Liposomal nanocarriers for plasminogen activators.

Several plasminogen activators (PAs) have been found effective in treating different thromboembolic diseases. However, administration of conventional thrombolytic therapy is limited by a low efficacy of present formulations of PAs. Conventional treatments using these therapeutic proteins are associated with several limitations including rapid inactivation and clearance, short half-life, bleeding complications or non-specific tissue targeting. Liposome-based formulations of PAs such as streptokinase, tissue-plasminogen activator and urokinase have been developed to improve the therapeutic efficacy of these proteins. Resulting liposomal formulations were found to preserve the original activity of PAs, promote their selective delivery and improve thrombus targeting. Therapeutic potential of these liposome-based PAs has been demonstrated successfully in various pre-clinical models in vivo. Reductions in unwanted side effects (e.g., hemorrhage or immunogenicity) as well as enhancements of efficacy and safety were achieved in comparison to currently existing treatment options based on conventional formulations of PAs. This review summarizes present achievements in: (i) preparation of liposome-based formulations of various PAs, (ii) development of PEGylated and targeted liposomal PAs, (iii) physico-chemical characterization of these developed systems, and (iv) testing of their thrombolytic efficacy. We also look to the future and the imminent arrival of theranostic liposomal formulations to move this field forward.

Polyethylene glycol-derivatized cysteine-substitution variants of recombinant staphylokinase for single-bolus treatment of acute myocardial infarction.

BACKGROUND: Thrombolytic therapy of acute myocardial infarction (AMI) is evolving toward bolus administration. Derivatization of proteins with polyethylene glycol (PEG) may reduce their clearance. METHODS AND RESULTS: A staphylokinase (SakSTAR) variant with 12 amino acid substitutions to reduce its antigenicity, SakSTAR (K35A, E65Q, K74R, E80A, D82A, T90A, E99D, T101S, E108A, K109A, K130T, K135R), and with Ser in position 3 mutated into Cys (code SY161), was derivatized with maleimide-PEG with M:(r) of 5,000 (P5), 10,000 (P10), or 20,000 (P20). The PEGylated variants recognized only one third of the antibodies elicited with wild-type SakSTAR in AMI patients. In experimental animals, plasma clearances were reduced 2. 5- to 5-fold with P5, 5- to 20-fold with P10, and 20-fold with P20, and bolus injection induced pulmonary plasma clot lysis at doses inversely related to their clearance. Intravenous bolus injection of 5 mg of the P5, P10, or P20 variants in AMI patients was associated with plasma half-lives (t(1/2alpha)) of 13, 30, and 120 minutes and clearances of 75, 43, and 8 mL/min, respectively, compared with 3 minutes and 360 mL/min for SakSTAR. Injection of 5 mg P5 variant restored TIMI-3 flow within 60 minutes in 14 of 18 AMI patients (78%, 95% CI 55% to 91%) and of 2.5 mg in 7 of 11 patients (63%, 95% CI 35% to 85%), both in the absence of fibrinogen degradation. The immunogenicity of the variants was significantly (P:<0.002) reduced. CONCLUSIONS: The staphylokinase variant SY161-P5, derivatized with one linear polyethylene glycol molecule of M:(r) 5000, is a promising fibrin-selective agent for single-bolus coronary thrombolysis.

Botulinum toxin A and B: a comparative dosing study for spasmodic dysphonia.

OBJECTIVE: The purpose of this study was to find the conversion factor, safety, and efficacy of type A to type B toxin for laryngeal muscles. METHODS: Thirty-two patients with adductor spasmodic dysphonia with stable doses of A toxin to manage their symptom were given type B toxin starting at a conversion of 1 U of BTX-A to 50 U of BTX-B. The patients were followed for 1 year, and doses adjusted according to response. RESULTS: The conversion factor was found to be 52.3 U:1 U. The onset of action of type B was more rapid (2.09 days vs 3.2 days [P = 0.028]), with a shorter duration of benefit (10.8 weeks vs 17 weeks [P = 0.002). The safety profile for A and B toxin appeared the same, with 3 patients receiving Myobloc reporting dry mouth. CONCLUSION: This study shows that a conversion factor of 52.3:1 Myobloc (BTX-B) to Botox (BTX-A) and that Myobloc is an effective alternative to Botox (BTX-A) for patients with spasmodic dysphonia.

Clinical development of PEGylated recombinant staphylokinase (PEG-Sak) for bolus thrombolytic treatment of patients with acute myocardial infarction.

