Cysteine proteases; CANP; Cathepsin C

Loxistatin Acid (E-64C) normalizes the increased activities of cathepsin B and cathepsin H in the pectoral muscle of dystrophic chickens.[2] After the middle cerebral artery in rats is blocked, E-64C prevents the ischemic degradation of cerebral proteins.[3]

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E-64d, a membrane permeant derivative of E-64c, a thiol protease inhibitor (Tamai et al. (1986) J. Pharmacobio-Dyn. 9, 672-677), was tested for ability to inhibit calpain activity in intact platelets. Calpain activity was measured by proteolysis of actin-binding protein and talin, two known substrates of calpain. Incubation of platelets with E-64c/Loxistatin Acid (not permeant) or E-64d before lysis prevented proteolysis after lysis. When the platelets were incubated with E-64c/Loxistatin Acid or E-64d and then washed to remove the drugs before lysis, only E-64d inhibited proteolysis. When platelets were incubated with E-64c or E-64d and then activated with A23187 plus calcium, a treatment that activates intraplatelet calpain, only E-64d inhibited proteolysis. These results indicate that E-64d can enter the intact cell and inhibit calpain.[1]

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The DTT sensitivity of the Z-Val-Lys-Met-MCA cleaving activity suggested involvement of cysteine proteases that often require reducing conditions. The cysteine protease inhibitor Loxistatin Acid/E64c (10 μM) completely inhibited the chromaffin vesicle β-secretase activity; activity was also inhibited by cystatin C and p-hydroxymercuribenzoate (PHMB), which inhibit cysteine proteases. Phenylmethylsulfonyl fluoride (PMSF), a serine protease inhibitor, provided partial inhibition (Hook et al., 2002). Inhibition by chymostatin implicated cysteine or serine protease activity. Aspartyl and metalloprotease inhibitors had little effect. β-Secretase activity in chromaffin vesicles was not inhibited by a statine-substituted peptide inhibitor (Hook et al., 2002) that represents the Swedish mutant β-secretase cleavage site (Sinha et al., 1999), indicating that the chromaffin vesicle β-secretase activity possesses specificity for cleaving the wild-type β-secretase site that is expressed in the majority of AD patients (Price et al., 1998; Selkoe, 1998, 2001). Clearly, the prominent inhibition by E64c/Loxistatin Acid suggests a role for cysteine proteases for β-secretase activity in chromaffin vesicles.[4]

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Demonstration of Cysteine Proteases as β-Secretase Activity: Production of Aβ in Regulated Secretory Vesicles and Cleavage of Cellular APP [4]
The predicted role for cysteine protease activity for Aβ(1–40) production was further illustrated by E64c/Loxistatin Acid inhibition of Aβ(1–40) production in isolated, intact secretory vesicles (Fig. 5a). In addition, when chromaffin cells were treated with the cell-permeable analogue of E64c/Loxistatin Acid, known as E64d, production of the 12–14 kDa β-secretase product of APP was inhibited (Fig. 5b; Hook et al., 2002). These results imply a role for cysteine proteases in β-secretase processing of APP in neuronal chromaffin cells [4].

The total activities of cathepsin B and cathepsin H in pectoral muscle of dystrophic chickens (Line 413) were about two times higher than in control chickens (Line 412), and cathepsin D activity was about 3 times higher in this muscle in the dystrophic chickens. When E-64-c/Loxistatin Acid, a synthesized potent thiol inhibitor was injected subcutaneously, in various doses, daily for 80 days into dystrophic chickens (L 413), the activities of cathepsin B and cathepsin H were reduced to the levels in control chickens (Line 412), but cathepsin D activity, which is insensitive to Loxistatin Acid/E-64-c in vitro, was not changed. [2]

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Microtubule-associated protein 2 (MAP2) levels in the left cerebral hemisphere decreased significantly 3 days after occlusion of the left middle cerebral artery in rats to 29 +/- 16.3% of control levels. Since MAP2 is one of the substrates of calpain, E-64c/Loxistatin Acid, a synthetic calpain inhibitor, was administered at a dose of 400 mg/kg twice a day for 3 days, with the first dose being given before the production of ischemia. This depletion was significantly inhibited in vivo by E-64c (P less than 0.05) to increase MAP2 levels to 55 +/- 25.7% of control levels. E-64c/Loxistatin Acid had no significant effect on the ischemia-induced depletion of myelin-associated glycoprotein. Sham-operated rats were used as controls. Our results suggest that calpain is partially involved in the degradation of MAP2, and that the use of calpain inhibitors can be a useful clinical approach to cerebral ischemia. [3]

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Loxistatin Acid (E-64C), as a thiol protease inhibitor, inhibits calpain activity in intact platelets.[1] E-64C shows promise as a treatment for Alzheimer's disease because it reduces the production of Aβ and inhibits β-secretase activity in the regulated secretory vesicles of neuronal chromaffin cells.[4]

Lack of Detection of Cysteine Protease Activity During Purification of BACE 1 With Absence of Reducing Agent in β-Secretase Assay [4]
It is of interest that the purification of BACE 1 from human brain tissue utilized assays that did not include reducing conditions (Sinha et al., 1999). The absence of reducing agents in the assay explains how purification from brain tissue yielded BACE 1, whereas studies of secretory vesicles under reducing conditions detected cysteine protease activities as β-secretases (Hook et al., 2002). Interestingly, studies with BACE 1 knockout mice showed lowered β-secretase activity in neurons. However, those studies measured β-secretase activity in the absence of reducing conditions and in the presence of high amounts of the cysteine protease inhibitor Loxistatin Acid/E64c (Roberds et al., 2001). As a consequence, those studies could not have identified cysteine proteases.

