PCSK9/Low Density Lipoprotein Receptor

Novel mRNA display screening technology was used to identify lead chemical matter, which was then optimized by applying structure-based drug design enabled by novel synthetic chemistry to identify macrocyclic peptide (MK-0616) with exquisite potency and selectivity for PCSK9. MK-0616 was derived from the lead compound previously reported as 44 in Tucker et al which was identified through mRNA display screening combined with structure-based design and iterative medicinal chemistry.

MK-0616 displayed high affinity (Ki = 5pM) for PCSK9 in vitro and sufficient safety and oral bioavailability preclinically to enable advancement into the clinic. In Phase 1 clinical studies in healthy adults, single oral doses of MK-0616 were associated with >93% geometric mean reduction (95% CI, 84-103) of free, unbound plasma PCSK9; in participants on statin therapy, multiple-oral-dose regimens provided a maximum 61% geometric mean reduction (95% CI, 43-85) in low density lipoprotein cholesterol from baseline after 14 days of once-daily dosing of 20 mg MK-0616.

Alexa Fluorescence Resonance Energy Transfer PCSK9 Binding Assay
The PCSK9 time-resolved fluorescence resonance energy transfer Alexa fluorescence resonance energy transfer Standard assay measures the interaction between PCSK9 and an AlexaFluor647 (AF) tagged cyclic peptide, Reagent A (KD = 83 nM). A solution containing 1 nM biotinylated PCSK9 + 2.5 nM Lance Streptavidin Europium (Strep-Eu) was made in 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) pH 7.4, 0.15 M NaCl, 5 mM CaCl2, 0.01% bovine serum albumin (BSA), and 0.01% Surfactant P20. A separate solution containing 40 nM of the AF tagged cyclic peptide was made in the same buffer system. An Echo Acoustic Liquid Handler (Beckman) was used to transfer 0.750 μl of test compound to an assay plate followed by the addition of 15 μl of PCSK9+Stept-Eu and 15 μl of AF peptide. The final assay volume was 30.750 μl containing 0.5 nM PCSK9, 1.25 nM Strep-Eu, and 20 nM AF cyclic peptide. The reaction was incubated at room temperature for at least 2 hours prior to fluorescence measurements using an Envision Multilabel Reader. IC50 values were determined by fitting data to a sigmoidal dose-response curve using nonlinear regression. Ki was then calculated from the IC50 and the KD of AF cyclic peptide. Counts (B-counts) of the europium-labeled PCSK9 were followed to observe if compounds were adversely affecting PCSK9. A reduction in B-counts would suggest a false positive of inhibition of binding.
In Vitro Titration of PCSK9 in Human Plasma
To evaluate the in vitro potency of PCSK9 inhibitors to bind with PCSK9 in human plasma (MRL Volunteer Blood Donor Program for research; n=20 human controls), increasing concentrations (0, 0.3, 1, 3, 5, 10, 50, 100, 500, and 1000 nM) of test compounds 4418 and MK-0616 (each starting at 0.1 mM in dimethylsulfoxide) were prepared in 150 μL pooled ethylenediaminetetraacetic acid (EDTA) anticoagulated plasma (human or cynomolgus monkey) using an HP D300 digital dispenser. The plasma samples titrated with compound 44 or MK-0616 were incubated for 30 minutes at 25°C and 50 μL aliquots of each sample were used to assess PCSK9 target engagement using the affinity capture, enzymatic digestion, and liquid chromatography with tandem mass spectrometry procedure described.[1]

Determination of passive permeability
Briefly, MDCKII cells were cultured in 96-well transwell culture plates. The area of membrane was 0.11 cm2. MDCKII cells were originally obtained from P. Borst, MD, The Netherlands Cancer Institute (Amsterdam, Netherlands) and used under a license agreement. Solutions with [3H]MK-0616 (5 µM), [3H]verapamil (1 μM) or [3H]mannitol (5 μM) were prepared in Hanks balanced salt solution with 0.1% weight/volume bovine serum albumin, 10 mM HEPES (pH 7.4), 10 μM cyclosporin (to inhibit transporter activity), and 1.2 μM dextran Texas red. Substrate solution (150 µL) was added to either the apical or the basolateral compartment of the culture plate and buffer (150 µL; Hanks balanced salt solution, with 0.1% weight/volume BSA, 10 mM HEPES, 10 µM cyclosporin, pH 7.4) was added to the opposite compartment. A 50 µL aliquot was taken from both sides at 3 hours, and radioactivity was determined by liquid scintillation counting in a MicroBeta Wallac Trilux scintillation counter. The experiment was performed in triplicate. A mass balance ≥70% was considered acceptable. Dextran Texas red was used as a marker of paracellular flux to confirm monolayer integrity. The passive permeability of verapamil (Papp = 28.5*10-6 cm/s) and mannitol (Papp = 2.1*10-6 cm/s) confirmed the functionality of the assay. The reported apparent permeation (Papp) is the average of the Papp for transport from apical to basolateral and Papp for transport from basolateral to apical at t = 3 hours and is expressed as 10−6 cm/s.

Orally Bioavailable Macrocyclic Peptide That Inhibits Binding of PCSK9 to the Low Density Lipoprotein Receptor. Circulation. 2023 Jul 11;148(2):144-158.

C82H110CLFN14O15

1586.29

1584.7947

C, 62.09; H, 6.99; Cl, 2.23; F, 1.20; N, 12.36; O, 15.13

2407527-16-4

2407527-16-4 (chloride) 2407527-14-2 (cation)

Typically exists as solids (or liquids in special cases) at room temperature

366Ų

KZFXQSLIAQMQRP-MQCRZNPVSA-O

InChI=1S/C81H104FN15O15S2/c1-50-73(101)87-63-40-54-15-10-14-52(35-54)13-8-9-31-112-68-22-27-94-72(68)77(105)90-71(51(2)98)76(104)89-64(39-53-16-19-61(109-7)20-17-53)79(107)95-26-11-24-81(95,3)80(108)83-25-34-114-49-56-36-55(48-113-33-23-69(99)86-66(75(103)85-50)43-84-70(100)47-111-30-12-29-110-32-28-97(4,5)6)37-60(38-56)96-46-59(91-92-96)45-93-44-57(41-65(78(94)106)88-74(63)102)62-42-58(82)18-21-67(62)93/h8-10,14-21,35-38,42,44,46,50-51,63-66,68,71-72,98H,11-13,22-34,39-41,43,45,47-49H2,1-7H3,(H7-,83,84,85,86,87,88,89,90,99,100,101,102,103,104,105,108)/p+1/b9-8+/t50-,51-,63+,64+,65+,66+,68+,71+,72+,81+/m1/s1

L-Proline, L-alanyl-3-[[1-oxo-6-(trimethylammonio)hexyl]amino]-D-alanyl-3-[[(2-hydroxyacetyl)amino]methyl]-L-phenylalanyl-1-[6-[[[4-(2-aminoethyl)phenyl]methyl](3-carboxy-1-oxopropyl)amino]hexyl]-5-fluoro-L-tryptophyl-(3S)-3-hydroxy-L-prolyl-L-threonyl-O-methyl-L-tyrosyl-2-methyl-, (4→1),(8→4)-dilactam, cyclic (3→5)-ether, chloride (1:1)

MK0616; MK-0616; MK 0616

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.)