Thyroid hormone beta receptor/(TR)-β

MB07811 is the liver-targeted prodrug of the novel TR agonist 3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl)-phenoxy methylphosphonic acid (MB07344) and has been shown to have oral cholesterol lowering activity in a variety of animal models (Erion et al., 2007). MB07344 exhibits a TRβ binding affinity Ki of 2.17 ± 0.03 nmol·L−1, and a Ki TRα/Ki TRβ ratio of 15.8, whereas MB07811 has low TR affinity (>12 µmol·L−1). We report that MB07811/MB07344 had adjunctive activity when given in combination with atorvastatin in all three species. These results are supportive of the potential for TR agonists, such as MB07811, to have clinical utility as a treatment to further lower cholesterol in those patients who do not successfully achieve cholesterol goals with statin treatment alone[1].

In this study, researchers evaluated the activity of a liver-targeted prodrug, MB07811, of a novel TH receptor beta agonist, MB07344, as monotherapy and in combination with atorvastatin in rabbits, dogs and monkeys.
Key results: In rabbits, MB07344 (i.v.) decreased total plasma cholesterol (TPC) comparable to that achieved with a maximally effective dose of atorvastatin (p.o.). The addition of MB07344 to atorvastatin resulted in a further decrease in TPC. Similarly, the addition of MB07811 (p.o.) to atorvastatin treatment decreased TPC beyond the level achieved with either agent as monotherapy. In dogs and monkeys, atorvastatin and MB07811 were administered as monotherapy or in combination. Consistent with the rabbit studies, the combination treatment caused a greater decrease in TPC than either MB07811 or atorvastatin administered as monotherapy.

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Activity of MB07344as monotherapy and in combination with atorvastatin in normal rabbits[1]
The objective of this rabbit study was to determine if MB07344 would have adjunctive activity to a maximally efficacious dose of atorvastatin. The four experimental groups (vehicle control, atorvastatin alone, MB07344 alone and MB07344 + atorvastatin combination) and timing of drug treatment are depicted in Figure 2A. As shown in Figure 2B, cholesterol levels in the model were quite stable as TPC in vehicle control animals remained within 10% of baseline levels for the duration of the 5 week protocol. As observed previously, animals treated with atorvastatin alone exhibited a significant (P < 0.05) and stable reduction in TPC of 30–35% compared with baseline levels. The animals in the combination group, prior to MB07344 administration (weeks 1–2), exhibited a similar decrease in cholesterol. Importantly, MB07344 treatment administered as an adjunct to atorvastatin (starting at week 2) in the combination group was associated with a further significant reduction in TPC to 55 ± 8% compared with baseline. As shown in Figure 2C, MB07344 treatment as monotherapy starting at week 2 in the MB07344-alone group resulted in a decrease of 33–36% compared with baseline, which was comparable to that achieved with atorvastatin alone (Figure 2B).

In Vitro Metabolism.[2]
MB07811 was efficiently converted to MB07344 and the glutathione conjugate 2 by liver microsomes prepared from male Sprague–Dawley (SD) rats. The Vmax, Km, and CLint (intrinsic clearance = Vmax/Km) values were 2.74 ± 0.12 nmol·min−1·mg−1, 18.8 ± 3.06 μM, and 145 ± 24.5 μl·min−1·mg−1, respectively. Conversion was inhibited by clotrimazole (100% at 1 μM, Ki = 24 nM), suggesting that CYP3A is the predominant CYP responsible for prodrug conversion. Neither MB07811 nor MB07344 inhibited CYP3A at 10 μM.

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LC-MS/MS analysis[1]
A 10 µL plasma extract aliquot was injected onto a Gemini C18 column (5 µm, 2 × 50 mm, Phenomenex) fitted with a Gemini C18 guard column (5 µm, 4.0 × 3.0 mm) and eluted with a gradient consisting of mobile phase A (20 mmol·L−1 N,N-dimethylhexylamine and 10 mmol·L−1 propionic acid in 20% methanol) and B (20 mmol·L−1 N,N-dimethylhexylamine and 10 mmol·L−1 propionic acid in 80% methanol) at a flow rate of 0.4 mL·min−1 (0 min, 60% B; 0–0.5 min, 60–100% B; 0.5–6 min, 100% B; 6–6.1 min, 100–60% B; 6.1–9 min, 60% B). The injector temperature was set at 10°C. Elution times for MB07344, MB07811, atorvastatin and hydroxyatorvastatin were approximately 2.7, 4.9, 2.3 and 2.2 min respectively. MB07811, MB07734, AT and hydroxyatorvastatin were detected using the MS/MS mode (513/63.1 for MB07811, 363.3/63.1 for MB07344, 557.5/278.4 for AT and 573.5/278.4 for hydroxyatorvastatin) and quantified by comparison of peak areas to standard curves obtained by adding known concentrations of the analytes to blank monkey plasma. Calibration curves ranging from 1 to 3000 ng·mL−1 for MB07344 and MB07811 (LOQ of 1 ng·mL−1) and from 0.1 to 3000 ng·mL−1 for atorvastatin (LOQ of 0.1 ng·mL−1) were generated. Although there is more than one possible isomer of hydroxyatorvastatin, this assay could not distinguish between them. As hydroxyatorvastatin standards were not available, plasma concentrations of this analyte were estimated using the atorvastatin calibration curve.

