Antibody mimetic drug conjugate manufactured by high-yield Escherichia coli expression and non-covalent binding system.

Antibody-drug conjugates (ADCs) are a major therapeutic tool for the treatment of advanced cancer. Malignant cells in advanced cancer often display multiple genetic mutations and become resistant to monotherapy. Therefore, a therapeutic regimen that simultaneously targets multiple molecules with multiple payloads is desirable. However, the development of ADCs is hampered by issues in biopharmaceutical manufacturing and the complexity of the conjugation process of low-molecular-weight payloads to biologicals. Here, we report antibody mimetic-drug conjugates (AMDCs) developed by exploiting the non-covalent binding property of payloads based on high-affinity binding of mutated streptavidin and modified iminobiotin. Miniprotein antibodies were fused to a low immunogenic streptavidin variant, which was then expressed in Escherichia coli inclusion bodies, solubilized, and refolded into functional tetramers. The AMDC developed against human epidermal growth factor receptor 2 (HER2) effectively killed cultured cancer cells using bis-iminobiotin conjugated to photo-activating silicon phthalocyanine. The HER2-targeting AMDC was also effective in vivo against a mouse KPL-4 xenograft model. This AMDC platform provides rapid, stable, and high-yield therapeutics against multiple targets.

The biotin interference within interference suppressed immunoassays.

BACKGROUND: Reports of false laboratory findings due to a biotin supplementation have raised concerns about the safety of immunoassays. According to current research, biotin is known to cause interference in immunoassays. Since up to 70% of medical decisions are based on laboratory results and the significantly increased intake of biotin supplements in the recent years, the reliability of immunoassays is essential. METHODS: To evaluate this reliability two experiments were conducted. In the first experiment 59 interference suppressed immunoassays of the manufacturer Roche Diagnostics were examined regarding their sensitivity to a biotin interference. In the second experiment the pharmacokinetic of biotin was examined by supplementing volunteers with biotin. RESULTS: A combination of the results of both experiments suggests that a biotin interference in laboratory findings is probable. Contrary to the current state of research on sandwich immunoassays, falsely elevated test results occur more frequently than falsely low results. CONCLUSION: The interference suppressed immunoassays have shown in the experiment that they are susceptible to a biotin interference. Therefore, laboratory institutions, medical staff and patients must be aware of the possibility of a biotin interference. As a result, Roche Diagnostics may consider reviewing the interference suppression and their indications of the tests.

Effects of magnesium biotinate supplementation on serum insulin, glucose and lipid parameters along with liver protein levels of lipid metabolism in rats.

The objective of this study was to investigate the effects of a novel form of biotin (magnesium biotinate) on serum glucose, lipid profile, and hepatic lipid metabolism-related protein levels in rats. Forty-two rats were divided into six groups and fed a standard diet-based egg white powdered diet supplemented with either d-biotin at 0.01, 1, or 100 mg/kg BW or magnesium biotinate at 0.01, 1, or 100 mg/kg BW for 35 days. Neither form of biotin influenced (p > 0.05) serum glucose or insulin concentrations. Serum total cholesterol and triglyceride decreased with biotin from both sources (p < 0.05). Concentrations were lower with magnesium biotinate when comparing the 1 mg/kg dose (p < 0.05). Serum, liver, and brain biotin and liver cyclic guanosine monophosphate (cGMP) concentrations were greater when rats were treated with magnesium biotinate versus d-biotin, particularly when comparing the 1 and 100 mg/kg dose groups (p < 0.05). Both biotin forms decreased the liver SREBP-1c and FAS and increased AMPK-α1, ACC-1, ACC-2, PCC, and MCC levels (p < 0.05). The magnitudes of responses were more emphasized with magnesium biotinate. Magnesium biotinate, compared with a commercial d-biotin, is more effective in reducing serum lipid concentrations and regulating protein levels of lipid metabolism-related biomarkers.

Toxicologic evaluation of a novel, highly soluble biotin salt, magnesium biotinate.

