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BACKGROUND: The units of different preparations of botulinum neurotoxin type A (BoNT-A) have different potencies, and dosing recommendations for each product are not interchangeable. Historically, there has been debate concerning the dose-equivalence ratio that should be used in clinical practice. METHODS: Published evidence was considered to establish an appropriate dose-conversion ratio for the two main commercially available preparations of BoNT-A--Dysport (Dp) and Botox (Bx). RESULTS: Four key areas of evidence were identified: nonclinical and preclinical studies; studies exploring the diffusion characteristics and effects of complexing proteins; comparative experimental data from human studies; and clinical studies. Nonclinical data indicate that the principal reasons for differences in unit potency between the two products are dilution artefacts in the mouse assay. Use of saline as a diluent, at high dilutions, results in significant loss of potency in the Bx assay, whereas use of gelatin phosphate buffer in the Dp assay procedure protects the toxin during dilution. The published data on mouse assays show a Dp : Bx unit ratio range of 2.3-2.5 : 1 in saline and 1.8-3.2 : 1 in gelatin phosphate buffer. Data indicate that complexing proteins or size of the complex, which is highly pH sensitive, play no role in toxin diffusion and that Dp and Bx have similar diffusion characteristics when used at comparable doses. Randomized, controlled clinical studies indicate that 3 : 1 is more appropriate than 4 : 1, but the two products are not equivalent at this ratio. Comparative human experimental studies using the extensor digitorum brevis test, facial lines and anhidrotic action halo tests support dose-conversion ratios less than 3 : 1. LIMITATIONS: Data comparing dose equivalence ratios from the non-clinical setting should be extrapolated into the clinical setting with some caution. CONCLUSIONS: Dose-conversion ratios between Dp and Bx of 4 : 1 and greater are not supported by the recent literature. [ABSTRACT FROM AUTHOR]

Here, we describe molecular engineering of monovalent ultra-long acting two-chain insulin-Fc conjugates. Insulin-Fc conjugates were synthesized using trifunctional linkers with one amino reactive group for reaction with a lysine residue of insulin and two thiol reactive groups used for re-bridging of a disulfide bond within the Fc molecule. The ultra-long pharmacokinetic profile of the insulin-Fc conjugates was the result of concertedly slowing insulin receptor-mediated clearance by (1) introduction of amino acid substitutions that lowered the insulin receptor affinity and (2) conjugating insulin to the Fc element. Fc conjugation leads to recycling by the neonatal Fc receptor and increase in the molecular size, both contributing to the ultra-long pharmacokinetic and pharmacodynamic profiles.

Here, we describe the molecular engineering of insulin icodec to achieve a plasma half-life of 196 h in humans, suitable for once-weekly subcutaneously administration. Insulin icodec is based on re-engineering of the ultra-long oral basal insulin OI338 with a plasma half-life of 70 h in humans. This systematic re-engineering was accomplished by (1) further increasing the albumin binding by changing the fatty diacid from a 1,18-octadecanedioic acid (C18) to a 1,20-icosanedioic acid (C20) and (2) further reducing the insulin receptor affinity by the B16Tyr → His substitution. Insulin icodec was selected by screening for long intravenous plasma half-life in dogs while ensuring glucose-lowering potency following subcutaneous administration in rats. The ensuing structure-activity relationship resulted in insulin icodec. In phase-2 clinical trial, once-weekly insulin icodec provided safe and efficacious glycemic control comparable to once-daily insulin glargine in type 2 diabetes patients. The structure-activity relationship study leading to insulin icodec is presented here.