High intracellular levels of MB07344 were detected in freshly isolated rat hepatocytes incubated with MB07811 (Fig. 2A), indicating that MB07811 distributes readily into hepatocytes and is converted to MB07344 (Cmax = 1.03 ± 0.00 nmol per 106 cells; tmax = 1.5 h). Hepatocytes incubated with MB07344 also exhibited high intracellular MB07344 levels (1.77 ± 0.05 nmol per 106 cells; tmax = 1 h) (Fig. 2A), suggesting that MB07344, like other negatively charged phosphonates, may enter hepatocytes via organic anion transporters[2].

Activity of MB07344 as monotherapy and in combination with atorvastatin in normal rabbits[1]
\nThe focus of this experiment was to determine whether parenteral administration of the active TR agonist, MB07344, would have adjunctive activity to atorvastatin in the normal rabbit model. In this study, 32 rabbits (2.6–3.7 kg) were acclimatized for 29 days prior to entrance into the protocol. Baseline daily food intake was monitored and blood samples were collected via a superficial ear vein into potassium EDTA tubes, once on day −7, and again on day 0 to obtain two points for measurement of baseline TPC. Rabbits were randomized into four treatment groups (n = 8 per group) such that the average TPC were similar among the groups. The groups were non-drug control, atorvastatin alone, atorvastatin + MB07344, and MB07344 alone. The duration of the experiment was 5 weeks with blood samples taken weekly from each animal. Animals in the control group were maintained on the normal chow diet for the entire 5 week period with vehicle administration (saline) for the last 3 weeks on the same schedule as the MB07344 treated animals. Animals in the atorvastatin alone group were switched to the atorvastatin containing diet on day 0 and maintained on the diet for the entire 5 week period. These animals received vehicle for the last 3 weeks. Animals in the atorvastatin + MB07344 group were also maintained on the atorvastatin diet from day 0 for 5 weeks, but received MB07344 (0.05 mg·kg−1) via an ear vein 3 times per week for the last 3 weeks. Animals in the MB07344 alone group were maintained on the normal chow diet for the entire 5 week period but were treated with MB07344 (0.05 mg·kg−1) 3 times per week for the last 3 weeks. Body weight and food intake were monitored daily for all animals. There were no significant effects on either body weight or food intake (measured daily) among the experimental groups.\n

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\n\nAdjunctive activity of MB07811 in combination with atorvastatin in normocholesterolaemic rabbits[1]
\nTo evaluate the oral activity of MB07811 when administered as adjunctive treatment to atorvastatin, an experimental design similar to the MB07344 ± atorvastatin study described above was employed. The experimental groups (n = 6/group) were non-drug control, atorvastatin alone (3 mg·kg−1·day−1), MB07811 alone (10 mg·kg−1·day−1) and atorvastatin (3 mg·kg−1·day−1) + MB07811 (10 mg·kg−1·day−1). The duration of atorvastatin exposure prior to initiation of MB07811 treatment was increased to 3 weeks to ensure that TPC levels were stable prior to administration of MB07811. All other aspects of the protocol were identical to the MB07344 ± atorvastatin study. As with the first combination study, there were no significant effects on either body weight or food intake among the experimental groups.

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\n\nRat Pharmacokinetics.[2]
\nPharmacokinetic parameters for MB07344 (i.v.) and MB07811 (i.v. and per os) in male SD rats were determined from the temporal profile of MB07811 and MB07344 in plasma, using the methods described in SI Appendix. First-pass hepatic extraction (EH) was determined by measuring MB07811 levels after oral administration of MB07811 (3 mg/kg) to catheterized male SD rats (n = 4–5 per group) and by using the equation EH = (AUCpv − AUCsys)/AUCpv, wherein AUCpv and AUCsys represent AUC values derived from the portal vein (pv) and carotid artery (sys) plasma concentration-time profiles. Biliary excretion and enterohepatic recirculation were assessed by measuring MB07344 levels in plasma and bile collected from naïve and bile duct-cannulated SD rats (n = 3 per group) administered MB07344 (10 mg/kg, i.v.). The tissue distribution of MB07811 was evaluated in male SD rats (n = 4 per group) administered [14C]-MB07811 (5 mg/kg, per os). Tissues harvested after killing the animals 3 and 24 h after dosing were processed and analyzed directly by liquid scintillation counting. Mass balance studies were conducted by administration of [3H]-MB07344 [2 mg/kg, 45.5 mCi (1 Ci = 37 GBq)/mmol] or [14C]-MB07811 (5 mg/kg, 20.6 mCi/mmol) to male SD rats (n = 6) and monitoring mean cumulative radioactivity excreted in urine, wash, and feces at 0, 12, 24, 48, 72, and 96 h. Detailed procedures and additional results for the above studies are reported in SI Appendix.