A novel, highly soluble biotin salt, magnesium biotinate (MgB), was assessed for general and genetic toxicity using several toxicologic tests. This battery of tests included in vitro bacterial reverse mutation test, in vitro mammalian micronucleus assay, and oral acute, 14-day, and 90-day repeat-dose toxicity in Sprague-Dawley (SD) rats. The results of the in vitro studies indicate that MgB is not mutagenic, clastogenic, or aneugenic. The acute oral toxicity study established an LD50 ≥ 5000 mg MgB/kg. In the 14-day oral toxicity study, doses of MgB up to 2500 mg MgB/kg/day produced no clinical signs or mortality. In the 90-day oral toxicity study, administration of 600 mg MgB/kg/day resulted in no clinical signs and was determined to be the no-observed-adverse-effect-level (NOAEL), which equates to 39 g biotin/day for a 70 kg human. Since MgB is composed of 93% biotin, the 600 mg NOAEL equates to approximately 1.3 million times the current recommended daily allowance of 30 µg biotin/day and 3900 times supplement levels of 10 mg biotin/day. Based on the toxicologic profile and lack of findings in various in vitro and in vivo studies, MgB may be considered safe for long-term human use.

Efficient induction of proximity-dependent labelling by biotin feeding in BMAL1-BioID knock-in mice.

Proximity-dependent biotin identification (BioID) is a useful method to identify unknown protein-protein interactions. Few reports have described genetically engineered knock-in mouse models for in vivo BioID. Thus, little is known about the proper method for biotin administration and which tissues are applicable. Here, we established a BioID knock-in mouse model of Brain and Muscle ARNT-Like 1 (BMAL1) and the BirA biotin ligase with R118G mutation (BirA*). The BMAL1-BioID mouse model was used to investigate the effect of biotin diet feeding on protein biotinylation in several tissues. The BMAL1-BirA* fusion protein-retained proper intracellular localization of BMAL1 and binding to CLOCK protein in HEK293T cells. A biotin labelling assay in mouse embryonic fibroblasts revealed the protein biotinylation activity of BMAL1-BirA* expressed in knock-in mouse cells depending on biotin supplementation. Lastly, feeding a 0.5% biotin diet for 7 days induced protein biotinylation in the brain, heart, testis and liver of BMAL1-BioID mice without adverse effects on spermatogenesis. In the kidney, the biotin diet increased biotinylated protein levels in BMAL1-BioID and control mice, suggesting the existence of endogenous biotinylation activity. These results provide valuable information to optimize the in vivo BioID procedure.

Amacrine Cells Forming Gap Junctions With Intrinsically Photosensitive Retinal Ganglion Cells: ipRGC Types, Neuromodulator Contents, and Connexin Isoform.

Purpose: Intrinsically photosensitive retinal ganglion cells (ipRGCs) signal not only centrally to non-image-forming visual centers of the brain but also intraretinally to amacrine interneurons through gap junction electrical coupling, potentially modulating image-forming retinal processing. We aimed to determine (1) which ipRGC types couple with amacrine cells, (2) the neuromodulator contents of ipRGC-coupled amacrine cells, and (3) whether connexin36 (Cx36) contributes to ipRGC-amacrine coupling. Methods: Gap junction-permeable Neurobiotin tracer was injected into green fluorescent protein (GFP)-labeled ipRGCs in Opn4Cre/+; Z/EG mice to stain coupled amacrine cells, and immunohistochemistry was performed to reveal the neuromodulator contents of the Neurobiotin-stained amacrine cells. We also created Opn4Cre/+; Cx36flox/flox; Z/EG mice to knock out Cx36 in GFP-labeled ipRGCs and looked for changes in the number of ipRGC-coupled amacrine cells. Results: Seventy-three percent of ipRGCs, including all six types (M1-M6), were tracer-coupled with amacrine somas 5.7 to 16.5 µm in diameter but not with ganglion cells. Ninety-two percent of the ipRGC-coupled somas were in the ganglion cell layer and the rest in the inner nuclear layer. Some ipRGC-coupled amacrine cells were found to accumulate serotonin or to contain nitric oxide synthase or neuropeptide Y. Knocking out Cx36 in M2 and M4 dramatically reduced the number of coupled somas. Conclusions: Heterologous gap junction coupling with amacrine cells is widespread across mouse ipRGC types. ipRGC-coupled amacrine cells probably comprise multiple morphologic types and use multiple neuromodulators, suggesting that gap junctional ipRGC-to-amacrine signaling likely exerts diverse modulatory effects on retinal physiology. ipRGC-amacrine coupling is mediated partly, but not solely, by Cx36.

