Plantilla de Especificación Lógica (LST)

One such drug cocktail under consideration for an ALS treatment is known as Endotherapia, a mixture of small molecules each conjugated to poly-L-lysine (PLL) (Geffard et al. 2010). The mixture of small molecules consists of fatty acids, amino acids, antioxidant compounds, and vitamins, thought to have neuroprotective, antioxidant, and immunomodulatory activity (Geffard et al. 2010). Additionally, while the role of chronic infection in neurodegeneration is controversial (Nicolson 2008), the fatty acids in Endotherapia may prevent bacterial adhesion to reduce chronic infections (Geffard et al. 2010). The multiple potential actions of Endotherapia could affect several pathways at once, and thus would have more chance of ameliorating complex neurodegenerative diseases. Endotherapia complexes have been tested in the neurodegenerative diseases multiple sclerosis (MS) and ALS, using an MS-tailored Endotherapia mixture of 19 compounds, named ‘GEMSP’ (Mangas et al. 2006), and an ALS-tailored Endotherapia mixture containing 25 compounds, named ‘Gemals’ (Nicaise et al. 2008); 12 compounds are common to both GEMSP and Gemals. The base components of Gemals, along with information on their functions, are listed in Table 3.1. The composition of formulated Gemals, where the components are linked to PLL in various combinations, is listed in Table 3.2.

______________________________________________________________________ 95 Table 3.1 Functional categories of Gemals components

Constituent Molecule type; function

Oleic acid Unsaturated fatty acid; antioxidant, anti-inflammatory Thioctic acid Dithiol; antioxidant

Myristic acid Saturated fatty acid Palmitic acid Saturated fatty acid

Lauric acid Saturated fatty acid; anti-inflammatory

Linoleic acid Unsaturated fatty acid; antioxidant, anti-inflammatory Palmitoleic acid Unsaturated fatty acid; anti-inflammatory

Caprylic acid Saturated fatty acid T-T-Farnesyl-L.Cysteine Amino acid derivative; Cholesterol Sterol; anti-inflammatory L. Cysteine Amino acid; antioxidant

Taurine Amino acid derivative; antioxidant, neuroprotective L. Methionine Amino acid; antioxidant, anti-inflammatory

L. Glutathione Tripeptide; antioxidant Alpha-tocopherol-succinate Vitamin E; antioxidant Ascorbic acid Vitamin C; antioxidant

Coenzyme Q10 Respiratory chain component; antioxidant, anti- inflammatory, neuroprotective

Retinoic acid Vitamin A derivative; antioxidant, anti-inflammatory, neuroprotection

Pantothenic acid Vitamin B5; antioxidant, anti-inflammatory, neuroprotection

Biotin Vitamin B7; essential coenzyme for carboxylases Uric acid Organic acid; antioxidant

Agmatine Amino acid derivative; antioxidant, anti-inflammatory, neuroprotective

Glucosamine Monosaccharide; antioxidant, anti-inflammatory, neuroprotective

______________________________________________________________________ 96 Table 3.2 Gemals components and their concentration in the current study

Constituent Concentration

Oleic acid – PLL – Thioctic acid 66µM

Oleic acid – PLL – Myristic acid 66µM

Oleic acid – PLL – Palmitic acid 66µM

Oleic acid – PLL – Lauric acid 66µM

Oleic acid – PLL – Linoleic acid 66µM

Oleic acid – PLL – Palmitoleic acid 66µM

Lauric acid – PLL – Caprylic acid 66µM

T-T-Farnesyl-L.Cysteine – PLL – Palmitic acid 66µM

Cholesterol – PLL – Oleic acid 66µM

L. Cysteine – RG – PLL 66µM L. Cysteine – GA – PLL 66µM Taurine – RG – PLL 66µM Taurine – GA – PLL 66µM L. Methionine – RG – PLL 66µM L. Methionine – GA – PLL 66µM L. Glutathione – RG – PLL 66µM Alpha-tocopherol-succinate – PLL 20µM Ascorbic acid – PLL 20µM

Oleic acid – PLL – Coenzyme Q10 20µM

Oleic acid – PLL – Retinoic acid 20µM

Pantothenic acid – PLL 20µM

Biotin – PLL 20µM

Uric acid – F – PLL 66µM

Agmatine – RG – PLL 66µM

Glucosamine – GA – PLL 66µM

PLL, conjugated to poly-L-lysine; RG, reduced glutaraldehyde linkage; GA, glutaric anhydride linkage; F, formaldehyde linkage.