The development of bolus thrombolytic agents, in conjunction with bolus anti-thrombotics (e.g. low molecular weight heparins), remains an ambitious but achievable goal of therapy for acute myocardial infarction-a disease which takes the lives of millions each year. This chapter summarizes the data accumulated over nearly a decade of investigation of recombinant staphylokinase (Sak) as a safe, cost-effective thrombolytic agent. The results of extensive animal investigations suggested this agent exhibited a uniquely fibrin-selective mechanism of action. Administration of various recombinant versions of the molecule to over 1000 patients on a global scale suggest this agent may be as effective as tissue-type plasminogen activator (rt-PA) in achieving prompt arterial recanalization of acutely occluded coronary arteries. The development of this protein as a single bolus agent is described in detail, and the results of recently completed international trials comparing this bolus agent to front-loaded rt-PA are summarized.

Double-blind, randomized, placebo-controlled pilot study of the safety and efficacy of Myobloc (botulinum toxin type B) for the treatment of palmar hyperhidrosis.

BACKGROUND: Palmar hyperhidrosis is a problem of unknown etiology that affects patients both socially and professionally. Botulinum toxin type B (Myobloc), approved by the Food and Drug Administration for use in the treatment of cervical dystonia in the United States in December 2000, has subsequently been used effectively in an off-label indication to treat hyperhidrosis. There are sparse data, however, in the literature evaluating the safety and efficacy of BTX-B for the treatment of palmar hyperhidrosis. OBJECTIVE: We evaluated the safety and efficacy of Myobloc in the treatment of bilateral palmar hyperhidrosis. This was a double-blind, randomized, placebo-controlled study to report on the safety and efficacy of Myobloc. METHODS: Twenty participants (10 men, 10 women) diagnosed with palmar hyperhidrosis were injected with either Myobloc (5,000 U per palm) or a 1.0 mL vehicle (100 mM NaCl, 10 mM succinate, and 0.5 mg/mL human albumin) into bilateral palms (15 Myobloc, 5 placebo). The participants were followed until sweating returned to baseline levels. The main outcome measures were safety, efficacy versus placebo, and duration of effect. RESULTS: A significant difference was found in treatment response at day 30, as determined by participant assessments, between 15 participants injected with Myobloc and 3 participants injected with placebo. The duration of action, calculated in the 17 participants who received Myobloc injections and completed the study, ranged from 2.3 to 4.9 months, with a mean duration of 3.8 months. The single most reported adverse event was dry mouth or throat, which was reported by 18 of 20 participants. The adverse event profile also included indigestion or heartburn (60%), excessively dry hands (60%), muscle weakness (60%), and decreased grip strength (50%). CONCLUSION: Myobloc proved to be efficacious for the treatment of palmar hyperhidrosis. Myobloc had a rapid onset, with most participants responding within 1 week. The duration of action ranged from 2.3 to 4.9 months, with a mean of 3.8 months. The adverse event profile included dry mouth, indigestion or heartburn, excessively dry hands, muscle weakness, and decreased grip strength.

Compromised atrial coronary anatomy is associated with atrial arrhythmias and atrioventricular block complicating acute myocardial infarction.

BACKGROUND: The relevance of the atrial coronary anatomy in the pathogenesis of atrial arrhythmias and atrioventricular (AV) block complicating acute myocardial infarction (AMI) remains unclear. OBJECTIVES: We evaluated the location of the infarct-related coronary lesion relative to the principal atrial branches (ie, sinoatrial nodal, AV nodal, left atrial circumflex) in 454 patients with ST-elevation AMI in the CAPTORS II trial. METHODS: Patients underwent systematic 60-minute postfibrinolytic angiograms, and coronary anatomy was correlated with evidence of atrial arrhythmias and AV block on sequential electrocardiograms. RESULTS: Patients with either sinoatrial nodal or left atrial circumflex compromise (n = 34) had a higher incidence of "early" (ie, up to 90 minutes postfibrinolysis) atrial arrhythmias vs those without (23.5% vs 7.1%; P = .004). Patients with AV nodal compromise (n = 207) had a higher incidence of "early" AV block vs those without (12.1% vs 3.6%; P = .001). CONCLUSION: These findings support the etiological role of acute atrial ischemia in the development of early atrial arrhythmias and AV block complicating AMI.

Botulinum toxin type B in piriformis syndrome.