Dogs: Research is conducted on 83 mongrel dogs weighing an average of 11.2 kg. Through intravenous sodium thiamylal (7 mg/kg), they are rendered unconscious. Group A (n = 17) receives an intravenous bolus of E-64c (100 mg/kg) dissolved in saturated sodium bicarbonate just prior to the occlusion and following reperfusion, while Group B (n = 17) only receives the vehicle solution during these periods. The LAD is permanently ligated at the same level in the 49 dogs that remain (Groups C and D). An intravenous bolus of either vehicle only (Group D; n = 25) or loxstatin acid (100 mg/kg) is administered just prior to and one hour following the ligation. The estimated intramyocardial Loxistatin acid molecular concentration is 1,000 times that of total mCANP, and the dose of E-64c is intended for potential use in clinical practice.

123664 Rat Intraperitoneal LD50 2140 mg/kg Behavior: somnolence (overall activity inhibition); Behavior: ataxia; Lung, chest or respiration: dyspnea. Pharmaceutical Research, 17(736), 1986
123664 Rat Intravenous LD50 >1 gm/kg. Pharmaceutical Research, 17(736), 1986
123664 Mouse Intraperitoneal LD50 2400 mg/kg Behavior: somnolence (overall activity inhibition); Behavior: ataxia; Lung, chest or respiration: dyspnea. Iyakuhin Kenkyu. Medical Supplies Research, 17(736), 1986
123664 Mouse Intravenous LD50 >1 gm/kg Iyakuhin Kenkyu. Medical Supplies Research, 17(736), 1986

[1]. Biochem Biophys Res Commun . 1989 Jan 31;158(2):432-5.

[2]. J Biochem . 1981 Sep;90(3):893-6.

[3]. Brain Res . 1990 Aug 27;526(1):177-9.

[4]. J Neurosci Res . 2003 Nov 1;74(3):393-405.

E-64c is a leucine derivative. This article focuses on the production and secretion of β-amyloid (Aβ) peptides in the regulatory secretory pathway and its relationship to the accumulation of toxic Aβ in Alzheimer's disease. New findings indicate that the majority of Aβ is produced and secreted in an activity-dependent manner via the regulatory secretory pathway in neurons. Only a small number of cells secrete Aβ through the basal, constitutive secretory pathway. Therefore, regulatory secretory vesicles contain the major β-secretases responsible for producing the majority of secreted Aβ. Studies of β-secretase activity in neuronal chromaffin cell regulatory secretory vesicles show that cysteine proteases account for the majority of β-secretase activity. BACE1 is present in regulatory secretory vesicles but provides only a small fraction of β-secretase activity. Furthermore, cysteine protease activity tends to cleave wild-type β-secretase sites, which are associated with most Alzheimer's disease (AD) cases. Conversely, BACE1 tends to cleave Swedish mutant β-secretase sites, which are expressed in a minority of AD patients. These new findings lead to a unified hypothesis: cysteine proteases are the major β-secretases that regulate Aβ production in the major secretory pathway, while BACE1 is the major β-secretase that regulates Aβ production in the minor constitutive secretory pathway. These results suggest that inhibiting multiple proteases may be a strategy to reduce Aβ production and treat Alzheimer's disease. [4]

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Evidence of cysteine proteases as β-secretases in regulating Aβ production in secretory vesicles (a) and neuronal chromaffin cells (b). a: Cysteine protease inhibitor E64c/loxistatin inhibits Aβ (1–40) production in chromaffin cell vesicles. Lysed chromaffin cell vesicles were incubated at 37°C (pH 5.5, adjusted with DTT) for different times with (solid dots) or without (hollow dots) cysteine protease inhibitor E64c (10 μM). The content of Aβ (1–40) at different incubation times was determined by radioimmunoassay. b: Cysteine protease inhibitors reduced APP-derived β-secretase products in chromaffin cells. 12–14 kDa 35S-COOH-terminal APP fragments produced by β-secretase were detected by immunoprecipitation and autoradiography on SDS-PAGE gels using anti-Aβ(1–40) antibody. Phorbol myristate (PMA) stimulated the production of 12–14 kDa COOH-terminal fragments compared to the unstimulated control group (lane 1) (lane 2). However, E64d, a cell-permeable analog of E64c/loxistatin, reduced the production of APP COOH-terminal products produced by β-secretase (lane 3). [4]

C15H26N2O5

314.38

314.184

C, 57.31; H, 8.34; N, 8.91; O, 25.45

76684-89-4

123664

White to off-white solid powder

1.2±0.1 g/cm3

596.4±50.0 °C at 760 mmHg

314.5±30.1 °C

0.0±3.6 mmHg at 25°C

1.504

1.36

3

5

9

22

422

3

O=C([C@H]1O[C@@H]1C(N[C@H](C(NCCC(C)C)=O)CC(C)C)=O)O

SCMSYZJDIQPSDI-SRVKXCTJSA-N

InChI=1S/C15H26N2O5/c1-8(2)5-6-16-13(18)10(7-9(3)4)17-14(19)11-12(22-11)15(20)21/h8-12H,5-7H2,1-4H3,(H,16,18)(H,17,19)(H,20,21)/t10-,11-,12-/m0/s1

(2S,3S)-3-[[(2S)-4-methyl-1-(3-methylbutylamino)-1-oxopentan-2-yl]carbamoyl]oxirane-2-carboxylic 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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.62 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.62 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (6.62 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: Saline: 2mg/mL

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