Mediation of highly concentrative uptake of pregabalin by L-type amino acid transport in Chinese hamster ovary and Caco-2 cells.

Pregabalin (PGB) is a novel drug under development for the treatment of epilepsy, neuropathic pain, fibromyalgia, and generalized anxiety disorder. In this study, we investigated PGB transport in rats, mammalian cell lines, and Xenopus laevis oocytes. In contrast to gabapentin (GBP), PGB absorption in rats showed unique linear pharmacokinetics. PGB entered CHO and Caco-2 cells predominately via Na(+)-independent processes. Uptake of PGB was mutually exclusive with leucine, GBP and 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid, the substrates preferential for system L. The preloaded PGB in CHO cells was exchangeable with leucine, but at a lower exchange rate than that of leucine and GBP. Dixon plots showed competitive inhibition of leucine uptake by PGB, with a K(i) value very close to the K(m) value for PGB uptake (377 versus 363 microM). At an extracellular concentration of 300 microM, the intracellular PGB concentration in CHO cells reached 1.5- and 23-fold higher than that of GBP and leucine, respectively. In contrast, at clinically relevant concentrations, PGB seemed not to interact with GABA transport in GAT1, GAT2, and GAT3 cell lines, system y(+), b(0,+), B(0,+), and B(0) transport activities in Caco-2 and NBL-1 cells, and the b(0,+)-like transport activity in rBAT cRNA-injected X. laevis oocytes. Taken together, these results suggest that L-type transport is the major transport route for PGB and GBP uptake in mammalian cells. The differential affinity of PGB and GBP at L-type system leads to more concentrative accumulation of PGB than GBP, which may facilitate PGB transmembrane absorption in vivo.

Brain microdialysis and PK/PD correlation of pregabalin in rats.

Pregabalin [PGB, (S)-3-isobutyl GABA, CI-1008] is a derivative of the inhibitory neurotransmitter g-aminobutyric acid (GABA). It has shown anticonvulsant, analgesia and anxiety activity in animal models. In this report, blood-brain barrier (BBB) influx and efflux of PGB were investigated with microdialysis at efficacious doses in rats. BBB influx (CLin) and efflux (CLout) permeability for pregabalin were 4.8 and 37.2 microL/min/g brain, respectively, following an intravenous infusion to rats. The results indicate that PGB is brain penetrable, supporting its anti-epilepsy and other CNS pharmacology. Significant anticonvulsant action of PGB was detected between 2 and 8 hr post oral dose, which is lag behind ECF drug concentrations lees. A PK/PD link model was used to describe the counter-clockwise hysteresis relationship between pregabalin brain ECF concentration and the anticonvulsant effect in rats. The resulting Ce (concentration in effect compartment) versus effect profile exhibits a sigmoidal curve and the calculated ECe50 and Keo values were 95.3 ng/mL and 0.0092 min-1, respectively. The small Keo value suggests that the effect is not directly proportional to the amount of pregabalin in the ECF compartment possibly due to inherent delay.

Neuronal N-type calcium channel blockers: a series of 4-piperidinylaniline analogs with analgesic activity.

Several novel N-type voltage sensitive calcium channel blockers showed high affinity in the IMR32 assay and efficacy in the anti-writhing model. Herein, we describe the design, synthesis, SAR studies, biological data, physicochemical properties and pharmacokinetics of this 4-piperidinylaniline series.

The discovery of [1-(4-dimethylamino-benzyl)-piperidin-4-yl]-[4-(3,3-dimethylbutyl)-phen yl]-(3-methyl-but-2-enyl)-amine, an N-type Ca+2 channel blocker with oral activity for analgesia.

Our drug discovery efforts for N-type calcium channel blockers in the 4-piperidinylaniline series led to the discovery of an orally active analgesic agent 26.1-[4-Dimethylamino-benzyl)-piperidin-4-yl]-[4-(3,3-dimethyl-but yl)-phenyl]-(3-methyl-but-2-enyl)amine (26) showed high affinity to functionally block N-type calcium channels (IC50=0.7 microM in the IMR32 assay) and exhibited high efficacy in the anti-writhing analgesia test with mice (ED50=12 mg/kg by po and 4 mg/kg by iv). In this report, the rationale for the design, synthesis, biological evaluation, and pharmacokinetics of this series of blockers is described.

Allometric pharmacokinetic scaling: towards the prediction of human oral pharmacokinetics.