A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior. The tertiary structure was obtained using nuclear magnetic resonance and X-ray crystallography. The biological activity of HMWP was determined using 2 in vitro assays, and its influence on fibrillation was investigated using Thioflavin T assays. The dimer's tertiary structure was nearly identical to that of the noncovalent insulin dimer, and it was able to form hexamers in the presence of zinc. The dimer exhibited reduced propensity for self-association in the absence of zinc but significantly postponed the onset of fibrillation in insulin formulations. Consistent with its dimeric state, the tested species of HMWP showed little to no biological activity in the used assays. This study is the first detailed characterization of a specific type of human insulin HMWP formed during storage of a marketed pharmaceutical formulation. These results indicate that this specific type of HMWP is unlikely to antagonize the physical stability of the formulation, as HMWP retained a tertiary structure similar to the noncovalent dimer and participated in hexamer assembly in the presence of zinc. In addition, increasing amounts of HMWP reduce the rate of insulin fibrillation., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Hyperhidrosis affects 2.8% of the population and has severe negative influence on life quality. The disease is represented in many specialties but can unfortunately be incorrectly treated or not treated at all. Primary hyperhidrosis is the most common form. Secondary hyperhidrosis is most often excluded by a few anamnestic data. Botulinum toxin and anticholinergics are adequate treatment options when aluminium chloride is insufficient. This article describes the disease from the physician's as well as the patient's perspective. Furthermore, examination procedures and treatment procedures are presented.

Paediatric onset multiple sclerosis (POMS) is characterized by high inflammatory activity. No disease modifying treatment has been approved for POMS. The objective of this report was to report the use of rituximab, a B cell depleting monoclonal anti-CD20-antibody, in POMS. This is a retrospective case series at four specialized MS centres in Sweden. Participants were identified through the Swedish MS-registry and our own patient stocks. Data were collected through medical charts review. We identified 14 POMS patients treated with i.v. rituximab 500-1000 mg every 6th to 12th months. Median age at disease onset was 14.7 years, median age at rituximab treatment initiation was 16.5 years, and median treatment duration was 23.6 months. No relapses were reported, and the EDSS scores remained stable or decreased in 13 of 14 cases during rituximab treatment. Beyond 6 months from initiating rituximab treatment, only one new lesion was detected on MRI. No serious AEs were reported. The drug survival was 86%. Our data indicate that rituximab treatment is safe, effective and well tolerated in children with MS. Nine POMS cases treated with rituximab have previously been published. They had higher disease activity pre-rituximab, but similar safety and efficacy outcomes after treatment. An RCT of rituximab in POMS is warranted.

Purpose: To identify High Molecular Weight Products (HMWP) formed in human insulin formulation during storage., Methods: Commercial formulation of human insulin was stored at 37°C for 1 year and HMWP was isolated using preparative size exclusion chromatography (SEC) and reverse phase (RP) chromatography. The primary structure of the isolated species was analysed using liquid chromatography mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS). To test the hypothesis that amino groups of insulin are involved in HMWP formation, the HMWP content of various formulations spiked with amine compounds or formulations of insulin with modified amino groups was measured., Results: More than 20 species of HMWP were observed and 16 species were identified using LC-MS. All identified species were covalent dimers of human insulin linked via A21Asn and B29Lys, formed via the formation of an anhydride intermediate at A21Asn. Two types of HMWP were identified, with the covalent link in the open or closed (succinimidyl) form. Some species also contained single deamidation at B3 or the desPhe(B1)-N-oxalyl-Val(B2) modification. Reduced rate of HMWP formation was observed after addition of L-lysine, L-arginine or piperazine or when insulin analogues with methylated N-terminals and side chain amines and A21Gly mutation were used. Formulations of human insulin without zinc and m-cresol were found to contain a different pool of HMWP., Conclusions: HMWP formed in formulation of human insulin at pH 7.4 with zinc and m-cresol consists primarily of covalent dimers linked via A21Asn and B29Lys. Insulin formulation properties determine the amount and identity of formed HMWP.

