N-acetyl-L-leucine for Niemann-Pick type C: a multinational double-blind randomized placebo-controlled crossover study.

BACKGROUND: Niemann-Pick disease type C (NPC) is a rare autosomal recessive neurodegenerative lysosomal disease characterized by multiple symptoms such as progressive cerebellar ataxia and cognitive decline. The modified amino acid N-acetyl-leucine has been associated with positive symptomatic and neuroprotective, disease-modifying effects in various studies, including animal models of NPC, observational clinical case studies, and a multinational, rater-blinded phase IIb clinical trial. Here, we describe the development of a study protocol (Sponsor Code "IB1001-301") for the chronic treatment of symptoms in adult and pediatric patients with NPC. METHODS: This multinational double-blind randomized placebo-controlled crossover phase III study will enroll patients with a genetically confirmed diagnosis of NPC patients aged 4 years and older across 16 trial sites. Patients are assessed during a baseline period and then randomized (1:1) to one of two treatment sequences: IB1001 followed by placebo or vice versa. Each sequence consists of a 12-week treatment period. The primary efficacy endpoint is based on the Scale for the Assessment and Rating of Ataxia, and secondary outcomes include cerebellar functional rating scales, clinical global impression, and quality of life assessments. DISCUSSION: Pre-clinical as well as observational and phase IIb clinical trials have previously demonstrated that IB1001 rapidly improved symptoms, functioning, and quality of life for pediatric and adult NPC patients and is safe and well tolerated. In this placebo-controlled cross-over trial, the risk/benefit profile of IB1001 for NPC will be evaluated. It will also give information about the applicability of IB1001 as a therapeutic paradigm for other rare and common neurological disorders. TRIAL REGISTRATIONS: The trial (IB1001-301) has been registered at www. CLINICALTRIALS: gov (NCT05163288) and www.clinicaltrialsregister.eu (EudraCT: 2021-005356-10). Registered on 20 December 2021.

A master protocol to investigate a novel therapy acetyl-L-leucine for three ultra-rare neurodegenerative diseases: Niemann-Pick type C, the GM2 gangliosidoses, and ataxia telangiectasia.

BACKGROUND: The lack of approved treatments for the majority of rare diseases is reflective of the unique challenges of orphan drug development. Novel methodologies, including new functionally relevant endpoints, are needed to render the development process more feasible and appropriate for these rare populations and thereby expedite the approval of promising treatments to address patients' high unmet medical need. Here, we describe the development of an innovative master protocol and primary outcome assessment to investigate the modified amino acid N-acetyl-L-leucine (Sponsor Code: IB1001) in three separate, multinational, phase II trials for three ultra-rare, autosomal-recessive, neurodegenerative disorders: Niemann-Pick disease type C (NPC), GM2 gangliosidoses (Tay-Sachs and Sandhoff disease; "GM2"), and ataxia telangiectasia (A-T). METHODS/DESIGN: The innovative IB1001 master protocol and novel CI-CS primary endpoints were developed through a close collaboration between the Industry Sponsor, Key Opinion Leaders, representatives of the Patient Communities, and National Regulatory Authorities. As a result, the open-label, rater-blinded study design is considerate of the practical limitations of recruitment and retention of subjects in these ultra-orphan populations. The novel primary endpoint, the Clinical Impression of Change in Severity© (CI-CS), accommodates the heterogenous clinical presentation of NPC, GM2, and A-T: at screening, the principal investigator appoints for each patient a primary anchor test (either the 8-m walk test (8MWT) or 9-hole peg test of the dominant hand (9HPT-D)) based on his/her unique clinical symptoms. The anchor tests are videoed in a standardized manner at each visit to capture all aspects related to the patient's functional performance. The CI-CS assessment is ultimately performed by independent, blinded raters who compare videos of the primary anchor test from three periods: baseline, the end of treatment, and the end of a post-treatment washout. Blinded to the time point of each video, the raters make an objective comparison scored on a 7-point Likert scale of the change in the severity of the patient's neurological signs and symptoms from video A to video B. To investigate both the symptomatic and disease-modifying effects of treatment, N-acetyl-L-leucine is assessed during two treatment sequences: a 6-week parent study and 1-year extension phase. DISCUSSION: The novel CI-CS assessment, developed through a collaboration of all stakeholders, is advantageous in that it better ensures the primary endpoint is functionally relevant for each patient, is able to capture small but meaningful clinical changes critical to the patients' quality of life (fine-motor skills; gait), and blinds the primary outcome assessment. The results of these three trials will inform whether N-acetyl-L-leucine is an effective treatment for NPC, GM2, and A-T and can also serve as a new therapeutic paradigm for the development of future treatments for other orphan diseases. TRIAL REGISTRATION: The three trials (IB1001-201 for Niemann-Pick disease type C (NPC), IB1001-202 for GM2 gangliosidoses (Tay-Sachs and Sandhoff), IB1001-203 for ataxia telangiectasia (A-T)) have been registered at www.clinicaltrials.gov (NCT03759639; NCT03759665; NCT03759678), www.clinicaltrialsregister.eu (EudraCT: 2018-004331-71; 2018-004406-25; 2018-004407-39), and https://www.germanctr.de (DR KS-ID: DRKS00016567; DRKS00017539; DRKS00020511).

