GABABR/GABAB receptor

Lactate dehydrogenase (LDH) activity was significantly reduced in striatal cells (HD19 or HD43) expressing wild-type or heterogeneous huntingtin after being exposed to 1, 10 μM of baclofen for 24 hours. This suggests that the cells were more viable. In HD43 cells, baclofen dramatically boosts both cell survival and chymotrypsin-like pancreatic body activity [3].

In YAC128 HD buttons, baclofen (i.p. ; 10 μg/g; twice daily for three days) improves motor deficits [3].

Proteasome activity determination [3]
Proteasome activity was determined by measuring the fluorescence of 7-amido-4-methylcoumarin (AMC; excitation 380 nm, emission 460 nm) generated from peptide-AMC linked substrates. Reactions were conducted in a final volume of 200 μl of buffer including 50 mM Tris–HCl (pH 7.5) and 1 mM EDTA. After adding samples to the reaction mixtures, reactions were initiated by adding the substrate Suc-Leu-Leu-Val-Try-AMC (65 μM) to measure chymotrypsin-like activity. Reactions progressed for 360 min at 25 °C. Enzymatic activity was expressed as fluorescence units (FU)/mg/min of protein.

Cell culture and in vitro baclofen treatment [3]
Tet-off inducible wild type (HD19, 26 CAG repeats) and mutant (HD43, 105 CAG repeats) striatal cells were cultured at 33 °C in Dulbecco’s Modified Eagle’s Medium (DMEM; Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (Hyclone), 2 mM l-glutamine (Sigma, St. Louis, MO), penicillin, and streptomycin. Expression of the huntingtin gene was induced by administration of doxycycline (1 μg/ml) for 24 h. The selective GABAB receptor agonist, baclofen (RS-baclofen; Tocris Cookson, Ellisville, MO), was administered to cultured cells at the indicated concentrations. After 24 h of administration, cell culture medium was collected for cell viability assays. Cells were harvested and lysed in a homogenization buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100) containing the following protease and phosphatase inhibitors: 10 μg/ml aprotinin, 25 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml phenylmethanesulfonyl fluoride, 50 mM sodium fluoride, 50 mM sodium orthovanadate) for protein sample preparation.
Cell viability determination[3]
We determined cell viability by performing a lactate dehydrogenase (LDH) assay on the collected cell medium, as per the manufacturer’s instructions (Roche, Mannheim, Germany). LDH activities of control or baclofen-treated striatal cells were measured by absorbance at 490 nm.

Animal/Disease Models: Wild type (WT) and mutant (MT) male YAC128 mice 13-18 months old [3]
Doses: 10 μg/g
Route of Administration: IP; twice (two times) daily at 9:00 AM and 5:00 PM , for 3 days; then a single dose was administered at 9:00 am on the fourth day.
Experimental Results: Motor deficits in YAC128 HD transgenic mice were improved. Increased proteasome activity and diminished neuronal intranuclear inclusions (NII) in YAC128 HD transgenic mice.

Absorption, Distribution and Excretion
Baclofen's oral bioavailability is 70% to 85%. After oral administration, it is rapidly absorbed through the gastrointestinal tract, reaching peak plasma concentrations 2 to 3 hours after ingestion. Peak effect is reached approximately 4 hours after intrathecal injection. Absorption is dose-dependent; higher doses result in greater absorption. Inter-individual absorption varies. Compared to fasting, oral administration of baclofen suspension with a high-fat meal reduces AUC by 9% and Cmax by 33%. Approximately 70-80% of baclofen is excreted unchanged via the kidneys within 72 hours of administration. Approximately 5% of the dose is excreted via the kidneys as metabolites. Inter-individual excretion varies. The volume of distribution of baclofen is 0.7 L/kg. Because baclofen is primarily soluble in water, it does not readily cross the blood-brain barrier. The concentration of baclofen in cerebrospinal fluid is approximately 1/8.5 of its plasma concentration.
After oral administration, the systemic clearance (CL/F) is 180 mL/min, and the renal clearance is 103 mL/min.
Metabolism/Metabolites