PURPOSE: To evaluate (1) allometric scaling of systemic clearance (CL) using unbound drug concentration, (2) the potential usage of brain weight (BRW) correction in allometric scaling of both CL and oral clearance (CL/F). METHODS: Human clearance was predicted allometrically (CLu = a x W(biv)) using unbound plasma concentration for eight Parke-Davis compounds and 29 drugs from literature sources. When the exponent b(iv) was higher than 0.85, BRW was incorporated into the allometric relationship (CLu*BRW = a x W(biv)). This approach was also applied to the prediction of CLu/F for 10 Parke-Davis compounds. Human oral t1/2, Cmax, AUC, and bioavailability were estimated based on allometrically predicted pharmacokinetic (PK) parameters. RESULTS: Human CL and CL/F were more accurately estimated using unbound drug concentration and the prediction was further improved when BRW was incorporated into the allometric relationship. For Parke-Davis compounds, the predicted human CL and CL/F were within 50-200% and 50-220% of the actual values, respectively. The estimated human oral t1/2, Cmax, and AUC were within 82-220%, 56-240%, and 73-190% of the actual values for all 7 compounds, suggesting that human oral PK parameters of those drugs could be reasonably predicted from animal data. CONCLUSIONS: Results from the retrospective analysis indicate that allometric scaling of free concentration could be applied to orally administered drugs to gain knowledge of drug disposition in man, and to help decision-making at early stages of drug development.

Synthesis of a series of 4-benzyloxyaniline analogues as neuronal N-type calcium channel blockers with improved anticonvulsant and analgesic properties.

In this article, the rationale for the design, synthesis, and biological evaluation of a series of N-type voltage-sensitive calcium channel (VSCC) blockers is described. N-Type VSCC blockers, such as ziconotide, have shown utility in several models of stroke and pain. Modification of the previously reported lead, 1a, led to several 4-(4-benzyloxylphenyl)piperidine structures with potent in vitro and in vivo activities. In this series, the most interesting compound, (S)-2-amino-1-{4-[(4-benzyloxy-phenyl)-(3-methyl-but-2-enyl)-amino]-p iperidin-1-yl}-4-methyl-pentan-1-one (11), blocked N-type calcium channels (IC(50) = 0.67 microM in the IMR32 assay) and was efficacious in the audiogenic DBA/2 seizure mouse model (ED(50) = 6 mg/kg, iv) as well as the antiwrithing model (ED(50) = 6 mg/kg, iv). Whole-cell voltage-clamp electrophysiology experiments demonstrated that compound 11 blocked N-type Ca(2+) channels and Na(+) channels in superior cervical ganglion neurons at similar concentrations. Compound 11, which showed superior in vivo efficacy, stands out as an interesting lead for further development of neurotherapeutic agents in this series.

Quantitation of a novel metalloporphyrin drug in plasma by atomic absorption spectroscopy.

A bioanalytical method to quantify cobalt mesoporphyrin (CoMP), a novel therapeutic agent, in plasma has been developed and validated. The approach involves atomic absorption spectroscopy to determine total cobalt in a sample and a back-calculation of the amount of compound present. Endogenous plasma cobalt concentrations were small ( <0.2 ng/ml(-1) Co in rat plasma) in comparison to the quantitation limit (4.5 ng/ml(-1) Co). The inter-day imprecision of the method was 10.0% relative standard deviation (RSD) and the inter-day bias was +/- 8.0% relative error (RE) over a standard curve range of 4.5- 45.0 ng/ml(-1) Co. Because it quantifies total cobalt, the method cannot differentiate between parent drug and metabolites, but negligible metabolism allows reliable estimates of the actual parent drug concentration. A correlation study between the atomic absorption method and 14C-radiometry demonstrated excellent agreement (r = 0.9868, slope = 1.041 +/- 0.028, intercept = 223.7 +/- 190.0) and further substantiated the accuracy of the methods. Methodology was successfully applied to a pharmacokinetic study of CoMP in rat, with pharmacokinetic parameter estimation. The elimination half-lives, after intra-muscular and subcutaneous administration, were 7.7 and 8.8 days, respectively.

Disposition of the antipsychotic agent CI-1007 in rats, monkeys, dogs, and human cytochrome P450 2D6 extensive metabolizers. Species comparison and allometric scaling.