[The cellular factors of innate immunity in nonpsychotic patients at high risk for schizophrenia]. / Kletochnye faktory estestvennogo immuniteta u bol'nykh s nepsikhoticheskimi psikhicheskimi rasstroistvami, otrazhayushchimi vysokii risk razvitiya shizofrenii.

AIM: Changes in the parameters of innate immunity in patients with schizophrenia are observed already in the first episode. The study was performed to find out whether these changes take place prior to disease manifestation, and what role do they play in the pathogenesis of schizophrenia. MATERIAL AND METHODS: Thirty-five male nonpsychotic patients at high risk of psychosis, aged between 17 to 23 years, were examined. Phagocyte activity (PA) of neutrophils in the blood serum was evaluated by the number of active neutrophils, i.e. phagocytic index (PhI), and phagocytic number (PhN), which was determined by counting latex particles absorbed with a single phagocytic cell. Cytotoxic activity of natural killer lymphocytes (NK CA) was evaluated by the number of cell targets K-562, which remained non-degraded after the contact with natural killer cells. The influence of monocytes on NKCA was determined as well. RESULTS AND CONCLUSION: Compared to controls, patients had the lower PhI level (p<0.001) which was compensated by the increase in PhN levels, and the lower NK CA level which was increased due to the influence of monocytes. Negative correlations between PhI and PhN (r= -0.83, p<0.01) and between the level of NKCA and PhI (r= -0.83, p<0.05) as well as the positive correlation between PhN and SOPS scores (r=0.69, p<0.01) were found. After treatment, there was the decreasein the severity of mental disorders (p<0.001). The level of PhAN was normalized in 61.9% of patients compared to 36.7% before treatment. After treatment, the proportion of patients with normal levels of NK CA was the same as before treatment (40 and 35%, respectively). The immune disturbances revealed in the study may play a role in the pathogenesis of the disease and have predictive value for schizophrenia.

[The activity of complement system in children with autistic spectrum disorders]. / Aktivnost' sistemy komplementa u detei s rasstroistvami autisticheskogo spektra.

AIM: To evaluate the state of complement system (CS) activity in children with autistic spectrum disorders (ASD) and children with schizophrenia on the basis of development and implementation of a new method of CS determination. MATERIAL AND METHODS: A study included 249 patients, aged from 3 to 14 years. The control group consisted of 279 age-matched children. The authors developed a method for integral evaluation of CS activity based on the changes in the death of free swimming ciliata Tetrahymena pyriformis measured with the apparatus BioLat (Moscow, Russia). The integral CS activity (T50) was estimated as the time of death of 50% of ciliata in the blood serum (serum concentration was 5%). RESULTS AND CONCLUSION: A comparative analysis of CS activity showed statistical differences in median T50 values between patients and controls (p<0.005). Based on CS activity levels, three groups of patients were determined: 1) with the levels lower than the lowest value of the control group (n=112 (39%)); 2) higher than the highest level of the control group (n=103 (36%)); intermittent between low and high values of the control group (n=72 (25%)). Significant differences in T50 between the psychotic autism group and children schizophrenia group were identified (p<0.005). The CS activity was lower in patients with ASD compared to children with schizophrenia.

[Interaction effect of serotonin transporter gene and brain-derived neurotrophic factor on the platelet serotonin content in stroke patients].