Endogenous insulin secretion exposes the liver to three times higher insulin concentrations than the rest of the body. Because subcutaneous insulin delivery eliminates this gradient and is associated with metabolic abnormalities, functionally restoring the physiologic gradient may provide therapeutic benefits. The effects of recombinant human insulin (HI) delivered intraportally or peripherally were compared with an acylated insulin model compound (insulin-327) in dogs. During somatostatin and basal portal vein glucagon infusion, insulin was infused portally (PoHI; 1.8 pmol/kg/min; n = 7) or peripherally (PeHI; 1.8 pmol/kg/min; n = 8) and insulin-327 (Pe327; 7.2 pmol/kg/min; n = 5) was infused peripherally. Euglycemia was maintained by glucose infusion. While the effects on liver glucose metabolism were greatest in the PoHI and Pe327 groups, nonhepatic glucose uptake increased most in the PeHI group. Suppression of lipolysis was greater during PeHI than PoHI and was delayed in Pe327 infusion. Thus small increments in portal vein insulin have major consequences on the liver, with little effect on nonhepatic glucose metabolism, whereas insulin delivered peripherally cannot act on the liver without also affecting nonhepatic tissues. Pe327 functionally restored the physiologic portal-arterial gradient and thereby produced hepato-preferential effects., (© 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Objectives: The units of different botulinum toxin products are not identical, and the dose equivalence has been debated for several years. In the year 2000, our clinic changed the recommended botulinum toxin product from Botox to Dysport for the treatment of cervical dystonia. Based on published reports, where dose conversion ratios from 1:1 to 1:6 (Botox:Dysport) had been used, and our own clinical experience, the dose conversion ratio was set to 1:2. The objective of this study was to retrospectively monitor the used doses of each product and the subsequent clinical effect., Methods: A retrospective study, using casebook notes from 75 patients, was done to investigate treatment doses, subjective clinical effect, and the appearance of adverse events., Results: The median dose conversion ratio that had been used at the product switch was 1:2.3 (Botox:Dysport). After clinical adjustment, the ratio was 1:2.1 at the next 3 treatments. There was a tendency for a more effective treatment and more adverse events after the product switch. A follow-up was performed 6.5 years later using casebook notes from 53 of the same patients. By this time, the doses had been reduced, and the median dose conversion ratio had decreased to 1:1.7 (Botox:Dysport). The adverse events reported at this point were fewer for the patients treated., Conclusions: In this study, the most appropriate dose conversion ratio to use when switching from Botox to Dysport was 1:1.7.

Rationale: The collision cross-section (Ω) of a protein or protein complex ion can be measured using traveling-wave (T-wave) ion mobility (IM) mass spectrometry (MS) via calibration with compounds of known Ω. The T-wave Ω-values depend strongly on instrument parameters and calibrant selection. Optimization of instrument parameters and calibration standards are crucial for obtaining accurate T-wave Ω-values., Methods: Human insulin and the fast-acting insulin aspart under native-like conditions (ammonium acetate, physiological pH) were analyzed on Waters SYNAPT G1 and G2 HDMS instruments. The calibrated T-wave Ω-values of insulin monomer, dimer, and hexamer ions were measured using many different combinations of denatured and native-like calibrants (masses between 2.85 and 256 kDa) and T-wave conditions. Drift-tube Ω-values were obtained on a modified SYNAPT G1., Results: Insulin T-wave Ω-values were measured at 26 combinations of T-wave velocity and amplitude. Optimal sets of calibrants were identified that yield Ω-values with minimal dependence on T-wave conditions and calibration plots with high R(2)-values. The T-wave Ω-values determined under conditions satisfying these criteria had absolute errors <2%. Structural differences between human insulin and fast-acting insulin aspart were probed with IM-MS. Insulin aspart monomers have increased flexibility, while hexamers are more compact than human insulin., Conclusions: Accurate T-wave Ω-values that are indistinguishable from drift-tube values are obtained when using (1) native-like calibrants with masses that closely bracket that of the analyte, (2) T-wave velocities that maximize the R(2) of the calibration plot for those calibrants, and (3) at least three replicates at T-wave velocities that yield calibration plots with high R(2)., (Copyright © 2012 John Wiley & Sons, Ltd.)

