Calpain II (ID50 = 34 nM); Calpain I (ID50 = 40 nM); Calpain I (ID50 = 52 nM); Papainb (ID50 = 138 nM)

Calpeptin inhibits thrombin, ionomycin, or collagen-stimulated platelets' 20K phosphorylation in a dose-related manner.[1] By inhibiting Calpain activity, capeptin stimulates neurite elongation in PC12 cells that are differentiating.[2] Calpeptin functions as a functional neuroprotectant in rat retinal ganglion cells by reducing apoptosis and preserving the normal whole-cell membrane potential.[3]

In a feline right ventricular (RV) PO (RVPO) model, calpeptin (0.6 mg/kg, i.v.) blocks the activation of calpain and caspase-3, cleavage of their substrates, and cardiomyocyte programmed cell death. In a rat focal cerebral ischemia–reperfusion injury model, Calpeptin reduces the neuronal apoptosis in hippocampal CA1 sector via inhibition of the expression of Caspase-3.

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Calpain activation is linked to the cleavage of several cytoskeletal proteins and could be an important contributor to the loss of cardiomyocytes and contractile dysfunction during cardiac pressure overload (PO). Using a feline right ventricular (RV) PO model, we analyzed calpain activation during the early compensatory period of cardiac hypertrophy. Calpain enrichment and its increased activity with a reduced calpastatin level were observed in 24- to 48-h-PO myocardium, and these changes returned to basal level by 1 wk of PO. Histochemical studies in 24-h-PO myocardium revealed the presence of TdT-mediated dUTP nick-end label (TUNEL)-positive cardiomyocytes, which exhibited enrichment of calpain and gelsolin. Biochemical studies showed an increase in histone H2B phosphorylation and cytoskeletal binding and cleavage of gelsolin, which indicate programmed cardiomyocyte cell death. To test whether calpain inhibition could prevent these changes, we administered calpeptin (0.6 mg/kg iv) by bolus injections twice, 15 min before and 6 h after induction of 24-h PO. Calpeptin blocked the following PO-induced changes: calpain enrichment and activation, decreased calpastatin level, caspase-3 activation, enrichment and cleavage of gelsolin, TUNEL staining, and histone H2B phosphorylation. Although similar administration of a caspase inhibitor, N-benzoylcarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VD-fmk), blocked caspase-3 activation, it did not alleviate other aforementioned changes. These results indicate that biochemical markers of cardiomyocyte cell death, such as sarcomeric disarray, gelsolin cleavage, and TUNEL-positive nuclei, are mediated, at least in part, by calpain and that calpeptin may serve as a potential therapeutic agent to prevent cardiomyocyte loss and preserve myocardial structure and function during cardiac hypertrophy.[4]

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To demonstrate the protective effects of Calpeptin as the Calpain inhibitor against focal cerebral ischemia-reperfusion injury in rats and to explore it's possible mechanism. 96 rats were randomly divided into four groups. The model of middle cerebral artery occlusion was used for the research of focal cerebral ischemia. Using this animal model, the effects of Calpeptin on the neurological functions, infarction volume and infarction volume percentage of brain, Caspase-3 expression and neuronal apoptosis in hippocampal CA1 sector after focal cerebral ischemia-reperfusion injury in rats were investigated. The current results confirmed that Calpeptin as the Calpain inhibitor might paly an important role for neuroprotection against focal cerebral ischemia-reperfusion injury. Calpeptin could reduce the neuronal apoptosis in hippocampal CA1 sector when the rats was subjected to the focal cerebral ischemia-reperfusion, the potential mechanism might be related to the inhibition of the expression of Caspase-3 by Calpeptin. However, it is still unknown to what the exact mechanism of Calpeptin inhibits the activation of Caspase-3 in this process. Therefore, further research needs to be done to unravel the underlying mechanisms in the future [5].

Calpeptin (0.6 mg/kg, i.v.) inhibits the activation of calpain and caspase-3, cleavage of their substrates, and cardiomyocyte programmed cell death in a feline right ventricular (RV) PO (RVPO) model.[4] Through the inhibition of Caspase-3 expression, Calpeptin lowers the neuronal apoptosis in the hippocampal CA1 sector in a rat focal cerebral ischemia–reperfusion injury model.[5]

Calpeptin inhibited lung fibroblasts' ability to produce TGF-b1, IL-6, angiopoietin-1, and collagen. Calpeptin also inhibited the migration of cells that was dependent on angiopoietin-1 and the proliferation of cells that was dependent on IL-6. This may be the fundamental mechanism underlying Calpeptin's protective effect against pulmonary fibrosis.

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Apoptosis of retinal ganglion cells (RGCs) impairs vision in glaucoma patients. RGCs are also degenerated in multiple sclerosis (MS), resulting in loss of visual perception in MS patients. We examined the involvement of calpain and caspase cascades in apoptosis of the rat retinal ganglion cell line RGC-5 following 24 h of exposure to 250 nM ionomycin (IMN) or 300 units/ml interferon-gamma (IFN-gamma) and then evaluated functional neuroprotection with 2 microM calpeptin (CP, a calpain-specific inhibitor). Morphological and biochemical features of apoptosis were detected in RGC-5 cells following exposure to IMN or IFN-gamma. Fura-2 assay determined significant increases in intracellular free [Ca2+] following exposure to IMN or IFN-gamma. Pretreatment with CP for 1 h prevented Ca2+ influx, proteolytic activities, and apoptosis in RGC-5 cells. Western blot analyses showed an increase in activities of calpain and caspase-12, upregulation of Bax:Bcl-2 ratio, release of cytochrome c from mitochondria, and increase in caspase-9 and caspase-3 activities during apoptosis. Increased caspase-3 activity was also confirmed by a colorimetric assay. Activation of caspase-8 and cleavage of Bid to tBid in RGC-5 cells following exposure to IFN-gamma indicated co-operation between extrinsic and intrinsic pathways of apoptosis. Patch-clamp recordings showed that pretreatment with CP attenuated apoptosis and maintained normal whole-cell membrane potential, indicating functional neuroprotection. Taken together, our results demonstrated that Ca2+ overload could be responsible for activation of calpain and caspase cascades leading to apoptotic death of RGC-5 cells and CP provided functional neuroprotection [3].