Platelet serotonin content in patients in the acute period of stroke is an important index of clinical changes during the post stroke period as well as a predictor of development of mental disorders. We studied the association between two polymorphisms (5-HTTLPR and Val66Met BDNF) and the platelet serotonin content in 47 patients with stroke. We also investigated the moderating effect of genetic variants on the association between platelet serotonin content and development of affective and anxiety disorders in stroke patients in the acute period of stroke. The interaction effect of two polymorphisms on levels of platelet serotonin was found. The lowest level was observed in patients with the diplotype LL*ValVal, the highest level--in the group of patients with the LL genotype and genotypes containing at least one copy of a Met allele. No moderating effect of genetic variants on the relationship between serotonin content and affective or anxiety disorder was found.

Analysis of serotonin transporter in human platelets by immunoblotting using site-specific antibodies.

We have produced a panel of site-specific antibodies recognizing different regions of the human serotonin transporter (SERT). This panel included: 1) monoclonal antibodies 23C5 (mAbs 23C5) to the C-terminal region (amino acid residues 597-630); 2) polyclonal antibodies (pAbs) to the N-terminal region (amino acid residues 69-83); 3) pAbs to the region (amino acid residues 86-100) in the beginning of the first transmembrane domain (TMD). The antibodies were produced using recombinant proteins and synthetic peptides (containing certain sequences of SERT) as antigens. These antibodies were purified by affinity chromatography, conjugated to horseradish peroxidase (HRP), and used for immunoblotting analysis of SERT in extracts of human platelets. Sodium dodecyl sulfate extracts were prepared under conditions preventing non-specific proteolytic degradation of the proteins. In platelet extracts, all antibodies were able to detect the 67 kD protein, apparently corresponding to full-length SERT molecule (its theoretical mass is about 70 kD). These antibodies also detected several polypeptides of smaller size (56, 37, 35, 32, 22, and 14 kD), apparently corresponding to N-terminal, C-terminal, and non-terminal SERT fragments. Specificity of immunostaining was confirmed by preincubation of HRP-labeled anti-SERT antibodies with excess of corresponding antigen, which resulted in disappearance of protein band staining. It is suggested that SERT undergoes a programmed proteolytic cleavage (processing) resulting in formation of several SERT-derived polypeptides of smaller size. It is possible that one of the cleaved SERT species is required for serotonin transport activity. Possible sites for specific proteolysis may be located in the region near TMD1 and in the intracellular loop between TMD4 and TMD5.

The state of the serotonin transporter protein in the platelets of patients with somatoform [correction of somatiform] disorders.

The role of the serotonin transporter protein (STP) in the development of somatoform [corrected] disorders was addressed in a correlational study of the levels of immunoreactive STP (IR-STP) using site-specific antibodies against the least conserved (among a group of other cotransporters) epitope at the C-terminal of STP and the level of anxiety symptoms in patients with somatoform [corrected] disorders. A total of 22 patients were studied, with DSM-IV diagnoses of somatoform [corrected] disorders, along with 32 mentally healthy subjects of comparable age and sex. Immunoblotting of IR-STP from patients from healthy donors produced a diffuse band between 68 and 105 kDal and a clear narrow band at 43 kDal. The 43-kDal IR-STP protein was almost completely absent from most patients, as compared with the levels of this protein in healthy donors. This result suggests an abnormality of STP processing or, perhaps, alternative splicing of the gene encoding STP in patients with somatoform [corrected] disorders, and this appears to reflect the dysfunction in serotoninergic transmission in the CNS in these patients.

[The status of serotonin protein -- a serotonin transporter in thrombocytes in patients with somatoform disorders]. / Sostoiane belka -- perenoschika serotonina v trombotsitakh u bol'nykh s somatoformnymi zabolevaniiami.

The role of serotonin transporter (SERT) protein in the development of somatoform disorders (SD) was investigated. An association study was performed in terms of the evaluation of the level of SERT immunoreactive (IR-SERT) protein using site-specific antibodies directed at SERT C-terminus fragment, poorly conserved among the other cotransporters. The level of the anxious symptomatology was also estimated in the patients with SD. 22 patients, who met DSM-IV criteria for somatoform disorders, and 32 normals were examined. In platelets from normals, IR-SERT protein migrated as a difuse band between 68 and 105 kDa, and a major sharper band at 43 kDa. Almost complete disappearance of platelet 43 kDa IR-SERT protein band was observed in most of the patients with SD. These findings permitted to suggest a possibility of either biosynthetic or processing abnormality of SERTs in the affected population, that might reflect a dysfunction of serotonin neurotransmission in CNS of the patients with SD.

BIO-SPEAD: a parallel computing environment to accelerate development of biologic signal processing algorithms.