The disposition of CI-1007 (I), an antipsychotic dopamine agonist, was studied after iv or po administration to rats, monkeys, and dogs and po administration to human cytochrome P450 2D6 extensive metabolizers (EMs). I was extensively metabolized after po administration, with high hepatic clearance (CL) values and negligible urinary excretion. Values for systemic plasma CL (28-40 ml/min/kg) suggested hepatic plasma flow-limited CL. The oral CL of I was similar among the species. Strong correlation was achieved in interspecies scaling for CL. After oral administration of [14C]I, the major route of 14C elimination in rats was in the bile (64%), followed by feces (29%) and urine (3.2%). Fecal excretion (64%) was the major route of 14C elimination in monkeys, followed by urine (14%). Three hydroxy metabolites, i.e. PD 147693 (II), PD 149394 (III), and PD 155144 (IV), and two sulfates, i.e. PD 163637 (VI) and PD 163639 (VIII), were identified in monkey plasma, urine, or feces. VIII was the major metabolite excreted in monkey urine, and VI was the major component in feces. Trace amounts of II, VI, and VIII were detected in the plasma and urine of human EMs but not in rats or dogs. II is an active metabolite that was present in all species. After oral administration, observed maximal plasma concentration and AUC values for II were higher than the corresponding values for I in dog plasma, approximately 20-40% of the values for I in monkeys and human EMs, and <5% of the values for I in rat plasma. Although the metabolic profiles differ among species, strong correlation was achieved in allometric scaling because the elimination of I from the body is mainly limited by hepatic blood flow.

Disposition kinetics of cobalt mesoporphyrin in mouse, rat, monkey and dog.

1. Radiometric and UV analyses indicated > 95% unchanged cobalt mesoporphyrin (CoMP) in plasma after i.v. or i.m. administration. Blood clearance of CoMP is < 2% of hepatic blood flow in mouse and rat, and < 0.5% of hepatic blood flow in monkey and dog. CoMP elimination t1/2 ranged from 3.1 to 9.9 days in animals after i.v. administration. 2. CoMP is highly (> 99.5%) bound to plasma proteins, but has low affinity for blood cells (Kp < 0.15). The volume of CoMP distribution (Vss < 0.91/kg) is reflective of a distribution to total body water following i.v. administration to mouse, rat, monkey and dog. 3. [14C]CoMP reached highest levels in rat tissue between 1 and 4 days following i.m. injection. Liver, kidney cortex, lymph node, adrenal and spleen demonstrated greatest uptake of radiolabel. Concentration in tissues was readily detectable at 60 days post-dose. 4. CoMP was slowly absorbed after i.m. administration showing dose-dependent pharmacokinetics. The major route of radiolabel elimination was faecal excretion (54% of dose) in rat after an i.m. dose of [14C]CoMP. Approximately 1% of the 14C dose was recovered in the urine over 7 days post-dose. 5. As a polar metalloporphyrin, CoMP has low clearance, restricted tissue distribution and long elimination t1/2 in the laboratory animals.

Initial safety, tolerability pharmacodynamics, and pharmacokinetics of CI-1007 in patients with schizophrenia.

CI-1007 is a novel dopamine autoreceptor agonist and partial dopamine D2/D3 agonist that is currently under development for the treatment of schizophrenia. This single-blind, rising, multiple-dose, inpatient bridging study was designed to evaluate the safety and tolerability of CI-1007 in consecutive panels of patients with schizophrenia. Following a 4-day placebo washout period, 16 patients (4 per panel) were assigned to receive one of four fixed-dosage regimens of CI-1007 (5, 10, 15, or 20 mg q12h for 9 doses). CI-1007 was generally well tolerated over the dose range evaluated. Adverse events, including mild to moderate sporadic orthostatic hypotension and/or nausea and vomiting, were most commonly observed after the initial drug dose and decreased after repeated dosing. Serum concentrations of growth hormone (GH) increased following the administration of CI-1007, confirming its central dopamine agonist activity. Changes in serum prolactin were not related to dose. The pharmacokinetics of CI-1007 and its active metabolite appear linearly related to dose. The results of this study suggest that patients with schizophrenia tolerate slightly higher initial doses of CI-1007 than do healthy subjects.

Sample pooling to expedite bioanalysis and pharmacokinetic research.