OBJECTIVE: To evaluate the clinical safety and efficacy of botulinum toxin type B (MYOBLOC) in reducing myofascial pain associated with piriformis syndrome. DESIGN: This was a single-center, outpatient, open-label study of patients with piriformis syndrome. Subjects were treated unilaterally or bilaterally, depending on their symptoms. Evaluations and procedures were performed by a single examiner who was not blinded, and there were no control subjects in this case series. Each piriformis muscle was infiltrated from one injection site under electromyographic guidance with 5000 units of botulinum toxin type B. RESULTS: A total of 20 patients were enrolled in this study. Significant reductions in mean visual analog scale scores for buttock and hip pain were noted at weeks 4, 12, and 16 and for low back pain at weeks 2, 12, and 16. Visual analog scale scores for general and low back pain, pain radiating into lower limbs, and tingling were significantly lower at week 2 after injection, suggesting early onset. A total of 95% of patients reported fair to excellent improvement in pain. Botulinum toxin type B was considered to have fair to excellent efficacy in 90% of patients, as evaluated by the investigator rating of overall efficacy. Botulinum toxin treatment was well tolerated. Dry mouth was the most common treatment emergent adverse event, reported in 6 of 20 patients. CONCLUSIONS: Findings suggest the possibility that botulinum toxin type B may be of potential benefit in the treatment of pain attributed to piriformis syndrome.

Mouse macrophage metalloelastase gene delivery by HVJ-cationic liposomes in experimental antiangiogenic gene therapy for murine CT-26 colon cancer.

We previously demonstrated that gene replacement of mouse macrophage metalloelastase (MME) into murine melanoma cells that grow rapidly and are MME deficient suppresses the primary tumor growth in vivo by halting angiogenesis. The aim of the present study was to evaluate the effectiveness of gene therapy against cancer using a cDNA-encoding MME gene. In a subcutaneous tumor model of CT-26 mouse colon cancer cells that are MME deficient, syngeneic mice repetitively treated with direct injections into the tumors of MME- hemagglutinating virus of Japan (HVJ), a type of HVJ-cationic liposome encapsulating a plasmid expressing MME, developed smaller tumors (210 +/- 47.2 mm(3) versus 925 +/- 156 mm(3) mean +/- SEM; p = 0.0004) with fewer microvessels (10.25 +/- 1.03 vs. 17.25 +/- 2.14; p = 0.03) than control mice. TUNEL staining revealed a significant increase of apoptotic cells in the MME-HVJ liposomes-treated tumors compared with control tumors. MME was effectively expressed in the s.c. tumors treated with MME-HVJ liposomes, inducing angiostatin generation in those tumors, as demonstrated by Western blot analysis. In conclusion, our study demonstrated that repeated in vivo transduction of the MME gene directly into the tumors using HVJ-cationic liposomes suppressed the tumor growth by an antiangiogenic mechanism, providing, then, a feasible strategy for gene therapy of cancer.

Pharmacokinetic and thrombolytic properties of cysteine-linked polyethylene glycol derivatives of staphylokinase.

Recombinant staphylokinase (SakSTAR) variants obtained by site-directed substitution with cysteine, in the core (lysine 96 [Lys96], Lys102, Lys109, and/or Lys135) or the NH(2)-terminal region that is released during activation of SakSTAR (serine 2 [Ser2] and/or Ser3), were derivatized with thiol-specific (ortho-pyridyl-disulfide or maleimide) polyethylene glycol (PEG) molecules with molecular weights of 5,000 (P5), 10,000 (P10), or 20,000 (P20). The specific activities and thrombolytic potencies in human plasma were unaltered for most variants derivatized with PEG (PEGylates), but maleimide PEG derivatives had a better temperature stability profile. In hamsters, SakSTAR was cleared at 2.2 mL/min; variants with 1 P5 molecule were cleared 2-to 5-fold; variants with 2 P5 or 1 P10 molecules were cleared 10-to 30-fold; and variants with 1 P20 molecule were cleared 35-fold slower. A bolus injection induced dose-related lysis of a plasma clot, fibrin labeled with 125 iodine ((125)I-fibrin plasma clot), and injected into the jugular vein. A 50% clot lysis at 90 minutes required 110 microg/kg SakSTAR; 50 to 110 microg/kg of core-substitution derivatives with 1 P5; 25 microg/kg for NH(2)-terminal derivatives with 1 P5; 5 to 25 microg/kg with derivatives with 2 P5 or 1 P10; and 7 microg/kg with P20 derivatives. Core substitution with 1 or 2 P5 molecules did not significantly reduce the immunogenicity of SakSTAR in rabbits. Derivatization of staphylokinase with a single PEG molecule allows controllable reduction of the clearance while maintaining thrombolytic potency at a reduced dose. This indicates that mono-PEGylated staphylokinase variants may be used for single intravenous bolus injection.