Tables 1 and 2 list the mean pharmacokinetic parameters calculated from the individual concentration-time curves of each analyte (MB07811, MB07344, atorvastatin, and hydroxyatorvastatin). There were no significant differences in the pharmacokinetics of MB07811 or atorvastatin and their respective metabolites when used in combination compared with monotherapy. In addition, the AUC_last ratios of MB07344/MB07811 and hydroxyatorvastatin/atorvastatin observed after monotherapy (29 and 1.7, respectively) remained essentially unchanged in male and female animals after combination therapy (28 and 1.3, respectively). These data suggest that the major absorption, metabolism, and elimination pathways of MB07811 and atorvastatin are largely unaffected by the combination of the two drugs. [1] Pharmacokinetic studies. [2] Following intravenous injection, MB07344 showed moderate clearance in male SD rats (0.28 ± 0.00 L·kg⁻¹·h⁻¹, volume of distribution 0.39 ± 0.05 L/kg, plasma half-life 1.27 ± 0.26 h (Table 1)). In contrast, MB07811 (SD rats, intravenous injection) was rapidly cleared (11.6 ± 1.9 L·kg⁻¹·h⁻¹), with a volume of distribution of 14.8 ± 4.0 L/kg and a half-life of 1.23 ± 0.15 h. The relatively high oral clearance of MB07811 significantly improved its bioavailability (39%) compared to MB07344 (<1%, data not shown). After oral administration of MB07811, the first-pass extraction rate in the liver was significantly increased (≈55%; Fig. 2B and Supplementary Information (SI) Appendix Fig. 6). [2]
In SD rats, after oral administration of 5 mg/kg [14C]-MB07811, 28% and 93% of the radioactivity were excreted at 3 hours and 24 hours, respectively. Mass balance studies showed that MB07344 (2 mg/kg, intravenous bolus) was mainly excreted via the biliary system, with only 2% of the total radioactivity recovered in urine after 96 hours, while 98% was recovered in feces. Bile samples collected from rats after intravenous injection of MB07344 via bile duct cannulation showed that MB07344 was excreted via bile, with 50% of the dose excreted within 1 hour (Figure 2C). Furthermore, plasma MB07344 levels in bile-splitting rats (area under the curve (AUC0–24 h) = 48.1 ± 13.7 mg·h/L) were similar to those in normal rats (AUC0–24 h = 35.1 ± 8.5 mg·h/L), indicating that MB07344 was not affected by enterohepatic circulation (SI Appendix, Figure 7). [2] High-performance liquid chromatography (HPLC) separation of extractable radiometabolites showed that 63 ± 7% and 54 ± 5% of the radioactive material co-eluted in plasma and liver, respectively, 3 hours after oral administration of [14C]-MB07811. MB07811 levels in both tissues were below the limit of quantitation. The tissues with the highest radioactivity concentrations were the stomach, small intestine, large intestine, mesenteric lymph nodes, and liver. Concentrations were lower in the spleen, adrenal glands, kidneys, and heart (Figure 2D), while concentrations were extremely low in bone† and the other 16 tested tissues (<5% of liver concentration) (SI Appendix, Table 3). After 24 hours, the liver had the highest radioactivity concentration, followed by fat, pancreas, skin, and kidneys; all other non-gastrointestinal tissues had radioactivity concentrations <3% of liver concentration.

[1]. Thyroid hormone beta receptor activation has additive cholesterol lowering activity in combination with atorvastatin in rabbits, dogs and monkeys. Br J Pharmacol. 2009 Feb;156(3):454-65.
[2]. Targeting thyroid hormone receptor-beta agonists to the liver reduces cholesterol and triglycerides and improves the therapeutic index. Proc Natl Acad Sci U S A . 2007 Sep 25;104(39):15490-5.

