Calcineurin (CN)
(up to 3-fold greater inhibition of calcineurin activity compared to cyclosporine A) [1]
Calcineurin (CN) [2]

Voclosporin is the name of this novel semisynthetic calcineurin (CN) inhibitor (ISATX247). Using an in vitro calcineurin assay, the immunosuppressive effectiveness of vocasporin was investigated [1]. In vitro, the calcineurin inhibitor vocosporin (ISATX247) is more potent than cyclosporine [2].

---

In a human whole blood calcineurin inhibition assay, Voclosporin was significantly more potent than cyclosporine A. [1]
In vitro, Voclosporin exhibited up to a 3-fold greater inhibition of calcineurin activity compared to cyclosporine A. [1]

All of the animals showed good tolerance to cyclosporine A (CsA) and voclosporin (ISATX247). Serious adverse effects are absent from both drugs. During the trial, all but one animal in the Voclosporin group experienced diarrhea of varying length (mean 2.3 days, range 2 to 7 days). In contrast, no animals experienced diarrhea in the CsA or control groups. The Voclosporin group saw an average weight reduction of 3.4%, 2.0%, and 1.0% at the end of the research, respectively, which was slightly higher than that of the CsA and control groups [2].

---

In a rat heterotopic heart transplant model, at equivalent doses (1.75 mg/kg), Voclosporin prolonged graft survival 3-fold compared to cyclosporine A. [1]
In cynomolgus monkeys treated orally twice daily for 7 days with Voclosporin (25 or 50 mg/kg) or cyclosporine A (25 mg/kg): On Day 7 at 3 hours post-dose, Voclosporin inhibited lymphocyte proliferation significantly more (approximately 80-90% by ³H-thymidine incorporation and PCNA flow cytometry) than cyclosporine A (65-75% inhibition). At 14-hour trough (C_14hr), both drugs suppressed proliferation by about 50%. Voclosporin produced greater or similar inhibition of T-cell surface activation antigens (CD25, CD71, CD11a, CD95, CD154) compared to cyclosporine A, despite lower blood levels. For intracellular cytokine production (IL-2, IFN-γ, TNF-α), maximal inhibition was about 75%; at C_14hr on Day 7, cyclosporine A showed greater suppression of IL-2 and TNF-α than Voclosporin, but this difference was not present 24 hours later. [2]

Whole blood mitogen-stimulated lymphocyte proliferation and function assays were used, optimized for cynomolgus monkey blood. Lymphocyte proliferation was assessed by ³H-thymidine incorporation: 1:10 diluted whole blood was stimulated with concanavalin A (7.5 μg/ml) and incubated for 72 hours at 37°C in 5% CO₂, then ³H-thymidine was added and incorporation measured. Proliferation was also measured by flow cytometry for proliferating cell nuclear antigen (PCNA) expression in S/G₂M phase cells.

T-cell surface activation antigens (CD25, CD71, CD11a, CD95, CD154) were quantified by flow cytometry. Whole blood diluted 1:10 was stimulated with concanavalin A (7.5 μg/ml) for 72 hours. Cells were stained with fluorescently labeled monoclonal antibodies (anti-CD71-FITC + anti-CD3-PE + anti-CD25-PerCP-Cy5.5; anti-CD11a-FITC + anti-CD95-PE + anti-CD3-PerCP-Cy5.5; anti-CD3-FITC + anti-CD154-PE), then red blood cells were lysed, and leukocytes analyzed. The percentage of CD3+ lymphocytes expressing each antigen was calculated.
Intracellular T-cell cytokine production (IL-2, IFN-γ, TNF-α) was measured by flow cytometry. Undiluted whole blood was stimulated with PMA (30 ng/ml) and ionomycin (750 ng/ml) for 5 hours at 37°C in the presence of brefeldin A (10 μg/ml). Cells were fixed with formaldehyde, permeabilized with saponin, stained with anti-CD3-PerCP-Cy5.5 and anti-cytokine-FITC/PE antibodies, and analyzed. The percentage of CD3+ cells positive for each cytokine was determined. [2]

