A novel calcimimetic agent, evocalcet (MT-4580/KHK7580), suppresses the parathyroid cell function with little effect on the gastrointestinal tract or CYP isozymes <i>in vivo</i> and <i>in vitro</i>

<div><p>Cinacalcet hydrochloride (cinacalcet), an oral calcimimetic agent has been widely used for the management of secondary hyperparathyroidism (SHPT) in chronic kidney disease (CKD). In sharp contrast to vitamin D receptor activators, cinacalcet suppresses SHPT without inducing hypercalcemia or hyperphosphatemia. Nevertheless, some patients remain refractory to SHPT with this agent, as the dose cannot be sufficiently increased due to gastrointestinal symptoms. In order to resolve this issue, we have developed a newly synthesized calcimimetic agent, evocalcet (MT-4580/KHK7580). In a rat model of CKD induced by 5/6 nephrectomy, oral administration of evocalcet efficiently suppressed the secretion of parathyroid hormone (PTH). With regard to the gastro-intestinal effects, cinacalcet induced a significant delay in gastric emptying in rats, while evocalcet did no marked effects on it. Evocalcet also demonstrated the less induction of emesis compared to cinacalcet in common marmosets. The pharmacological effects of evocalcet were observed at lower doses because of its higher bioavailability than cinacalcet, which may have contributed to the reduced GI tract symptoms. In addition, evocalcet showed no substantial direct inhibition of any CYP isozymes in <i>in vitro</i> liver microsome assay, suggesting a better profile in drug interactions than cinacalcet that inhibits cytochrome P450 (CYP) 2D6. These findings suggest that evocalcet can be a better alternative to cinacalcet, an oral calcimimetic agent, with a wider safety margin.</p></div

The chemical structure of evocalcet and cinacalcet.

<p>The chemical structure of evocalcet and cinacalcet.</p

The effects of evocalcet and cinacalcet on emesis in common marmosets.

<p>The effects of evocalcet and cinacalcet on emesis in common marmosets.</p

The effect of evocalcet on gastric emptying for 30 minutes in rats.

<p>Vehicle, evocalcet (0.3, 1, or 3 mg/kg), or cinacalcet (10, 30, or 100 mg/kg) were orally administered to rats. The data are presented as the mean + S.E. n = 8/group. ***<i>P</i> < 0.001 vs. vehicle group (Dunnett’s test).</p

Plasma concentrations of evocalcet after repeated oral administration to 5/6 nephrectomized rats.

<p>Plasma concentrations of evocalcet after repeated oral administration to 5/6 nephrectomized rats.</p

Pharmacokinetic parameters of evocalcet after oral administration to male rats.

<p>Pharmacokinetic parameters of evocalcet after oral administration to male rats.</p

A novel calcimimetic agent, evocalcet (MT-4580/KHK7580), suppresses the parathyroid cell function with little effect on the gastrointestinal tract or CYP isozymes <i>in vivo</i> and <i>in vitro</i> - Fig 3

<p><b>The pharmacological effects of evocalcet and cinacalcet on the serum PTH (A; evocalcet, C; cinacalcet) and calcium (B; evocalcet, D; cinacalcet) levels in normal rats.</b> Vehicle, evocalcet (0.03, 0.1, 0.3, or 1 mg/kg), or cinacalcet (1, 3, 10, or 30 mg/kg) were orally administered to the rats. The data are presented as the mean + S.E. n = 10/group. ^a<i>P</i> < 0.05, ^b<i>P</i> < 0.01, and ^c<i>P</i> < 0.001 vs. Vehicle group (Steel test).</p

The agonistic activity of evocalcet on hCaR-expressing HEK293 (hCaR-HEK293) cells.

<p>The transient changes in the cytoplasmic calcium concentration (Δ[Ca²⁺]i) induced by receptor activation were measured. (A) The effect of evocalcet on the Δ[Ca²⁺]i value in hCaR-HEK293 cells. (B) The effect of evocalcet on the response rates elicited by increasing the extracellular calcium level in hCaR-HEK293 cells. The response rate was calculated using the following formula: </p><p></p> Δ[Ca²⁺]i: Subtraction of basal [Ca²⁺]i from maximum [Ca²⁺]i observed after addition of evocalcet; Max Δ[Ca²⁺]i: Δ[Ca²⁺]i at the maximum Ca concentration (3 mmol/L).<p></p

Utility of NucleoCounter for the chondrocyte count in the collagenase digest of human native cartilage

In cartilage tissue engineering, viable cell numbers should be correctly counted in the collagenase digest of the biopsied cartilage. However, this is a difficult task due to the presence of matrix debris, cell ghosts and their aggregates. To search for the correct cell counting method in this situation, we evaluated the utility of an automatic cell counting device, the NucleoCounter, and compared it with conventional staining using the LIVE/DEAD® kit. We first measured the cell numbers of a standard chondrocyte sample by the NucleoCounter, which showed a high accuracy (R2 = 0.9999) and reproducibility (%CV: 2.00–8.66). We then calculated the cell numbers and viability in some collagenase digests of native cartilage using either the NucleoCounter or LIVE/DEAD® kit, revealing that the total cell numbers, viable ones and viability were highly correlated between them (R2 = 0.9601, 0.9638 and 0.917, respectively). However, both the intrapersonal and interpersonal variabilities in the NucleoCounter was significantly decreased to about 1/20–1/5, compared to that of the LIVE/DEAD® kit. The NucleoCounter was regarded as a useful tool for simple, rapid, and highly reproducible cell counts, which may not only provide constant experimental data in a certain laboratory, but also contribute to the high reproducibility of the clinical results of cartilage tissue engineering among multiple institutions