Relapses in Patients Treated with High-Dose Biotin for Progressive Multiple Sclerosis.

High-dose biotin (HDB) is a therapy used in non-active progressive multiple sclerosis (PMS). Several reports have suggested that HDB treatment may be associated with an increased risk of relapse. We aimed to determine whether HDB increases the risk of clinical relapse in PMS and describe the characteristics of the patients who experience it. We conducted a French, multicenter, retrospective study, comparing a group of PMS patients treated with HDB to a matched control group. Poisson regression was applied to model the specific statistical distribution of the annualized relapse rate (ARR). A propensity score (PS), based on the inverse probability of treatment weighting (IPTW), was used to adjust for indication bias and included the following variables: gender, primary PMS or not, age, EDSS, time since the last relapse, and co-prescription of a DMT. Two thousand six hundred twenty-eight patients treated with HDB and 654 controls were analyzed with a follow-up of 17 ± 8 months. Among them, 148 validated relapses were observed in the group treated with biotin and 38 in the control group (p = 0.62). After adjustment based on the PS, the ARR was 0.044 ± 0.23 for the biotin-treated group and 0.028 ± 0.16 for the control group (p = 0.18). The more relapses there were before biotin, the higher the risk of relapse during treatment, independently from the use of HDB. While the number of relapses reported for patients with no previous inflammatory activity receiving biotin has gradually increased, the present retrospective study is adequately powered to exclude an elevated risk of relapse for patients with PMS treated with HDB.

Thiamine Treatment and Favorable Outcome in an Infant with Biallelic TPK1 Variants.

Episodic encephalopathy due to mutations in the thiamine pyrophosphokinase 1 (TPK1) gene is a rare autosomal recessive metabolic disorder. Patients reported so far have onset in early childhood of acute encephalopathic episodes, which result in a progressive neurologic dysfunction including ataxia, dystonia, and spasticity. Here, we report the case of an infant with TPK1 deficiency (compound heterozygosity for two previously described pathogenic variants) presenting with two encephalopathic episodes and clinical stabilization under oral thiamine and biotin supplementation. In contrast to other reported cases, our patient showed an almost normal psychomotor development, which might be due to an early diagnosis and subsequent therapy.

Mineral composition and microstructure of the abaxial hoof wall in dairy heifers after biotin supplementation.

The aim of this study was to evaluate the effect of daily biotin supplementation on the mineral composition and microstructure of the abaxial hoof wall in dairy heifers. The heifers were housed on a concrete floor and fed for weight gain more than 800 g per day, which is a challenging environment for the hoof. Twelve crossbred dairy heifers (Jersey × Holstein) were divided into two treatment groups. Animals in the control group (n = 6) received a diet without supplemental biotin, while the heifers in the biotin-supplemented feed group (n = 6) each received 20 mg of biotin daily for 120 days. Samples of the abaxial hoof wall were collected from the outer claw of the fore and hind limb, before and after supplementation. The samples were evaluated by X-ray fluorescence spectrometry, computed microtomography, atomic force microscopy and confocal laser scanning microscopy. Biotin supplementation increased the sulphur content and decreased the calcium and potassium content in the abaxial hoof wall. Biotin treatment also increased the percentage of horn tubules with smaller diameter marrow (17-51 µm). However, biotin did not influence the surface relief of the hoof wall, suggesting that its action is limited to the inner layers of the stratum corneum. Daily supplementation with 20 mg of biotin promoted changes in the mineral composition and microstructure of abaxial hoof wall of crossbred dairy heifers. These findings suggest biotin supplementation improves hoof quality and may help to understand the function of biotin in the stratum corneum.

Cell type-selective secretome profiling in vivo.

