reduction in the score for progression of joint space narrowing (JSN) and the total score (a combination of erosion and JSN) in all treatment groups. Least-squares mean changes in the Genant erosion score from baseline to 24 weeks were significantly reduced after treatment with IL-1Ra at 30 mg/day and for all IL-1Ra treatment groups combined. The changes corresponded to a reduction of 38% in erosion, 58% in JSN, and 47% in total score. Patients treated with IL-1Ra at 75 mg/day had a significant reduction in the Larsen erosive joint count (LEJC), and all IL-1RA-treated groups combined showed a 45% reduction. Correlations (r) between the Genant total and Larsen scores were 0.84 at baseline, 0.83 at week 24, and 0.83 at week 48 (P < 0.0001); correlations between the Genant erosion score and the LEJC were 0.83 (P < 0.0001) at all visits; correlations between the Genant total and the Larsen scores were 0.32 and 0.49 (P < 0.0001) for progression from baseline to week 24 and from baseline to week 48, respectively; correlations between the Genant erosion score and the LEJC were 0.36 and 0.41 (P < 0.0001) for progression to weeks 24 and 48, respectively. CONCLUSION: IL-1Ra reduced radiologic progression of RA. Scores by the 2 methods correlated strongly for each individual time point, but much less strongly for assessments of disease progression.

Treatment of rheumatoid arthritis with recombinant human interleukin-1 receptor antagonist.

OBJECTIVE: To evaluate the efficacy and safety of interleukin-1 receptor antagonist (IL-1Ra) in patients with rheumatoid arthritis (RA). METHODS: Patients with active and severe RA (disease duration <8 years) were recruited into a 24-week, double-blind, randomized, placebo-controlled, multicenter study. Doses of nonsteroidal antiinflammatory drugs and/or oral corticosteroids (< or =10 mg prednisolone daily) remained constant throughout the study. Any disease-modifying antirheumatic drugs that were being administered were discontinued at least 6 weeks prior to enrollment. Patients were randomized to 1 of 4 treatment groups: placebo or a single, self-administered subcutaneous injection of IL-1Ra at a daily dose of 30 mg, 75 mg, or 150 mg. RESULTS: A total of 472 patients were recruited. At enrollment, the mean age, sex ratio, disease duration, and percentage of patients with rheumatoid factor and erosions were similar in the 4 treatment groups. The clinical parameters of disease activity were similar in each treatment group and were consistent with active and severe RA. At 24 weeks, of the patients who received 150 mg/day IL-1Ra, 43% met the American College of Rheumatology criteria for response (the primary efficacy measure), 44% met the Paulus criteria, and statistically significant improvements were seen in the number of swollen joints, number of tender joints, investigator's assessment of disease activity, patient's assessment of disease activity, pain score on a visual analog scale, duration of morning stiffness, Health Assessment Questionnaire score, C-reactive protein level, and erythrocyte sedimentation rate. In addition, the rate of radiologic progression in the patients receiving IL-1Ra was significantly less than in the placebo group at 24 weeks, as evidenced by the Larsen score and the erosive joint count. IL-1Ra was well tolerated and no serious adverse events were observed. An injection-site reaction was the most frequently observed adverse event, and this resulted in a 5% rate of withdrawal from the study among those receiving IL-1Ra at 150 mg/day. CONCLUSION: This study confirmed both the efficacy and the safety of IL-1Ra in a large cohort of patients with active and severe RA. IL-1Ra is the first biologic agent to demonstrate a beneficial effect on the rate of joint erosion.

A randomised trial of cyclophosphamide with and without low dose alpha-interferon in the treatment of newly diagnosed myeloma.

The role of alpha-interferon (IFN) in initial treatment of multiple myeloma is controversial. We have conducted a trial of cyclophosphamide with and without low dose IFN in newly diagnosed myeloma. Thirty four patients, mean age 63 were all given cyclophosphamide 600mg/m2 IV every 21 days while 17 were randomised to also receive IFN at a dose equivalent to 6mU per week. In the IFN group 8/15 assessable patients (53%) had a greater than 50% reduction in paraprotein compared with 4/16 (25%) of controls (p < 0.05). However the median duration of response was the same in the two groups (IFN 8 months, controls 8.5 months). After a median follow up of 28 months there is no significant difference in survival. Toxicity including myelosuppression was commoner in the IFN group. These findings suggest that the addition of IFN to cyclophosphamide is not likely to offer major benefits in treating myeloma.

von Willebrand factor as a marker of endothelial cell activation following BMT.

