Mechanism of Action

Carfilzomib irreversibly inhibited 20S-proteasome, the proteolytic core particle within the 26S proteasome, greatest inhibition of chymotrypsin-like activity with IC₅₀=4.4 ± 2.7 nM, which were 375-fold and 545-fold more potential to inhibit β1 and β2 subunit of 20S proteasome. ^[1,2]

Carfilzomib exhibited equal potency of antiproliferative and proapoptotic activity, but greater selectivity for the chymotrypsin-like activity of the proteasome compared with bortezomib.

Off-target screening against a panel of 21proteases, 67 receptors and 16 enzymes, Carfilzomib did not significantly bind or inhibit (>50%) any of these targets at 10 μM, except neurokinin NK1 and NK2, with 100-fold lower potency than towards the proteasome. ^[1]

In Vitro Efficacy

Carfilzomib led to cell death in a panel of cell lines for 72 hrs treatment and decreased the cell number at 72 hrs wash out post 1hr treatment due to irreversibly binding: ^[2]

●    Hematologic tumor cell lines: IC₅₀=2.4-10 nM for 72 hrs treatment, 31-170 nM at 72 hrs wash out post 1hr treatment.

●    Solid tumor cell lines: IC₅₀= 6.2-60 nM for 72 hrs treatment, 350-1200 nM at 72 hrs wash out post 1hr treatment.

●    Non-Transformed cell lines: IC₅₀= 7.2-14 nM for 72 hrs treatment, 455-389 nM at 72 hrs wash out post 1 hr treatment.

Carfilzomib showed cytotoxicity for multiple resistant type cell lines: ^[1,2,3]

●    Carfilzomib inhibited cell growth of multiple resistant MM cell lines: IC₅₀=15-100 nM.

●    Carfilzomib was able to partially overcome bortezomib-resistant multiple myeloma.

●    Carfilzomib was a substrate for P-gp proteins (≥100 nM), suggesting that it may be susceptible to multidrug resistance protein 1 (MDR1)-mediated drug resistance¹.

In Vivo Efficacy

Carfilzomib resulted in proteasome inhibition shortly after administration in vivo, which was sustained in blood and some tissues. ^[1]

Carfilzomib significantly decreased the tumor growth: ^[1,2]

●    HT-29 colorectal adenocarcinoma xenograft mice model at 5 mg/kg, biweekly at day 1/day 2.

●    RL B-cell lymphoma xenograft mice model at 5 mg/kg, biweekly at day 1/day 2.

●    HS-Sultan Burkitt’s lymphoma xenograft mice model at 5 mg/kg, biweekly at day 1/day 2.

Absorption of Carfilzomib

Both AUC and C_max increased dose proportionally from 2 to 4 mg/kg, AUC and C_max increased less than dose proportionally from 4 to 8 mg/kg following a single intravenous bolus administration in rats; AUC increased dose dependently following a single intravenous bolus administration from 1 to 4 mg/kg in monkeys, dose-dependent increases in C_max and AUC was absence following a single intravenous infusion administration from 11 to 27 mg/m² in patients.

The half-life of carfilzomib was very short in animals (≤ 20 min) and patients (< 40min).

High plasma clearance in rats, mice, monkeys and humans was comparable to liver blood flow, suggesting that carfilzomib was largely eliminated extrahepatically.

The volume of distribution at steady state was higher than blood volume in monkeys and patients (>0.9 L/kg), suggesting large distribution into peripheral tissues, but smaller in rats (0.3 L/kg).

Distribution of Carfilzomib

Plasma protein binding of carfilzomib was high (94 – 97 %) and carfilzomib was stable in plasma using the RED device at concentration range of 0.4 - 4 μM in rats, monkeys and humans.

Following administration to rats, carfilzomib was rapidly and widely distributed to most tissues, more so than blood, except for epididymis and eye, and did not cross the blood-brain barrier. Carfilzomib elimination was very slow, where in it was still present 168 hrs post-dose.

Metabolite of Carfilzomib

The hepatic clearance of carfilzomib was high in monkey (82%) and very high in human and mouse (93 - 98 %) from liver microsomal incubation with NADPH.