Secreted polypeptides are a fundamental axis of intercellular and endocrine communication. However, a global understanding of the composition and dynamics of cellular secretomes in intact mammalian organisms has been lacking. Here, we introduce a proximity biotinylation strategy that enables labeling, detection and enrichment of secreted polypeptides in a cell type-selective manner in mice. We generate a proteomic atlas of hepatocyte, myocyte, pericyte and myeloid cell secretomes by direct purification of biotinylated secreted proteins from blood plasma. Our secretome dataset validates known cell type-protein pairs, reveals secreted polypeptides that distinguish between cell types and identifies new cellular sources for classical plasma proteins. Lastly, we uncover a dynamic and previously undescribed nutrient-dependent reprogramming of the hepatocyte secretome characterized by the increased unconventional secretion of the cytosolic enzyme betaine-homocysteine S-methyltransferase (BHMT). This secretome profiling strategy enables dynamic and cell type-specific dissection of the plasma proteome and the secreted polypeptides that mediate intercellular signaling.

Safety and efficacy of MD1003 (high-dose biotin) in patients with progressive multiple sclerosis (SPI2): a randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: There is an unmet need to develop therapeutic interventions directed at the neurodegeneration that underlies progression in multiple sclerosis. High-dose, pharmaceutical-grade biotin (MD1003) might enhance neuronal and oligodendrocyte energetics, resulting in improved cell function, repair, or survival. The MS-SPI randomised, double-blind, placebo-controlled study found that MD1003 improved disability outcomes over 12 months in patients with progressive multiple sclerosis. The SPI2 study was designed to assess the safety and efficacy of MD1003 in progressive forms of multiple sclerosis in a larger, more representative patient cohort. METHODS: SPI2 was a randomised, double-blind, parallel-group, placebo-controlled trial done at 90 academic and community multiple sclerosis clinics across 13 countries. Patients were aged 18-65 years, had a diagnosis of primary or secondary progressive multiple sclerosis fulfilling the revised International Panel criteria and Lublin criteria, a Kurtzke pyramidal functional subscore of at least 2 (defined as minimal disability), an expanded disability status scale (EDSS) score of 3·5-6·5, a timed 25-foot walk (TW25) of less than 40 s, evidence of clinical disability progression, and no relapses in the 2 years before enrolment. Concomitant disease-modifying therapies were allowed. Patients were randomly assigned (1:1) by an independent statistician using an interactive web response system, with stratification by study site and disease history, to receive MD1003 (oral biotin 100 mg three times daily) or placebo. Participants, investigators, and assessors were masked to treatment assignment. The primary endpoint was a composite of the proportion of participants with confirmed improvement in EDSS or TW25 at month 12, confirmed at month 15, versus baseline. The primary endpoint was assessed in the intention-to-treat analysis set, after all participants completed the month 15 visit. Safety analyses included all participants who received at least one dose of MD1003. This trial is registered with ClinicalTrials.gov (NCT02936037) and the EudraCT database (2016-000700-29). FINDINGS: From Feb 22, 2017, to June 8, 2018, 642 participants were randomly assigned MD1003 (n=326) or placebo (n=316). The double-blind, placebo-controlled phase of the study ended when the primary endpoint for the last-entered participant was assessed on Nov 15, 2019. The mean time in the placebo-controlled phase was 20·1 months (SD 5·3; range 15-27). For the primary outcome, 39 (12%) of 326 patients in the MD1003 group compared with 29 (9%) of 316 in the placebo group improved at month 12, with confirmation at month 15 (odds ratio 1·35 [95% CI 0·81-2·26]). Treatment-emergent adverse events occurred in 277 (84%) of 331 participants in the MD1003 group and in 264 (85%) of 311 in the placebo group. 87 (26%) of 331 participants in the MD1003 group and 82 (26%) of 311 participants in the placebo group had at least one serious treatment-emergent adverse event. One (<1%) person died in the MD1003 group and there were no deaths in the placebo group. Despite use of mitigation strategies, MD1003 led to inaccurate laboratory results for tests using biotinylated antibodies. INTERPRETATION: This study showed that MD1003 did not significantly improve disability or walking speed in patients with progressive multiple sclerosis and thus, in addition to the potential of MD1003 for deleterious health consequences from interference of laboratory tests, MD1003 cannot be recommended for treatment of progressive multiple sclerosis. FUNDING: MedDay Pharmaceuticals.