Endothelial cell activation may play a role in thrombotic complications of BMT such as hepatic veno-occlusive disease (VOD), right atrial line thrombosis and microangiopathic haemolysis. To assess this, von Willebrand factor antigen (vWF:ag) was measured in 72 patients (25 allografts, 46 autografts and one syngeneic) during the first 6 weeks post-transplant. There was a significant rise in vWF:ag in both allografts and autografts but a greater increase was seen in the allografts. The changes in vWF:ag did not correlate with changes in C reactive protein showing that this was not merely an acute phase response. vWF multimers were normal in a subgroup of uncomplicated transplants showing that there was no large scale endothelial cell disruption. Patients with VOD did not have changes in vWF:ag that were consistently different from uncomplicated controls. Three of four patients who developed line thrombosis had higher levels of vWF:ag compared with control groups; multimeric structure of the vWF was again normal. These results show that there is endothelial cell activation post-BMT and that this is greater in allografts compared with autografts, thus suggesting a possible mechanism for the higher incidence of VOD in this group. There were no useful predictive markers of VOD or thrombosis in individual patients.

Recombinant alpha 2B interferon in combination with oral chemotherapy in late chronic phase chronic myeloid leukaemia.

The place of alpha interferon (IFN) therapy in the treatment of chronic myeloid leukaemia (CML) is under intensive investigation at present. It is now established that as a single agent it can provide good disease control in the chronic phase and that cytogenetic responses will occur in a minority of patients. However its impact on long term survival has been less certain. Optimal haematological and cytogenetic results have to date been seen when IFN is used in the early phase of the disease, i.e. within one year of diagnosis. We have performed a prospective single arm study on the effect on survival of the addition of low dose IFN (9 mU/week) to conventional oral chemotherapy in patients who were at a median of 19 months from the initial diagnosis at the time of study entry. Comparison of this cohort with a control group of CML patients treated with oral chemotherapy only at the same participating institutions gave an estimated 72% reduction in the risk of death as a result of IFN therapy. Median survival for the IFN group has not been reached at 43 months compared with a median survival of 33 months for the chemotherapy alone group. These results suggest that the introduction of low dose IFN at any stage in the chronic phase may produce a worthwhile improvement in survival.

Serum osteocalcin in the management of myeloma.

We have measured serum osteocalcin, a vitamin K-dependent glycoprotein synthesised by osteoblasts in 62 patients, 49 with myeloma, 26 at presentation and 23 previously treated, 7 with Waldenstrom's macroglobulinaemia (WM), and 6 with monoclonal gammopathy of uncertain significance (MGUS). Osteocalcin levels were normal in WM and MGUS. High values were found in 5/26 (19%) patients with myeloma at presentation. There was no relationship between serum osteocalcin and stage of disease. Osteocalcin was normal in all patients in plateau phase, falling to low levels in relapsed patients who failed to respond to further treatment. Serum osteocalcin may be a useful indicator of bone metabolism in myeloma.

Hydroxyethyl starch induced acquired von Willebrand's disease.

Hydroxyethyl starch (HES) (Hespan, DuPont) is a widely used synthetic volume expander which in standard doses of up to 1.5l in 24 h has no significant effect on coagulation (Munsch et al. 1988). However, the data sheet states that in large volumes HES may alter the coagulation mechanism. We now report a case of HES induced acquired von Willebrand's disease (vWD) in which severe bleeding occurred.

Pregnancy associated aplastic anaemia: a report of five cases and review of current management.

The occurrence of aplastic anaemia in pregnancy has been long recognized but its rarity has made it difficult to establish the relationship between the two conditions and the optimal management. We now report five cases of aplastic anaemia in pregnancy and offer some recommendations for treatment. In two patients the pregnancy was allowed to continue to term and the disease persisted post-partum leading to death in one case. The other three patients had their pregnancies terminated; one subsequently deteriorated and died, two had spontaneous remissions of their aplasia. We suggest that patients presenting with severe aplastic anaemia in early pregnancy should be offered termination because this may be followed by haematological improvement. If haematological improvement does not occur allogeneic bone marrow transplantation (BMT) may be considered. Aplastic anaemia presenting in late pregnancy should be treated with supportive care until delivery. On the basis of our experience, antilymphocyte globulin may safely be given during pregnancy.