Approximately 15% of the oral dose is metabolized in the liver, primarily through deamination. Deamination produces the major metabolite β-(p-chlorophenyl)-4-hydroxybutyric acid, which has no pharmacological activity.
Approximately 15% of the dose is metabolized in the liver, primarily through deamination. 70-80% of the dose is excreted unchanged or as a metabolite in the urine, with the remainder excreted in the feces. Baclofen is readily absorbed from the gastrointestinal tract after oral administration. Baclofen appears in the bloodstream within half an hour after oral administration. It is evenly distributed in most organs and body tissues. After oral administration of baclofen, approximately 85% is excreted unchanged in the urine and feces, with the remainder being oxidized and denitrogenated in the liver to produce β-(p-chlorophenyl)-γ-hydroxybutyric acid as the major metabolite (L1322). Elimination pathway: In a study using radiolabeled baclofen, approximately 85% of the dose was excreted unchanged in urine and feces. Baclofen is primarily excreted unchanged via the kidneys; 70-80% of the dose is excreted unchanged in the urine. The remainder is excreted unchanged in feces or as metabolites in urine and feces. Half-life: 2.5-4 hours. The half-life after oral administration is 2-6 hours, and after intrathecal administration, it is 1-5 hours. The apparent elimination half-life of baclofen oral suspension or granules is approximately 5.6 hours.

Toxicity Summary
Baclofen is a direct agonist of the GABAB receptor. The exact mechanism of action of baclofen is not fully understood. It inhibits monosynaptic and polysynaptic reflexes at the spinal cord level, possibly through hyperpolarization of afferent nerve endings, although its effects may also be present at supra-spinal sites, potentially contributing to its clinical efficacy. Toxicity Data
LD50: 45 mg/kg (intravenous, mouse) (A308) LD50: 78 mg/kg (intravenous, rat) (A308)

[1]. A deeper insight into how GABA-B receptor agonism via baclofen may affect alcohol seeking and consumption: lessons learned from a human laboratory investigation. Mol Psychiatry. 2018 Oct 31.

[2]. Baclofen prevents MDMA-induced rise in core body temperature in rats. Drug Alcohol Depend, 2004. 74(1): p. 89-96.

[3]. Baclofen, a GABAB receptor agonist, enhances ubiquitin-proteasome system functioning and neuronal survival in Huntington's disease model mice. Biochem Biophys Res Commun. 2014 Jan 10;443(2):706-11.

Pharmacodynamics
Baclofen is an antispasmodic drug that induces muscle relaxation. It reduces the release of excitatory neurotransmitters in presynaptic neurons and stimulates inhibitory neural signals in postsynaptic neurons. Oral baclofen formulations are the most commonly used dosage form. In a cross-sectional study, intrathecal baclofen was more effective than oral baclofen in directly relieving spasticity at the spinal cord level. Baclofen has central nervous system depressant effects, causing sedation (with tolerance), somnolence, ataxia, and respiratory and cardiovascular depression. Baclofen also has some analgesic effects and can stimulate gastric acid secretion. Baclofen has anti-inflammatory and neuroprotective effects: it inhibits the release of pro-inflammatory cytokines from microglia and astrocytes and reduces oxidative stress levels in rats.

C10H12CLNO2

213.66

213.055

C, 56.21; H, 5.66; Cl, 16.59; N, 6.56; O, 14.98

1134-47-0

(R)-Baclofen;69308-37-8;Baclofen-d4;1189938-30-4;(R)-Baclofen hydrochloride;63701-55-3;Baclofen hydrochloride;28311-31-1

2284

White to off-white soild

1.3±0.1 g/cm3

364.3±32.0 °C at 760 mmHg

208-210°C

174.1±25.1 °C

0.0±0.9 mmHg at 25°C

1.577

1.56

2

3

4

14

191

0

O=C(O)CC(C1=CC=C(Cl)C=C1)CN

KPYSYYIEGFHWSV-UHFFFAOYSA-N

InChI=1S/C10H12ClNO2/c11-9-3-1-7(2-4-9)8(6-12)5-10(13)14/h1-4,8H,5-6,12H2,(H,13,14)

4-amino-3-(4-chlorophenyl)butanoic acid

Baclofen Lioresal, Liofen, Gablofen Baclon Kemstro

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~4.81 mg/mL (~22.51 mM)
H2O : ~2 mg/mL (~9.36 mM)
0.1 M HCL: ~10 mg/mL (~46.8 mM)

Solubility in Formulation 1: 2.5 mg/mL (11.70 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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Solubility in Formulation 2: ~2.5 mg/mL (11.7 mM) in PBS

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 (Please use freshly prepared in vivo formulations for optimal results.)