Biotinidase deficiency characterized by skin and hair findings.

Biotinidase deficiency is a rare hereditary metabolic disease. Only a few cases have been reported in China, almost all of which have been in the pediatric population. We report a case of a girl with characteristic skin and hair findings with a negative family history, although her grandparents were consanguineous. The metabolites in the proband's blood and urine increased prominently, and the percentage of biotinase was 1.168%, much lower than normal. Genotyping identified two heterozygous mutations, which were C.1457T>A (p.L486Q) and C.1491dupT (p.L498Ffs*13) in the BTD gene. The diagnosis of biotinidase deficiency was established. No relevant reports about the missense mutation at the mutation site C.1457T>A (p.L486Q) of the BTD gene have been retrieved. Biotin replacement therapy was administered in the dose of 20 mg/d. The dermatitis subsided after 1 month, and the hair color was almost normal after 3 months. This reminds dermatologists to include biotinidase deficiency in their clinical differential when faced with children's intractable dermatitis, yellow hair, and alopecia.

Imaging Biotin Trafficking In Vivo with Positron Emission Tomography.

The water-soluble vitamin biotin is essential for cellular growth, development, and well-being, but its absorption, distribution, metabolism, and excretion are poorly understood. This paper describes the radiolabeling of biotin with the positron emission tomography (PET) radionuclide carbon-11 ([11C]biotin) to enable the quantitative study of biotin trafficking in vivo. We show that intravenously administered [11C]biotin is quickly distributed to the liver, kidneys, retina, heart, and brain in rodents-consistent with the known expression of the biotin transporter-and there is a surprising accumulation in the brown adipose tissue (BAT). Orally administered [11C]biotin was rapidly absorbed in the small intestine and swiftly distributed to the same organs. Preadministration of nonradioactive biotin inhibited organ uptake and increased excretion. [11C]Biotin PET imaging therefore provides a dynamic in vivo map of transporter-mediated biotin trafficking in healthy rodents. This technique will enable the exploration of biotin trafficking in humans and its use as a research tool for diagnostic imaging of obesity/diabetes, bacterial infection, and cancer.

Dietary Influence on Systolic and Diastolic Blood Pressure in the TwinsUK Cohort.

Nutrition plays a key role in blood pressure (BP) regulation. Here, we examine associations between nutrient intakes and BP in a large predominantly female population-based cohort. We assessed the correlation between 45 nutrients (from food frequency questionnaires) and systolic BP/diastolic BP (SBP/DBP) in 3889 individuals from TwinsUK not on hypertensive treatments and replicated in an independent subset of monozygotic twins discordant for nutrient intake (17-242 pairs). Results from both analyses were meta-analysed. For significant nutrients, we calculated heritability using structural equation modelling. We identified and replicated 15 nutrients associated with SBP, 9 also being associated with DBP, adjusting for covariates and multiple testing. 14 of those had a heritable component (h2: 27.1-57.6%). Strong associations with SBP were observed for riboflavin (Beta(SE) = -1.49(0.38), P = 1.00 × 10-4) and tryptophan (-0.31(0.01), P = 5 × 10-4), while with DBP for alcohol (0.05(0.07), P = 1.00 × 10-4) and lactose (-0.05(0.0), P = 1.3 × 10-3). Two multivariable nutrient scores, combining independently SBP/DBP-associated nutrients, explained 22% of the variance in SBP and 13.6% of the variance in DBP. Moreover, bivariate heritability analysis suggested that nutrients and BP share some genetic influences. We confirm current understanding and extend the panel of dietary nutrients implicated in BP regulation underscoring the value of nutrient focused dietary research in preventing and managing hypertension.

Relapses During High-Dose Biotin Treatment in Progressive Multiple Sclerosis: a Case-Crossover and Propensity Score-Adjusted Prospective Cohort.