For the non-human primate study: Male cynomolgus monkeys (Macaca fascicularis, 5-8 kg) were treated orally twice daily (8:00 AM and 6:00 PM) for 7 days. Voclosporin was administered at 25 mg/kg/dose (n=6) or 50 mg/kg/dose (n=6) using a liquid formulation containing D-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS), medium-chain triglyceride (MCT) oil, ethanol and Tween-40. Cyclosporine A (Neoral microemulsion, 25 mg/kg/dose, n=5) and vehicle (1 ml/kg, n=4) served as controls. The calculated volume was drawn into a syringe and administered directly into the stomach through an orogastric tube, followed by 20 ml lukewarm water rinse. Blood was collected from the femoral vein under ketamine sedation. Pharmacokinetic blood was collected daily before the morning dose (14-hour trough), and on Day 7 at 3 and 10 hours post-dose. Pharmacodynamic blood was collected on Day 7 before the morning dose, and at 3 and 24 hours post-dose. Hemogram and serum chemistry were performed before the first dose and on Day 7. [2]
For the rat heterotopic heart transplant model (from [1]): Lewis rats (n=8/group) were treated daily with oral Voclosporin (1.75 mg/kg) or cyclosporine A (1.75 mg/kg). Graft survival was monitored. No further details on formulation or procedure were provided. [1]

Absorption, Distribution and Excretion
When taken on an empty stomach, the median time to peak concentration (Tmax) of vorticol is 1.5 hours, but the range is 1–4 hours. The estimated AUC is 7693.6 ng/mLh, and the estimated Cmax is 955.5 ng/mL. Vorticol is primarily excreted in urine and feces, with approximately 88% detected in feces and approximately 2% in urine. The apparent volume of distribution of vorticol is 2154 L. Vorticol is widely distributed in erythrocytes; its distribution in whole blood and plasma depends on concentration and temperature. The mean apparent steady-state clearance of vorticol is 63.6 L/h. Hepatic and renal impairment significantly reduces the clearance of vorticol. Metabolism/Metabolites Vorticol is primarily metabolized by hepatic cytochrome CYP3A4. Its pharmacological activity is primarily attributed to the parent molecule. A major metabolite was detected in human whole blood, accounting for 16.7% of total exposure; the potency of this metabolite is approximately 1/8 that of the parent drug, vorticol.

---

Biological Half-Life
The mean terminal half-life of vorticol is approximately 30 hours (24.9 to 36.5 hours).

---

In cynomolgus monkeys after twice-daily oral dosing for 7 days: For Voclosporin 25 mg/kg: C_14hr (trough) = 68 ± 7 ng/ml; C_3hr (peak) = 303 ± 94 ng/ml; AUC_0-10hr = 1,979 ± 759 ng·h/ml. For Voclosporin 50 mg/kg: C_14hr = 69 ± 4 ng/ml; C_3hr = 375 ± 62 ng/ml; AUC_0-10hr = 2,496 ± 614 ng·h/ml. These values were significantly lower than those for cyclosporine A 25 mg/kg (C_14hr = 368 ± 21 ng/ml; C_3hr = 877 ± 202 ng/ml; AUC_0-10hr = 6,262 ± 1,340 ng·h/ml). No dose-proportional increase in exposure was observed between 25 and 50 mg/kg. [2]
Voclosporin has similar bioavailability and half-life as other calcineurin inhibitors. [1]

Hepatotoxicity
In a large randomized controlled trial of voricosporin, 3% of patients experienced a transient increase in serum transaminase levels, compared to a similar incidence (2%) in the control group receiving standard treatment. Similar to cyclosporine, voricosporin treatment also resulted in a slight increase in alkaline phosphatase and bilirubin levels, but these values were generally within the normal range. No clinically significant liver injury attributable to treatment was observed in premarketing studies of voricosporin. However, clinical experience with this drug is limited, and other calcineurin inhibitors have been found to cause rare liver injury, typically cholestatic, mild to moderate in severity, which resolves spontaneously upon discontinuation. Probability Score: E (Unlikely to cause clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no information regarding the excretion of voricosporin into breast milk. Because vorticoloporin has a molecular weight of 1214 Da and a plasma protein binding rate as high as 97%, it is unlikely to enter breast milk in large quantities. However, especially in breastfed newborns or premature infants, other medications may be preferred.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

---

Protein binding
The protein binding rate of vorticoloporin is approximately 97%.