We have created BIO-SPEAD (pronounced speed), a BIOlogical Signal Processing Environment for Algorithm Development. BIO-SPEAD is designed to accelerate development of complex algorithms which integrate information derived from single or multiple physiologic waveforms. BIO-SPEAD currently performs all of the basic analyses of several arterial blood pressure waveforms, and allows the user to utilize the results of those low-level analyses for development of more complex algorithms. We utilized a parallel programming architecture called the Process Trellis which keeps the different tasks, or processes, within BIO-SPEAD independent of each other. Additionally, we have developed a graphics interface to enable the user to visualize the waveform under analysis, the low-level system analysis, and the internal workings of the algorithm under development. The system has been used for several algorithm development projects and has demonstrated its utility.

The multi-trellis software architecture and the intelligent cardiovascular monitor.

A real-time, intelligent cardiovascular monitor is complex. It must process multiple waveforms, recognize artifacts, extract pertinent parameters, recognize a patient's clinical state, analyze the problem and formulate a response. This paper presents the multi-trellis (a collection of process trellises), a software architecture for building such a monitor. A process trellis is a uniform hierarchical framework for heterogeneous program modules. The multi-trellis extension allows one to compile several process trellis programs with widely varying run-time requirements into a single executable program that it is efficient, predictable and usable. Our prototype consists of two process trellises. The lower trellis contains processes to analyze three different analog signals: the blood pressure from a non-invasive monitor and an arterial catheter, and the ECG. The upper trellis contains processes to help detect evolving hemodynamic trends, identify abnormalities, and present a succinct summary to the clinician. Our prototype shows that the multi-trellis is a demonstrably useful software architecture for building these real-time, intelligent monitors.

A parallel computing interface to facilitate development of physiologic signal processing algorithms.

We have created a system to aid in the development of algorithms related to the blood pressure waveform. The system performs all of the basic analyses of the waveform, and allows the user to utilize the results of those analyses for the algorithm under development. We have used a parallel programming architecture which keeps the different tasks, or processes, within the system independent of each other. Additionally, we have developed a graphics interface to enable the user to visualize the waveform, the system analysis, and the internal workings of the algorithm under development.

A parallel software architecture for building intelligent medical monitors.

Intensive care units become more complicated each day as the number of devices developed to monitor various aspects of a patient's status continues to increase. Intelligent monitors attempt to reduce this complexity by interpreting the data and presenting a high level summary to the clinician. We propose an innovative parallel software architecture for constructing intelligent medical monitors: the process trellis. The process trellis is an explicitly parallel structure, and therefore can take advantage of the performance gains available from parallel computing hardware. It does not, however, presuppose any expertise in parallel programming on the part of the application programmer. A prototype cardiovascular monitor has been built using this parallel software architecture. Preliminary testing of the monitor has shown that real-time cardiovascular monitoring, including data calculations, symbolic classification, and interpretation can be accomplished in real-time.

DYNASCENE: an approach to computer-based intelligent cardiovascular monitoring using sequential clinical "scenes".

Hemodynamic abnormalities such as hypovolemia typically progress through a sequence of discrete clinical phases or "scenes" (e.g., intravascular volume depletion, vasoconstriction, hypotension). Each scene can be defined by a cluster of hemodynamic trends. A natural approach to modeling the process of hemodynamic monitoring involves identifying these scenes and the temporal relationships among them. This approach has been utilized in the development of DYNASCENE, a parallel programming implementation of a computer-based intelligent hemodynamic monitor. This paper discusses: (1) The rationale for utilizing sequential clinical scenes to represent knowledge of hemodynamic behavior, (2) the design of the DYNASCENE system, and (3) preliminary tests of the DYNASCENE system.

Physiologic trend detection and artifact rejection: a parallel implementation of a multi-state Kalman filtering algorithm.

Using a parallel implementation of the multi-state Kalman filtering algorithm, we have developed an accurate method of reliably detecting and identifying trends, abrupt changes, and artifacts from multiple physiologic data streams in real-time. The Kalman filter algorithm was implemented within an innovative software architecture for parallel computation: a parallel process trellis. Examples, processed in real-time, of both simulated and actual data serve to illustrate the potential value of the Kalman filter as a tool in physiologic monitoring.

Does platelet serotonin receptor supersensitivity accompany endogenous depression?