No unique human metabolites were identified, and none of the major metabolites inhibited in vitro proteasome activity significantly.

●    M4 was a major metabolite in rat hepatocytes; M16 was a major metabolite in monkey and human hepatocytes.

●    M14, M15 and M16 were the most abundant metabolites detected in human plasma. M14 and M16 were major metabolites in rats and monkeys plasma.

Carfilzomib was highly metabolized by two main mechanisms: peptide cleavage and epoxide hydrolysis, cytochrome P450 isoenzymes did not play a significant role in carfilzomib metabolism.

Excretion of Carfilzomib

Carfilzomib was rapidly (majority <4 hrs) cleared largely extrahepatically, with 30% being cleared via biliary excretion and 30% via the kidneys by 24 hrs post-dose, and with the majority being excreted within the first 4 hrs, following a single bolus intravenous administration in rats.

●    Renal excretion: M14>> M15≈M16>carfilzomib.

●    Biliary excretion: M15≈M14>M16>>carfilzomib.

Human excretion studies were not conducted for carfilzomib.

Drug-Drug Interaction

Carfilzomib was a direct, competitive, and irreversible/quasi-irreversible inhibitor of CYP3A4/5 enzymes with relatively moderate potency, also a direct, but weak inhibitor of CYP1A2, CYP2C9, CYP2C19 andCYP2D6, but not inhibit CYP2C8.

Carfilzomib did not induce CYP1A2 or CYP3A4 activity in primary human hepatocytes, indirectly decreased CYP1A2 activity at high doses (≥2.5 μM); directly inhibited CYP3A4 protein activity and down-regulated CYP3A4 mRNA expression at all doses examined (≥0.1 μM).

Carfilzomib was a P-glycoproteins (P-gp) substrate at all concentrations examined (0.1 ~ 3 μM), weakly inhibited P-gp transport at 3 μM.

Single-Dose Toxicity

Single dose intravenous administration of carfilzomib in different species:

Rats MTD: 7 mg/kg.

Monkeys MTD: 2 mg/kg.

Repeated-Dose Toxicity

Repeated dose intravenous administration of carfilzomib in different species from 1 to 36 weeks.

Multiple dose MTD, rats and monkeys: 1 mg/kg (6 and 12 mg/m², respectively).

Significant and consistent dose-dependent mortality and toxicities were observed in all studies conducted with carfilzomib.

At the significantly toxic doses there were signs of cardiac failure, preprenal azotemia, acute phase response and thrombocytopenia.

Notable carfilzomib toxicity and target organ effects: heart > kidney > liver > large intestine > neurotoxicity > lymph node > bone > spleen > lung > pancreas > intestinal fat.

Safety Pharmacology

hERG potassium current inhibition (IC₅₀=2.1 μM).

A single bolus intravenous injection of 3 mg/kg carfilzomib in monkeys resulted in increased ventricular premature complex, ST segments and T wave amplitudes in one male, and increased heart rate and troponin-T levels and decreased blood pressure, PR interval, QRS interval and QT interval in the other tested.

No effects on respiratory and neurological system.

Genotoxicity

Carfilzomib was not genotoxic in the reverse mutation bacterial test (Ames) and the mouse micronucleus test.

Carfilzomib caused an increase in chromosome structural aberrations in human peripheral blood cells at ≥0.04 μg/mL and at ≥2.4 μg/mL in vitro in the absence and presence of metabolic activation, respectively.

Reproductive and Developmental Toxicity

Carfilzomib caused no overt teratogenicity in pregnant rats at exposures (AUC) or in rabbits at doses that were lower than in patients receiving the recommended dose.

Embryo-lethality occurred below human exposures or doses based on findings of increased post-implantation loss from early resorptions in rats and rabbits.

The negative effects on implantation in both species, and fetal weight decreases in rabbits may be secondary due to maternal toxicities, such as body weight and feed consumption decreases.

Carcinogenicity

Carcinogenicity studies had not been conducted with carfilzomib.