In the progression from drug discovery to development, not only pharmacokinetic (PK) characterization needed for lead compound selection often becomes a rate-limiting step, but also high volume of routine sample analysis ensued from numerous required biodisposition studies for the lead compounds and their back-ups often place a burdensome hurdle to the throughput of IND and NDA development phases. Higher throughput of PK screening via cocktail dosing has been reported to accelerate PK screening in the discovery phase. However, concerns on drug-drug interactions and other limitations associated with the cocktail M-in-One dosing (multiple compounds per dose per animal) has prompted the present investigation of sample pooling alongside One-in-One dosing strategy (one compound per dose per animal) as an alternative to the cocktail dosing approach. Using traditional HPLC for bioanalysis as an example, the present study illustrate the concept and usefulness of sample pooling that could facilitate the throughput of PK screening and characterization in both discovery and development phases. Six proprietary dopamine D4 receptor antagonist preleads representing three different chemical classes, used as model compounds (C1-C6), were administered orally to rats. One rat received one compound and three rats were used for each compound. Six unknown plasma samples from six different rats at each time point were pooled. The pooled plasma samples were extracted by a one-step liquid-liquid extraction and concentrations of the six preleads were quantitated simultaneously. By sample pooling, a substantial amount of PK information was obtained at the same time for the six preleads, which requires much less workload than when bioanalysis is dealt with one compound at a time. For the first time in one aspect of innovative bioanalysis, the present investigation has demonstrated that sample pooling following One-in-One dosing can be utilized to enhance the throughput rate in PK screening in discovery phase. The sample pooling approach is likely to be useful in enhancing the throughput of PK characterization in development phase. With the advent of LC-MS and its becoming user-friendly, where separation of drug compounds is no longer an issue, the uniqueness of sample pooling may also pose a new way of thinking in regard to the old ways of handling bioanalysis for traditional PK research.

Determination of two CI-1007 sulfate metabolites in monkey plasma and urine.

Two HPLC assays were developed and validated for simultaneous quantitation of two sulfate metabolites, PD 163637 (VI) and PD 163639 (VIII), of an investigational antipsychotic drug CI-1007 (I) in monkey plasma and urine. VI and VIII were identified as major metabolites in monkey plasma, and both were excreted in urine. Monkey plasma samples were directly injected after deproteinization, and urine samples were analyzed after a clean-up procedure using methyl-tert.-butyl ether. Liquid chromatographic separation was achieved on a Zorbax RX C8 analytical column using gradient elution. Column effluent was monitored using fluorescence detection with excitation and emission wavelengths of 254 and 330 nm, respectively. Minimum quantitation limit was 50 ng/ml in plasma and 100 ng/ml in urine. Linearity was demonstrated up to 3000 ng/ml in plasma and urine. Recoveries of the analytes from plasma and urine were greater than 85%. The assay has been applied to the determination of VI and VIII in plasma and urine samples from monkeys receiving oral administration of I.

Pharmacokinetics and pharmacodynamics of an investigational antipsychotic agent, CI-1007, in rats and monkeys.

PURPOSE: To study the pharmacokinetics (PK) and pharmacodynamics (PD) of an investigational antipsychotic agent, CI-1007, in rats and monkeys. METHODS: CI-1007 and a pharmacologically active metabolite, PD 147693 (M1), were evaluated in animal antipsychotic tests (inhibition of dopamine neuron firing and spontaneous locomotor activity in rats, and inhibition of continuous avoidance in monkeys). Plasma concentrations of CI-1007 and M1 were determined using validated HPLC assays. Log-linear and link models were used for PK/PD analysis. RESULTS: CI-1007 and M1 have shown similar effects on dopamine neuron firing (2.5 mg/kg i.p.), and produced dose-related effects on spontaneous locomotor activity in rats (0.3-30 mg/kg p.o.) and on continuous avoidance in monkeys (0.6-1.2 mg/kg p.o.). After pharmacologically active CI-1007 doses, mean plasma CI-1007 Cmax increased from 19 to 200 ng/ml in Sprague-Dawley rats at doses of 3-30 mg/ kg, and from 8.1 to 34 ng/ml in squirrel monkeys at doses of 0.6-1.2 mg/kg, but corresponding plasma M1 Cmax values were near or below the limit of quantitation (5 ng/ml). CI-1007 EC50 was 31.1 ng/ml in rats, calculated from a long-linear regression. In monkeys, CI-1007 ECe50, gamma, and Keo at 0.6 and 1.2 mg/kg were 4.8 and 4.5 ng/ml, 1.9 and 2.0, and 0.47 and 0.48 hr-1, respectively, calculated by the link model. CONCLUSIONS: CI-1007 has shown dose-related pharmacokinetics and pharmacodynamics in rats and monkeys. Although M1 produces antipsychotic-like effects similar to CI-1007, the contribution of M1 to the activity of the parent drug may not be significant in rats and monkeys as based on plasma levels. CI-1007 plasma concentration correlates log-linearly with inhibition effect from the rat locomotor study. The counter-clockwise hysteresis relationship of CI-1007 plasma concentration and inhibition effect from the monkey avoidance test was described by a link model, and the resulting Ce (concentration in effect compartment) versus effect profile exhibits a sigmoidal curve.