---

Long-term multiple-dose toxicity studies in rats, rabbits, dogs, and primates showed that Voclosporin is significantly less toxic than cyclosporine A, even at doses up to 100-fold in excess of that required for immunosuppression. It caused fewer renal side effects, and no signs of interstitial fibrosis (consistent with chronic cyclosporine A nephrotoxicity) were found in the kidneys. [1]
In non-human primates treated once daily for 13 weeks with Voclosporin up to 300 mg/kg, no major side-effects were observed except for thickening or inflammation of the gingiva and diarrhea (also seen in the vehicle control group). [2]
In the 7-day twice-daily cynomolgus monkey study: All but one animal in the Voclosporin groups had diarrhea of varying duration (mean 2.3 days, range 2-7 days), while no diarrhea occurred in the cyclosporine A or control groups. Mean weight loss at the end of the study was 3.4% in the Voclosporin groups vs 2.0% (cyclosporine A) and 1.0% (control). Hemograms and serum chemistries showed no differences between groups; all values were within normal limits. [2]
In Phase 1 clinical trials (single dose up to 6.0 mg/kg, multiple dose 2 mg/kg/day BID for 7 days), no significant adverse events were noted. [1]

[1]. ISATX247: a novel calcineurin inhibitor. J Heart Lung Transplant. 2001 Feb;20(2):161.

[2]. In vivo evaluation of the novel calcineurin inhibitor ISATX247 in non-human primates. J Heart Lung Transplant. 2003 Dec;22(12):1343-52.

Voclosporin is a homocyclic peptide. Lupus nephritis (LN) is a type of glomerulonephritis that occurs in patients with systemic lupus erythematosus (SLE). LN is a major cause of kidney failure, morbidity, and mortality in SLE patients. Within 10 years of SLE diagnosis, 5-20% of LN patients will develop end-stage renal disease, a fatal condition. Early and accurate intervention for LN is crucial for improving clinical outcomes. Voclosporin, marketed as Lupkynis, is a calcineurin inhibitor used to treat LN. This cyclosporine A analogue received FDA approval on January 22, 2021, following encouraging results in clinical trials. Early intervention with Voclosporin, combined with renal response, is believed to prevent irreversible kidney damage and improve long-term clinical outcomes in LN patients. Compared to cyclosporine, Voclosporin exhibits more stable pharmacokinetic and pharmacodynamic relationships, higher potency, and better metabolic properties compared to older-generation calcineurin inhibitors. In July 2022, the European Medicines Agency's Committee for Medicinal Products for Human Use (CHMP) recommended approval of voclosporin in combination with mycophenolate mofetil for the treatment of active lupus nephritis in adults. Voclosporin is a calcineurin inhibitor, an immunosuppressant. Its mechanism of action is as a calcineurin inhibitor, a P-glycoprotein inhibitor, an organic anion transporter 1B1 inhibitor, and an organic anion transporter 1B3 inhibitor. Voclosporin is an oral calcineurin inhibitor and a potent immunosuppressant, used in combination with mycophenolate mofetil and glucocorticoids for the treatment of acute lupus nephritis. A small, transient increase in serum enzymes may occur during voclosporin treatment, but it has not been found to be associated with clinically significant acute liver injury with jaundice. Indications: Voclosporin is used in combination with a basic immunosuppressive regimen for the treatment of lupus nephritis. The safety of its use in combination with cyclophosphamide has not been established.
Lupkynis, in combination with mycophenolate mofetil, is indicated for the treatment of active class III, IV, or V lupus nephritis (LN) in adults (including mixed class III/V and IV/V).
Treatment of systemic lupus erythematosus (SLE)
Treatment of non-infectious uveitis