In a population of drug-free bipolar and unipolar depressed women, it was found that the concentration of serotonin sufficient to induce half-maximal shape change velocity in platelets is significantly (p less than 0.001) lower (0.13 +/- 0.0.04 microM) than that of closely matched controls (0.532 +/- 0.1 microM). This platelet concentration becomes higher after 1-3 months of antidepressant treatment (0.47 +/- 0.16 microM). Possible mechanisms for this up- or down-regulation of platelet serotonin receptor responsiveness are discussed.

Change in Bmax and Kd for [3H]flunitrazepam observed in the course washing rat brain tissue with distilled water.

A method for the removal of an endogenous inhibitor(s) of benzodiazepine receptor binding (extraction in distilled water at 20 degrees) was used to evaluate the possible influence of this inhibitor(s) on the affinity of the benzodiazepine receptor in vivo. The results indicate that the presence of an inhibitor(s) in the concentration which exists in the rat brain leads to a 30-fold increase in the Kd value as compared with the results obtained in vitro. The endogenous inhibitor(s) appeared to be a thermostable, proteinase K-resistant substance(s) with Mr between 500 and 10,000 daltons. Repeated washings of the brain tissue with distilled water were accompanied by a decrease in the Bmax for [3H]flunitrazepam. The [3H]flunitrazepam binding sites with Kd = 1.7 nM and Bmax = 61 fmoles/mg of protein were found in the supernatant collected after the second and third washings in distilled water. The presence of diazepam and the benzodiazepine antagonist Ro 15-1788 prevented in a dose-dependent manner the decrease in the Bmax value for [3H]flunitrazepam during the tissue washing with distilled water.

Increase of benzodiazepine binding to the membranes isolated in the presence of diazepam.

The preparation of brain cells in the presence of diazepam leads to a 60-70% increase in benzodiazepine receptor density. Diazepam may promote the dissociation of endogenous inhibitors (modulators) and/or retard the dissociation of benzodiazepin receptors from the membrane to the medium. Routinely used procedures for tissue preparation reveal only part of the benzodiazepine receptors originally present in the tissue.

Immunological responsiveness of frozen-thawed human lymphocytes.

Mononuclear cells (10--20 X 10(6)) obtained from human peripheral blood by a standard Ficoll-Hypaque technique were suspended in RPMI 1640 media at 4 degrees C containing 10% foetal calf serum and 7-5% dimethyl sulphoxide (DMSO). Two-millilitre aliquots were cooled at -1 degree C/min in a Cryoson BV-4 programmed freezing system to -30 degrees C, then -5 degrees C/min to -80 degrees C and stored in liquid nitrogen vapor. On the day of testing, cell suspensions were thawed rapidly in a 37 degree C water bath. DMSO was diluted slowly out of the sample and cells resuspended in fresh RPMI 1640. It was found that frozen stored human lymphocytes (FSHL) demonstrated all the characteristics of fresh unfrozen cells. These included their ability to form spontaneous rosettes with sheep erythrocytes ('E' rosettes) and sheep erythrocyte--antibody--complement rosettes ('EAC' rosettes). The presence of surface immunoglobulins and Fc receptors were shown by membrane immunofluorescence to be comparable. In addition, the results show that FSHL respond to mitogens, specific antigens; act as both stimulators and responders in the mixed lymphocyte culture reaction; and exhibit cell-mediated lymphocytotoxicity following in vitro sensitization, or against antibody-coated target cells.

Inhibition of herpes simplex virus type 2 replication by thymidine.

Replication of herpes simplex virus type 2 (HSV-2) was impeded in KB cells which were blocked in their capacity to synthesize DNA by 2 mM thymidine (TdR). The degree of inhibition was dependent upon the concentration of TdR. In marked contrast, HSV-1 is able to replicate under these conditions. The failure of HSV-2 to replicate is probably due to the inhibition of viral DNA synthesis; there was a marked reduction in the rate of DNA synthesis as well as the total amount of HSV-2 DNA made in the presence of 2 mM TdR. We postulated that the effect of TdR on viral replication occurs at the level of ribonucleotide reductase in a manner similar to KB cells. However, unlike KB cells, an altered ribonucleotide reductase activity, highly resistant to thymidine triphosphate inhibition, was found in extracts of HSV-2-infected KB cells. This activity was present in HSV-2-infected cells incubated in the presence or absence of TdR. Ribonucleotide reductase activity in extracts of HSV-1-infected KB cells showed a similar resistance to thymidine triphosphate inhibition. These results suggest that the effect of TdR on HSV-2 replication occurs at a stage of DNA synthesis other than reduction of cytidine nucleotides to deoxycytidine nucleotides.