Glycoscience Research Inc.

A safe, plentiful, source of GM1 ganglioside for therapeutic use for clinical trials in Huntingtonís disease (a fatal
progressive genetic neurologic disease) is needed. Prior development in the field was based on GM1 production from bovine brain collected at slaughter plants throughout Europe. In the 90ís Phase II clinical trials were in progress for spinal cord injury and Parkinsonís disease when Mad Cow disease (BSE) was diagnosed in cattle in England. With the discovery of BSE, raw material from non-source verified animals of any species could not be reliably used for ganglioside production for pharmaceutical use. GM1 ganglioside cannot be practically synthesized. Glycoscience Research Inc. has developed a unique genetic line of sheep that have been deemed an acceptable raw material source for GM1 ganglioside production for pharmaceutical use in a recent pre-Investigation New Drug review by the FDA.  While these sheep represent the only avenue for GM1 ganglioside production, the Pharmaceutical industry, venture capitalists and many in the National Institutes of Health are skeptical of the Sheep Industries ability to meet a demand for GM1 ganglioside. Therefore, the GM1 project has been unable to obtain the needed capital to complete the work required for an IND application.

Objective 1: Optimization of ovine raw material for GM1 production

Objective 2: Pre-clinical studies in transgenic HD mice

Efforts to bring ovine GM1 ganglioside to clinical trials is a collaborative effort with Dr. Steve Hersch, Mass. General Hospital, coordinating pre-clinical studies and clinical trial design, and Avanti Polar Lipids, Alabaster Alabama, coordinating GM1 manufacturing. Glycoscience Research Inc. is charged with expansion of a network of sheep producers to produce GM1 lambs to meet this pharmaceutical need. Objective 1 will include GM1 analysis of individual tissues to determine which tissues will be included in the final raw material. Also included in this objective is finalizing the cryo-grinding process that will be used in producing the final raw material. The second objective, based on recent responses from the FDA and NIH, focuses on collecting more preclinical data in order to be able to bridge the previously utilized bovine source of GM1 and the new ovine source. Although GM1 is a naturally occurring molecule in all mammals, FDA requires additional rodent experiments to bridge data from the bovine and ovine source. Previous experiments have shown that GM1 is able to reduce the toxic mutant Htt protein in transgenic mice by up to 50% when administered by intraperitoneal, subcutaneous and intranasal routes.


Progress Report:

Objective 2:  Preclinical studies of GM1 in transgenic HD mice

Huntington's disease (HD) is a neurodegenerative disorder characterized by progressive motor, psychiatric and cognitive decline. Currently there is no neuroprotective treatment available for this devastating and lethal disease. GM1 is a crucial neurotrophic brain ganglioside that was recently shown to be depleted in HD, to modulate huntingtin phosphorylation and clearance, to reduce brain levels of mutant huntingtin (mtHtt) when repleted, and to be highly neuroprotective. It is thus a leading candidate disease-modifying therapy for HD. However, there is no practical synthetic route for GM1, which also has poor oral bioavailability. GM1 was previously marketed as a parenteral therapeutic for other indications and it had been sourced from animal tissues, particularly from slaughterhouse bovine brain tissue. However safety concerns about the bovine sourcing and other factors led to its withdrawal from the market. We have developed an ovine source of GM1 based on a line of sheep with a point mutation deactivating GM1 metabolism and leading to its accumulation such that one lamb brain contains 40x the GM1 of a much larger cow brain.

 The overall goal of this study was to define the pharmacology of ovine GM1 by examining pk/PD markers in HD transgenic mice and define optimal doses and frequencies for human therapeutic testing.

 Experimental Plan: First, we tested ovine GM1 in R6/2 transgenic HD mice. The R6/2 mice express small N-terminal fragments (exon 1) of human huntintin and exhibit many progressive behavioral and neuropathological features observed in HD patients, including choreiform-like movements, involuntary stereotypic movements, tremor, weight loss and striatal cortical neurodegeneration. These mice have small life span of 3-4 months. Proof-of-concept studies in R6/2 mice have been the foundation of most human clinical trials testing medications being examined for their potential to slow the progression of HD.  GM1 was administered subcutaneously to 8 weeks old R6/2 HD mice at the dose rate of 50mg/kg twice daily for 5 days and the response of treatment on the mhtt levels was determined by FRET assay as described below. To ensure homogeneity of experimental cohorts, mice from the same F generation were systemically assigned to experimental groups such that age, weight, and CAG-repeat lengths are balanced. The mice were identified by the originally assigned code so that the subsequent studies were performed blind as to the genetic identity of the mice.

Measurement of soluble mhtt levels by FRET assay:  We have developed and validated a highly sensitive and quantitative assay for measuring soluble levels of mutant and total huntingtin in tissues and blood. The monoclonal antibodies used in this HTRF assay are specific for selected epitopes on the Htt molecule. The antibodies include: 2B7 monoclonal antibody (MAb) (Novartis, Switzerland) specific for the first N-17 amino acids (aa) of normal and mtHtt;11 MW1 MoAb15 (Developmental Studies Hybridoma Bank, University of Iowa) binds preferentially to expanded polyglutamine sequences (polyQ), hence binds to mtHtt and to a lesser extent to normal Htt;11 and 2166 MAb (Millipore Corp, Cat# MAB2166), which binds to the Htt epitope starting at aa 443-457 and recognizes both normal and mtHtt, hence tHtt16. In brief, brain tissue lysate is mixed with a reaction buffer containing fluorophore conjugated antibodies (2B7-Tb, MW1-Alexa488 and 2166-D2) and  the HTRF signal is read on VICTORX5 plate reader (Perkin Elmer) after 2 hrs of incubation at 40C. After excitation of the Tb donor at 320 nm, emission signals of Alexa488 and D2 is detected at 510 and 665 nm respectively. The signal resulted from the emission of the Tb is measured at 615 nm and is used for normalization of potential signal artifacts.  The relative mhtt and total htt concentration will be represented by the 510/615 nm and 665/615-nm ratios. The assay is performed and recorded according to a GLP compliant SOP.

 Results:   As anticipated, in our preliminary studies we found 33% reduction in mutant huntingtin levels with subcutaneously delivered ovine GM1.

 Ongoing studies/ Future plan: Currently, we are testing the Ovine GM1 in CAG 140 KI HD mice (n=5 each group). These knock-in mice express the full length human huntingtin gene, and have subtle behavioral and neuropathological phenotype with a normal lifespan and are more analogous to presymptomatic and early symptomatic disease in humans.  ICV administration of bovine GM1 in these mice has shown to improve the motor and cognitive function.   Similar to R6/2 study, GM1 was administered subcutaneously to 6 month old CAG 140 KI mice at the dose rate of 50mg/kg twice daily for 5 days and mice were sacrificed after 2 hrs of last injections; brains were removed and snap frozen in liquid nitrogen.   Analysis of these brain lysates for mutant huntingtin levels by FRET assay is in progress. 

 Based on results of these preliminary studies we will perform dose finding studies with higher and lower doses and will define the minimum effective dose and optimal dose by studying the dose response relationship. Time course pharmacodynamic studies will be performed to study the timing of the onset, duration and termination of effects of GM1 on mhtt levels. Establishing a relationship between the PD marker (mhtt levels) and GM1 exposure in preclinical models will be critical for successful translational of GM1 therapy to clinical studies.