---

Mechanism of Action
Voclosporin stabilizes podocytes in the kidneys by inhibiting calcineurin, blocking IL-2 expression and T cell-mediated immune responses. Voclosporin is a cyclosporine A analogue. Its structure is similar to cyclosporine A (CsA), differing in a modification of one amino acid region. This modification alters the binding of voclosporin to calcineurin. Cyclosporine inhibitors reversibly inhibit T lymphocytes. They also inhibit the production and release of lymphokines. Cyclosporine A exerts its inhibitory effect on T lymphocytes by binding to cyclophilic proteins. The formation of the cyclosporine-cyclosporine complex leads to the inhibition of both calcium-dependent and calmodulin-dependent serine/threonine phosphatase activities of calcineurin. In addition to inhibiting calcineurin, it also inhibits many transcription factors essential for inducing the expression of various cytokine genes, such as IL-2, IFN-γ, IL-4, and GM-CSF. This, in turn, reduces inflammation, thereby treating glomerulonephritis associated with systemic lupus erythematosus.

---

Pharmacodynamics
Walcrosporine inhibits calcineurin, suppressing T cell activation by blocking the transcription of early inflammatory cytokines. This reduces kidney inflammation, treats lupus nephritis, and prevents permanent kidney damage.

---

Voclosporin (ISATX247) is a novel semi-synthetic calcineurin inhibitor, an analogue of cyclosporine A with similar molecular weight (±5%) and structure. It acts by inhibiting calcineurin, thereby preventing NFAT translocation and impairing transcription of IL-2 and other lymphokines. It exhibits increased potency (up to 3-fold in vitro) and a broader safety margin/therapeutic index compared to cyclosporine A, making it a more desirable agent for transplantation and autoimmune diseases. Phase 2 trials were expected to commence in 2001. [1]
Voclosporin suppresses diverse T-cell functions (proliferation, activation antigen expression, cytokine production) more potently than cyclosporine A in non-human primates in vivo, despite lower blood levels and total exposure. This may be due to higher affinity for immunophilins or more effective inhibition of calcineurin isoforms specific for immune function. [2]

C63H111N11O12

1214.64

1213.84

515814-01-4

6918486

White to off-white solid powder

>129

4.315

5

12

16

86

2380

12

CC[C@H]1C(=O)N(CC(=O)N([C@H](C(=O)N[C@H](C(=O)N([C@H](C(=O)N[C@H](C(=O)N[C@@H](C(=O)N([C@H](C(=O)N([C@H](C(=O)N([C@H](C(=O)N([C@H](C(=O)N1)[C@@H]([C@H](C)C/C=C/C=C)O)C)C(C)C)C)CC(C)C)C)CC(C)C)C)C)C)CC(C)C)C)C(C)C)CC(C)C)C)C

BICRTLVBTLFLRD-PTWUADNWSA-N

InChI=1S/C63H111N11O12/c1-25-27-28-29-41(15)53(76)52-57(80)66-44(26-2)59(82)68(18)34-49(75)69(19)45(30-35(3)4)56(79)67-50(39(11)12)62(85)70(20)46(31-36(5)6)55(78)64-42(16)54(77)65-43(17)58(81)71(21)47(32-37(7)8)60(83)72(22)48(33-38(9)10)61(84)73(23)51(40(13)14)63(86)74(52)24/h25,27-28,35-48,50-53,76H,1,26,29-34H2,2-24H3,(H,64,78)(H,65,77)(H,66,80)(H,67,79)/b28-27+/t41-,42+,43-,44+,45+,46+,47+,48+,50+,51+,52+,53-/m1/s1

(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-ethyl-33-((1R,2R,E)-1-hydroxy-2-methylhepta-4,6-dien-1-yl)-6,9,18,24-tetraisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-nonamethyl-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-2,5,8,11,14,17,20,23,26,29,32-undecaone

ISATX247 LX211 LX 214 ISATX-247 LX-211 LX-214LuveniqLupkynis LX211Voclosporin ISA247 ISAtx-247 ISAtx247ISAtx 247

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 : ≥ 50 mg/mL (~41.17 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.06 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.

---

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