Background: Treatment of palmar hyperhidrosis with botulinum toxin (BTX) requires effective anesthesia, but previous methods have not provided enough pain relief or have resulted in a prolonged impaired hand function., Objective: This is a study of bilateral forearm intravenous regional anesthesia using prilocaine for BTX treatment of palmar hyperhidrosis., Methods: In all, 166 patients (100 female and 66 male) were treated bilaterally with intracutaneous BTX type A injections using intravenous regional anesthesia with prilocaine (5 mg/mL). In a subgroup of patients, forearm nerves were studied with neurophysiologic methods and blood concentrations of prilocaine were measured. Pain evaluation with a visual analog scale was accompanied with a questionnaire about the treatment., Results: In all, 95% of the patients answering the questionnaire (response rate 89%) were satisfied with the anesthetic effect. No serious adverse events occurred. There was a fast recovery of motor function (in median 6 minutes) and sensory function (in median 20 minutes). No subclinical signs of sensory nerve damage were found., Limitations: Recall and reporting bias are potential sources of limitations in this study., Conclusion: Bilateral forearm intravenous regional anesthesia provides an effective and well-tolerated anesthesia during BTX treatment of palmar hyperhidrosis., (Copyright 2009 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.)

Through binding to and signaling via the insulin receptor (IR), insulin is involved in multiple effects on growth and metabolism. The current model for the insulin-IR binding process is one of a biphasic reaction. It is thought that the insulin peptide possesses two binding interfaces (sites 1 and 2), which allow it to bridge the two alpha-subunits of the insulin receptor during the biphasic binding reaction. The sequential order of the binding events involving sites 1 and 2, as well as the molecular interactions corresponding to the fast and slow binding events, is still unknown. In this study we examined the series of events that occur during the binding process with the help of three insulin analogues: insulin, an analogue mutated in site 2 (B17A insulin), and an analogue in which part of site 1 was deleted (Des A1-4 insulin), both with and without a fluorescent probe attached. The binding properties of these analogues were tested using two soluble Midi IR constructs representing the two naturally occurring isoforms of the IR, Midi IR-A and Midi IR-B. Our results showed that in the initial events leading to Midi IR-insulin complex formation, insulin site 2 binds to the IR in a very fast binding event. Subsequent to this initial fast phase, a slower rate-limiting phase occurs, consistent with a conformational change in the insulin-IR complex, which forms the final high-affinity complex. The terminal residues A1-A4 of the insulin A-chain are shown to be important for the slow binding phase, as insulin lacking these amino acids is unable to induce a conformational change of IR and has a severely impaired binding affinity. Moreover, differences in the second phase of the binding process involving insulin site 1 between the IR-A and IR-B isoforms suggest that the additional amino acids encoded by exon 11 in the IR-B isoform influence the binding process.

Botulinum toxin is used in various fields of medicine, including in the treatment of hyperhidrosis. Three products containing botulinum toxin are commercially available in Sweden; Botox, Dysport and Neurobloc. In the literature dose-response has varied with respect to these 3 products. We hypothesized that the dilution level of botulinum toxin is of importance for the effect and we therefore investigated anhidrosis after intradermal injections of each product in 3 different concentrations. Nine healthy subjects received 0.1 ml injections in the back. The anhidrotic areas were identified by an iodine-starch test after 3 weeks. When the 3 products were diluted to 100 U/ml level the achieved mean anhidrotic areas were approximately the same. This is in strong contrast with the large dose conversion factors suggested for intramuscular injections of the products. Furthermore, the lowest used concentrations for Botox(R) (25 U/ml) and Neurobloc (100 U/ml) led to the largest anhidrotic mean area per unit, respectively. The optimal concentration in this study was 25 U/ml for Botox, 100 U/ml for Dysport and 100 U/ml for Neurobloc, but for Botox and Neurobloc the optimal concentrations may be even lower.