High-dose biotin (HDB) is a therapy used in non-active progressive multiple sclerosis (PMS). Some reports have suggested that HDB treatment may be associated with an increased risk of relapse. We evaluate the relationship between exposure to HDB for treating PMS and the risk of relapse. We screened for PMS patients prospectively registered in a French regional cohort being part of the OFSEP national registry. In a case-crossover design among patients who received HDB, we first compared number of relapses before and after initiation of HDB. Second, time to the first clinical relapse was compared between patients who received HDB (biotin group) and a control group using a Cox survival analysis after a propensity score (PS) matching (1:1) and inverse probability of treatment weighting (IPTW) method. In the 42 PMS patients who received HDB, the number of relapses was statistically and clinically significant higher after biotin initiation than before biotin initiation (incident rate ratio [IRR] 7.4, 95% confidence interval [CI] 3.5-15.9, p < 0.0001). With the PS matching method, the risk of relapse was significantly higher in the biotin group compared to the control group (hazard ratio [HR] 4.3, 95% CI 1.4-13.3, p = 0.01). The IPTW method with 440 control patients revealed consistent results (HR 5.1, 95% CI 2.3-11.3, p < 0.0001). In our non-randomized study, HDB treatment for PMS was associated with an increased risk of relapse. The follow-up of PMS patients initiating HDB should include careful assessment of clinical and radiological activity to monitor the potential pro-inflammatory effect of biotin.

Heat Stress and Pulsed Unfocused Ultrasound: The Viability of these Physical Approaches for Drug Delivery into Testicular Seminiferous Tubules.

AIM: To investigate the application of Scrotal Heat Stress (SHS) and Pulsed Unfocused Ultrasound (PuFUS) to explore Blood-Testis Barrier (BTB) permeability in adult mice. BACKGROUND: The BTB provides a stable microenvironment and a unique immune barrier for spermatogenesis. Meanwhile, it blocks macromolecular substances access, including therapeutic agents and antibodies, thereby it decreases the therapeutic or immunocontraception effects. OBJECTIVES: To determine the viability of these physical approaches in delivering macromolecular substances into seminiferous tubules. MATERIALS & METHODS: Mice were subjected to receive single SHS intervention at 39°C, 41°C, or 43°C for 30 min. Whereas, mice received the PuFUS intervention at 1.75w/cm2, 1.25w/cm2, and 2.5w/cm2 for 2 min, 5 min, and 10 min, respectively. The Biotin and macromolecular substances (IgG, IgM, and exosomes) were separately injected into the testicular interstitium at different times following SHS or PuFUS interventions, to observe their penetration through BTB into seminiferous tubules. RESULTS: As detected by Biotin tracer, the BTB opening started from day-2 following the SHS and lasted for more than three days, whereas the BTB opening started from 1.5h following PuFUS and lasted up to 24h. Apparent penetration of IgG, IgM, and exosomes into seminiferous tubules was observed after five days of the SHS at 43°C, but none at 39°C, or any conditions tested with PuFUS. CONCLUSION: The current results indicate that SHS at 43°C comparatively has the potential for delivering macromolecular substances into seminiferous tubules, whereas the PuFUS could be a novel, quick, and mild approach to open the BTB. These strategies might be useful for targeted drug delivery into testicular seminiferous tubules. However, further studies are warranted to validate our findings.

Prevalence of detectable biotin in The Netherlands in relation to risk on immunoassay interference.

Despite the increasing awareness about biotin interference with immunoassays, so far, only two studies have quantified the prevalence of elevated biotin in patient populations. In a US study, over 7% had biotin concentrations exceeding 10 ng/mL, whereas in an Australian study only 0.8% of ED samples contained biotin exceeding 10 ng/mL. At present, representative data for the European population are lacking. In this study, we investigated biotin prevalence in The Netherlands in a representative cohort of routine laboratory requests in our laboratory using an LC-MS/MS assay for quantification of biotin in human plasma. In our study, we found 0.2% of samples exceeding 10 ng/mL of biotin, a finding more or less in line with the Australian data. Even though the biotin prevalence appears to be low, with concomitant low to moderate biotin concentrations, it is by no means a rare phenomenon. Laboratories like ours are likely to experience biotin positive samples on a daily basis with variable impact on patient care depending on the analytical bias from the immunoassay platform used. Our simple and robust LC-MS/MS assay for quantification of biotin in human samples may contribute to better understanding of the systemic concentrations seen after moderate- and high-dose biotin supplementation and the extent of immunoassay interference.