Identification, characterization and pharmacological profile of three metabolites of (R)-(+)-1,2,3,6-tetrahydro-4-phenyl-1-[(3-phenylcyclohexen-1- yl)methyl]pyridine (CI-1007), a dopamine autoreceptor agonist and potential antipsychotic agent.

Liquid chromatographic-mass spectrometric (LC-MS) analysis of plasma taken from cynomolgus monkeys dosed orally with (R)-(+)-1,2,3,6-tetrahydro-4-phenyl-1-[(3-phenylcyclohexen-1- yl)methyl]pyridine (1), a dopamine (DA) autoreceptor agonist and potential antipsychotic agent, revealed several metabolites. The molecular masses of three major metabolites suggested that they were mono- and dihydroxylated derivatives of 1. We synthesized compounds 2 and 3, the two possible mono-p-hydroxyphenyl derivatives of 1, along with the bis-p-hydroxyphenyl derivative 4. These compounds coeluted by HPLC with the three hydroxylated metabolites of 1. Compounds 2-4 all had high affinities for DA D2 and D3 receptors and moderate affinities for D4 receptors. Like 1, compound 2 decreased DA synthesis and neuronal firing in rat brain, indicative of DA autoreceptor activation. Compound 2 inhibited exploratory locomotor activity in rodents and was active in the Sidman avoidance test in squirrel monkeys, predictive of antipsychotic activity in humans. Compounds 3 and 4 showed weak activity in all these tests. After squirrel monkeys were dosed with 1 orally at the ED100 dose of the Sidman avoidance test, the plasma concentration of 2 was below the limit of quantitation. Therefore, these metabolites are unlikely to contribute greatly to the potent activity seen with 1 in the Sidman avoidance test.

Sensitive high-performance liquid chromatographic method for a dopamine receptor agonist, CI-1007, and its metabolite PD 147693 in monkey plasma.

A sensitive gradient high-performance liquid chromatographic (HPLC) method for the simultaneous quantitation of a dopamine autoreceptor agonist CI-1007 (I) and its metabolite PD 147693 (II) is described. Monkey plasma samples were purified by liquid-liquid extraction using hexane. Liquid chromatographic separation was achieved on two C18 analytical columns (installed in series) using gradient elution. Column effluent was monitored using a fluorescence detector programmed to change wavelengths at specified times. Minimum quantitation limits of I and II were 3.0 and 5.0 ng/ml, respectively, for a plasma sample volume of 0.100 ml. Linearity was demonstrated up to 300 ng/ml. The assay has been applied to the analysis of I and II in plasma from monkeys following intravenous and oral doses of I.

Effect of verapamil on the uptake and efflux of etoposide (VP16) in both sensitive and resistant cancer cells.

The effect of calcium antagonist verapamil on the uptake and efflux of Etoposide (VP16), a semi-synthetic derivative of podophylotoxin and a broad spectrum antineoplastic agent, has been investigated and compared in sensitive (UM-UC-2) and resistant (UM-UC-9) human bladder cancer cells, and L1210 leukemia cells, by using both radioisotope (3[H]-VP16) liquid scintillation and high performance liquid chromatography assay with electrochemical detection. The uptake of VP16 was rapid in all three cell lines, showing an initial rapid linear phase followed by a second slower phase, but at steady state the ratios of intracellular to extracellular VP16 concentrations were only 0.004-0.006. No significant difference in drug uptake was observed in sensitive UM-UC-2 and resistant UM-UC-9 cells at all concentrations studied. Verapamil at a concentration of 10 microM enhanced the intracellular VP-16 levels in all sensitive and resistant cell lines. The increments were 21.5% for UM-UC-2, 11.8% for UM-UC-9, and 31.0% for L1210 cells after 30 minutes incubation with 1 microM VP16. A slower efflux of VP16 was observed in verapamil treated cells in all three cell lines. There was a small increase in the nonexchangeable components in verapamil treated cells, although only 5-10% of VP16 was retained. No peak other than that of VP16 was detected in the HPLC chromatogram of extracts from both cell pellet and influx or efflux medium.