C57BL/6 female mice (Eight-week-old)[3]
0.04 mg/mouse.
Intraperitoneally three times weekly for 28 days (together with Bleo).

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In vivo administration of drugs.[4]
Calpain and caspase inhibitor studies were performed in 24-h-PO cats. Calpeptin (25 mg) was dissolved in 1 ml of DMSO and further diluted in physiological saline (250 μg/ml). Z-VD-fmk was dissolved in 0.05 M Tris·HCl (pH 8.5, 10 mg/ml). The pH of the drug solutions was adjusted to 7.2 before they were administered. Each drug was given by bolus intravenous injections twice, 15 min before and 6 h after induction of PO. The initial and final doses of calpeptin were 0.6 mg/kg. The first dose of Z-VD-fmk was 20 mg/kg, and the subsequent dose was 10 mg/kg.

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Separating groups [5]
Ninety-Six healthy adult SD rats were randomly divided into four equal groups. MCAO Group (n = 24): reperfusion after occlusion of the left middle cerebral artery for 2 h; Calpeptin Group (n = 24): received intra-cerebroventricle injection of Calpain inhibitor Calpeptin with 50 μg (dissolved in 5 μl DMSO) before the left middle cerebral artery occluded for 30 min; DMSO Group (n = 24): received intra-cerebroventricle injection of dimethyl sulfoxide with 5 μl before the left middle cerebral artery occluded for 30 min; Sham Group (n = 24): didn’t insert occluded string when the animal model was performed, but other steps were similar to the experiment group. After 2 h of left middle cerebral artery occluded, following by 12, 24 or 48 h of reperfusion.

[1]. Biochem Biophys Res Commun . 1988 Jun 30;153(3):1201-8.

[2]. Neurosci Lett . 1994 Mar 28;170(1):91-3.

[3]. Brain Res . 2006 Apr 21;1084(1):146-57.

[4]. Am J Physiol Heart Circ Physiol . 2008 Jul;295(1):H314-26.

[5]. Mol Biol Rep . 2011 Feb;38(2):905-12.

Calpeptin is an amino acid amide.

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N-terminal of Leu-norleucinal or Leu-methioninal was modified to obtain a cell penetrative peptide inhibitor against calpain. Benzyloxycarbonyl (Z) derivatives had less active against papain than phenylbutyryl derivatives and leupeptin. Z-Leu-nLeu-H (calpeptin) was more sensitive to calpain I than Z-Leu-Met-H and leupeptin. Calpeptin was most potent among synthesized inhibitors in terms of preventing the Ca2+-ionophore induced degradation of actin binding protein and P235 in intact platelets. After 30 min incubation with intact platelets, calpeptin completely abolished calpain activity in platelets but no effect was observed in case of leupeptin. Calpeptin also inhibited 20K phosphorylation in platelets stimulated by thrombin, ionomycin or collagen. Thus calpeptin was found to be a useful cell-penetrative calpain inhibitor. [1]

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Calpain activity of nerve growth factor (NGF)-induced rat pheochromocytoma (PC12) cells shows a transient diminution in the early phase of differentiation. Calpain activity can be further decreased by a cell-permeable calpain inhibitor, calpeptin, which enhances the effect of NGF by stimulating neurite elongation. The number of neurites sprouted by one cell is not increased by calpeptin. A possible role of calpain inhibition during PC12 cells' early differentiation is discussed. [2]

C20H30N2O4

362.46

362.22

C, 66.27; H, 8.34; N, 7.73; O, 17.66

117591-20-5

73364

White to off-white solid powder

1.1±0.1 g/cm3

550.7±45.0 °C at 760 mmHg

60-75 °C

286.8±28.7 °C

0.0±1.5 mmHg at 25°C

1.508

4.37

2

4

12

26

434

2

O=C(OCC1=CC=CC=C1)N[C@H](C(N[C@H](C=O)CCCC)=O)CC(C)C

PGGUOGKHUUUWAF-ROUUACIJSA-N

InChI=1S/C20H30N2O4/c1-4-5-11-17(13-23)21-19(24)18(12-15(2)3)22-20(25)26-14-16-9-7-6-8-10-16/h6-10,13,15,17-18H,4-5,11-12,14H2,1-3H3,(H,21,24)(H,22,25)/t17-,18-/m0/s1

benzyl N-[(2S)-4-methyl-1-oxo-1-[[(2S)-1-oxohexan-2-yl]amino]pentan-2-yl]carbamate

Calpain; N-Cbz-leu-nleu-al; calpeptin; 117591-20-5; N-Cbz-leu-nleu-al; Benzylcarbonyl-leu-nleu-H; UNII-18X9FR245W; 18X9FR245W; N-Benzyloxycarbonyl-L-leucylnorleucinal; CHEMBL92708; Benzylcarbonyl-leu-nleu-H

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: ≥ 2.5 mg/mL (6.90 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 25.0 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: ≥ 2.5 mg/mL (6.90 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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