Background: Intradermal botulinum toxin (Btx) produces long-lasting relief of focal hyperhidrosis, but its mechanism of action is poorly understood., Objective: To study the effect of Btx A on the size and innervation of sweat glands in patients with palmar hyperhidrosis., Methods: Palmar skin biopsy was performed in 26 hyperhidrotic patients before scheduled Btx treatment and in 11 controls. Twelve of the patients also underwent biopsy 1 to 6 months after the Btx injections. Sweat gland morphology was investigated by light microscopy; the cross-sectional area of the secretory tubule and its lumen was measured by image analysis. Immunofluorescence (IF) with antibodies to the neural markers protein gene product 9.5 (PGP 9.5) and growth-associated protein 43 (GAP 43), and to vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP), was used to analyze the periglandular innervation., Results: The gross morphology of the sweat glands was similar in patients and controls, with no significant differences in tubular and luminal areas between the groups. After Btx treatment, the tubular dimensions remained unchanged, but the lumen tended to be smaller ( P = .07). Around the glands, increased GAP 43 staining indicating sprouting was seen within 3 months after Btx treatment ( P = .016); whereas the PGP 9.5 staining was decreased in most specimens ( P = .09) indicating lack of functional nerve growth. No change in VIP or CGRP immunoreactivity was observed., Conclusions: The sweat glands appear structurally normal in hyperhidrotic patients before Btx therapy, whereas after therapy the luminal area of the gland is frequently diminished. The IF data GAP 43/PGP 9.5 suggest that Btx therapy induces long-standing functional denervation of the sweat glands, which might explain its anti-transpiratory efficacy.

Background: Botulinum toxin (Btx A) has recently been used in the treatment of focal hyperhidrosis. Hyperhidrosis is also an aggravating factor in nearly 40% of patients with dyshidrotic hand eczema., Objective: The objective of this study was to evaluate the effect of intradermal injections of Btx A on dermatitis in patients with vesicular hand dermatitis., Methods: Ten patients with vesicular dermatitis were treated on one hand with intradermal Btx A (mean, 162 U BOTOX, Allergan Pharmaceuticals, Irvine, Calif) with the untreated side as a control., Results: Self-assessment at follow-up 5 to 6 weeks after injection on a 5-point scale (none, slight, moderate, good, or very good effect) showed that 7 of 10 patients experienced good or very good effect. A decrease in itching was shown with a visual linear analogue scale (VAS) for itching, with mean 39% on the treated side compared with an increase by 52% on the untreated side. These findings were supported by the evaluation of clinical signs. Six of 7 patients who experienced good or very good effect also had aggravating hand sweating or worsening during the summer., Conclusion: Btx A can be a valuable alternative for patients with treatment-refractory hand eczema of the vesicular type, especially with hyperhidrosis or worsening during the summer.

The chloroplast Albino3 (Alb3) protein is a chloroplast homolog of the mitochondrial Oxa1p and YidC proteins of Escherichia coli, which are essential components for integrating membrane proteins. In vitro studies in vascular plants have revealed that Alb3 is required for the integration of the light-harvesting complex protein into the thylakoid membrane. Here, we show that the gene affected in the ac29 mutant of Chlamydomonas reinhardtii is Alb3.1. The availability of the ac29 mutant has allowed us to examine the function of Alb3.1 in vivo. The loss of Alb3.1 has two major effects. First, the amount of light-harvesting complex from photosystem II (LHCII) and photosystem I (LHCI) is reduced >10-fold, and total chlorophyll represents only 30% of wild-type levels. Second, the amount of photosystem II is diminished 2-fold in light-grown cells and nearly 10-fold in dark-grown cells. The accumulation of photosystem I, the cytochrome b(6)f complex, and ATP synthase is not affected in the ac29 mutant. Mild solubilization of thylakoid membranes reveals that Alb3 forms two distinct complexes, a lower molecular mass complex of a size similar to LHC and a high molecular mass complex. A homolog of Alb3.1, Alb3.2, is present in Chlamydomonas, with 37% sequence identity and 57% sequence similarity. Based on the phenotype of ac29, these two genes appear to have mostly nonredundant functions.