______________________________________________________________________ 97 3.1.2.1 Endotherapia as a treatment for multiple sclerosis

The therapeutic effects of Endotherapia have been examined in a rat model of MS and in MS patients. The Endotherapia mixture, GEMSP, was able to reduce brain leukocyte infiltration across the blood-brain barrier (BBB) after induction of acute experimental autoimmune encephalomyelitis (EAE) in rats, but did not diminish the short-term neurological symptoms of acute EAE (Mangas et al. 2006). However, in a chronic EAE rat model, GEMSP treatment abolished clinical symptoms of nerve damage (Mangas et al. 2008), indicating that GEMSP is capable of modulating clinical outcomes in rats in a chronic EAE model of multiple sclerosis.

The use of GEMSP has also been extended beyond rat models of MS, with two open clinical trials conducted using Endotherapia compounds. In the first, small, open clinical trial, most MS patients treated with GEMSP for 6 months experienced a stabilisation (55% of patients) or an amelioration (18% of patients) of disease symptoms (Geffard et al. 2010). In the second, larger, open clinical trial, over two thirds of MS patients receiving an Endotherapia mixture of PLL-conjugated compounds (exact composition unknown, patent pending as of Geffard et al. 2010) showed positive outcomes, with 35% showing ameliorated disability scores, 17% showing stabilisation of disease, and 20% showing a lower rate of disease progression than the expanded disability status score (EDSS) worldwide reference rate (Geffard et al. 2010). Thus, Endotherapia compounds appear to show beneficial effects in slowing disease progression in MS patients, with the caveat that these studies did not use an untreated control group. As GEMSP and Endotherapia contain multiple compounds, it is unclear whether Endotherapia acts through immunomodulatory or direct neuroprotective mechanisms. 3.1.2.2 Endotherapia as a treatment for ALS

Endotherapia compounds have also been tested in a rat model of ALS and in ALS patients. An Endotherapia mixture of PLL-conjugated compounds known as Gemals (Table 3.2) delayed disease onset, delayed body weight decline, delayed the onset of limb paralysis and increased survival time when administered pre-symptomatically in a SOD1 rat model of ALS (Nicaise et al. 2008). Gemals-treated SOD1 rats also tended to maintain motor performance, maintain compound muscle action potentials, and showed lower spontaneous muscle activity than vehicle-treated SOD1 rats (Nicaise et al. 2008). However, the effect of Gemals when administration was started from the onset of disease symptoms has not been investigated.

______________________________________________________________________ 98 In addition, a Gemals-like Endotherapia compound (exact composition unknown, patent pending as of Geffard et al. 2010) was tested in a small, open clinical trial of 12 ALS patients (Geffard et al. 2010). Endotherapia-treated ALS patients showed a decreased loss of functional capacity on the ALS assessment questionnaire (ALSAQ-40) compared with the worldwide ALSAQ-40 reference score (Geffard et al. 2010). Thus, preliminary studies of Endotherapia suggest that the drug mixture is not detrimental in ALS patients and may even have beneficial effects – however, the absence of a control group from the study relies on the assumption that the worldwide ALSAQ-40 reference score has not changed over time. Given that these preliminary studies hint that Endotherapia may have some positive effects, a randomised, controlled trial would provide stronger evidence for a protective effect in ALS patients. Despite its evaluation in SOD1 rat models and in ALS patients, the Endotherpia mixture ‘Gemals’ is yet to be tested in the commonly-used SOD1 mouse model of ALS.

3.1.3 Aims and hypothesis

The aim of this chapter was to examine the ability of Gemals compound (Nicaise et al. 2008) to extend the survival of SOD1 mice when administered after symptom onset, compared against untreated control mice.

Hypothesis: Administration of Gemals to SOD1 mice after disease onset would slow disease progression and increase survival time.

A cohort of SOD1 mice were treated from 100 days of age with Gemals or a vehicle control, and were compared in terms of survival, body weight, rotarod performance and grip strength over time.

______________________________________________________________________ 99

3.2

Methods