Background and Objectives: Thyroid hormone receptor (TR) agonists are currently in clinical trials for the treatment of hypercholesterolemia. Since statins are the standard of care, any new therapy must provide adjunctive therapy when used in combination with statins. As is known in the mechanism of action of statins, the cholesterol-lowering effect of TR activation involves increased expression of the low-density lipoprotein receptor. We used animal models to test whether TR activation has additive cholesterol-lowering activity in the presence of an effective dose of statin. Methods: We evaluated the activity of MB07811, a liver-targeted prodrug of a novel thyroid hormone receptor beta agonist MB07344, in rabbits, dogs, and monkeys as a monotherapy and in combination with atorvastatin. Primary Results: In rabbits, intravenous administration of MB07344 reduced total plasma cholesterol (TPC) to a level comparable to the maximum effective oral dose of atorvastatin. Adding MB07344 to atorvastatin treatment further reduced TPC levels. Similarly, the addition of oral MB07811 to atorvastatin treatment reduced TPC to levels below those of monotherapy or monotherapy. In dogs and monkeys, atorvastatin and MB07811 were administered as monotherapy or in combination. Consistent with the results in rabbits, combination therapy reduced TPC more than monotherapy (MB07811 or atorvastatin). Conclusions and implications: We conclude that MB07811 and atorvastatin have an additive effect in reducing cholesterol in animals. These results will encourage and support the clinical demonstration of similar efficacy enhancements in combination therapy compared to monotherapy. [1]

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Despite efforts over the past four decades, the therapeutic potential of thyroid hormone receptor (TR) agonists as lipid-lowering and anti-obesity drugs in humans remains largely unexplored due to dose-limiting cardiac effects and their influence on the thyroid hormone axis (THA), muscle metabolism, and bone turnover. TR agonists that selectively act on TRβ subtypes exhibit some cardioprotective effects in rodents and primates, but they fail to lower blood lipids without inducing TRβ-mediated THA inhibition. In this article, we describe a cytochrome P450-activated phosphonate-containing TR agonist prodrug that exhibits higher TR activation in the liver compared to extrahepatic tissues and has a higher therapeutic index. Rat pharmacokinetic studies showed that the prodrug (2R,4S)-4-(3-chlorophenyl)-2-[(3,5-dimethyl-4-(4'-hydroxy-3'-isopropylbenzyl)phenoxy)methyl]-2-oxo-[1,3,2]-dioxaphosphonate (MB07811) underwent first-pass liver extraction and cleaved to generate the negatively charged TR agonist (3,5-dimethyl-4-(4'-hydroxy-3'-isopropylbenzyl)phenoxy)methylphosphonic acid (MB07344), which was poorly distributed in most tissues and rapidly eliminated from bile. The enhanced liver targeting of MB07811 was further confirmed by comparing its effects on the expression of TR agonist-responsive genes in the liver and six extrahepatic tissues with those of the 3,5,3'-triiodothyronine (T3) and non-hepatic-targeted thyroid hormone receptor (TR) agonist 3,5-dichloro-4-(4-hydroxy-3-isopropylphenoxy)phenylacetic acid (KB-141). In normal rats, MB07811 showed enhanced cardioprotective effects, reflecting the pharmacological effects of liver targeting; in diet-induced obese mice, unlike KB-141, MB07811 reduced cholesterol and serum and hepatic triglyceride levels at doses that did not affect body weight, blood glucose, or total hepatic artery (THA). These results suggest that targeting the liver with TR agonists has the potential to reduce cholesterol and triglyceride levels with good safety. [2]

C₁₉H₂₅O₅P

364.37

364.144

C, 62.63; H, 6.92; O, 21.95; P, 8.50

852947-39-8

15941848

Typically exists as solid at room temperature

4.237

3

5

6

25

451

0

P(COC1C=C(C)C(=C(C)C=1)CC1=CC=C(C(=C1)C(C)C)O)(=O)(O)O

SVXLLCKJKRYATC-UHFFFAOYSA-N

InChI=1S/C19H25O5P/c1-12(2)17-9-15(5-6-19(17)20)10-18-13(3)7-16(8-14(18)4)24-11-25(21,22)23/h5-9,12,20H,10-11H2,1-4H3,(H2,21,22,23)

[4-[(4-hydroxy-3-propan-2-ylphenyl)methyl]-3,5-dimethylphenoxy]methylphosphonic acid

852947-39-8; MB-07344; Phosphonic acid, p-((4-((4-hydroxy-3-(1-methylethyl)phenyl)methyl)-3,5-dimethylphenoxy)methyl)-; ((4-(4-Hydroxy-3-isopropylbenzyl)-3,5-dimethylphenoxy)methyl)phosphonic acid; UNII-R1ZW9H43ZJ; R1ZW9H43ZJ; MB07344; [4-[(4-hydroxy-3-propan-2-ylphenyl)methyl]-3,5-dimethylphenoxy]methylphosphonic acid;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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