Defective fibrinolysis in Behçet's syndrome: significance and possible mechanisms.

Reduced fibrinolytic activity has frequently been reported in Behcet's syndrome, but both the underlying mechanism and its relation to the development of thrombosis are unclear. The fibrinolytic activity was studied in seven patients with Behçet's syndrome and 12 patients with idiopathic oral ulceration. The patients with Behçet's disease had significantly reduced resting tissue plasminogen activator(t-PA) both by euglobulin fibrin plate lysis and enzyme linked immunosorbent assay (ELISA), but the tissue plasminogen activator inhibitor(t-PAI) was not significantly different from the control value. After stimulation by venous occlusion the euglobulin fibrin plate lysis results showed three good responders and four poor responders, but neither t-PA antigen nor t-PAI concentrations showed any clear difference between the two groups. Interestingly, three out of the four patients with a history of thrombosis were good responders. The group with oral ulceration showed no significant difference from the controls for any of these measurements. These findings suggest that the reduced resting fibrinolytic activity is usually due to decreased production of t-PA, but that the defective response to stimulation may be multifactorial in origin. The fibrinolytic abnormalities appear unrelated to the development of thrombosis.

Smoking--a major cause of polycythaemia.

The importance of smoking in the aetiology of polycythaemia has been assessed in a group of patients referred to a general haematology clinic. All patients with true and relative polycythaemia (excluding those with polycythaemia rubra vera) were studied. Of the 21 such patients evaluated, 14 were smokers and had raised carboxyhaemoglobin levels and had no other demonstrable cause for their polycythaemia. The commonest physiological abnormality in these patients was a raised red cell mass combined with a low plasma volume. Six of the 14 patients were able to reduce their smoking with subsequent improvement in their haematocrits. These results suggest that smoking is a major cause of polycythaemia in an unselected series of referrals to a general haematology clinic. The early identification of these patients may be useful in planning therapy.

Myelodysplasia presenting as erythroderma.

Erythroderma has not previously been reported to be a feature of myelodysplasia. We report two cases of myelodysplasia presenting with erythroderma, one of which was associated with skin infiltration by blast cells.

Polycythaemia rubra vera transforming to acute lymphoblastic leukaemia.

Two patients who developed acute lymphoblastic leukaemia following polycythaemia rubra vera (PRV) are described. In both cases the diagnosis was made using cytochemistry and immunological markers. One patient's cells marked as T-cell acute lymphoblastic leukaemia (T-ALL), the other as unclassified (null) ALL. These cases support the concept that PRV is a stem cell disorder.

Protein phosphorylation in rat mammary acini and in cytosol preparations in vitro. Phosphorylation of acetyl-CoA carboxylase is unaffected by cyclic AMP.

Phosphorylation of soluble proteins in rat mammary acinar cells was investigated. When phosphorylation proceeded in intact cells, in the presence of [32P]Pi, the major non-casein phosphoproteins, including acetyl-CoA carboxylase, were unresponsive to incubation conditions that caused major increases in the intracellular concentration of cyclic AMP. The overall 32P specific radioactivity (c.p.m./microgram of protein) of acetyl-CoA carboxylase, assessed after affinity purification of the enzyme with avidin-Sepharose, was unchanged by incubation under such conditions. Furthermore, the distribution of 32P among tryptic phosphopeptides of the enzyme, resolved by reversed-phase h.p.l.c., was not altered by cyclic AMP-increasing treatments of the acinar cells. When cytosol fractions were incubated with [gamma-32P]ATP, some phosphoproteins responded to the addition of micromolar concentrations of dibutyryl cyclic AMP or cyclic AMP by undergoing an enhancement of phosphate incorporation. In these experiments in vitro, protein phosphatase activity did not make a major contribution to the net phosphorylation of individual phosphoproteins, and acetyl-CoA carboxylase was not prominent among the phosphoproteins identified after short (less than 1 min) incubations of cytosols with [gamma-32P]ATP. The resistance of protein phosphorylation to variations in the cyclic AMP concentration in intact mammary epithelial cells, demonstrated by this work, is one of several mechanisms that ensure the pleiotropic refractoriness of those cells to agents which normally cause a stimulation of adenylate cyclase activity in hormone-sensitive cells.