Gregory S. Gorman

Pharmacodynamics and pharmacokinetics of SQ109, a new diamine‐based antitubercular drug

1 SQ109 is a novel [1,2]‐diamine‐based ethambutol (EMB) analog developed from high‐throughput combinatorial screening. The present study aimed at characterizing its pharmacodynamics and pharmacokinetics. 2 The antimicrobial activity of SQ109 was confirmed in vitro (Mycobacterium tuberculosis‐infected murine macrophages) and in vivo (M. tuberculosis‐infected C57BL/6 mice) and compared to isoniazid (INH) and EMB. SQ109 showed potency and efficacy in inhibiting intracellular M. tuberculosis that was similar to INH, but superior to EMB. In vivo oral administration of SQ109 (0.1–25 mg kg−1 day−1) to the mice for 28 days resulted in dose‐dependent reductions of mycobacterial load in both spleen and lung comparable to that of EMB administered at 100 mg kg−1 day−1, but was less potent than INH at 25 mg kg−1 day−1. Monitoring of SQ109 levels in mouse tissues on days 1, 14 and 28 following 28‐day oral administration (10 mg kg−1 day−1) revealed that lungs and spleen contained the highest concentration of SQ109, at least 10 times above its MIC. 3 Pharmacokinetic profiles of SQ109 in mice following a single administration showed its Cmax as 1038 (intravenous (i.v.)) and 135 ng ml−1 (p.o.), with an oral Tmax of 0.31 h. The elimination t1/2 of SQ109 was 3.5 (i.v.) and 5.2 h (p.o.). The oral bioavailability was 4%. However, SQ109 displayed a large volume of distribution into various tissues. The highest concentration of SQ109 was present in lung (>MIC), which was at least 120‐fold (p.o.) and 180‐fold (i.v.) higher than that in plasma. The next ranked tissues were spleen and kidney. SQ109 levels in most tissues after a single administration were significantly higher than that in blood. High tissue concentrations of SQ109 persisted for the observation period (10 h). 4 This study demonstrated that SQ109 displays promising in vitro and in vivo antitubercular activity with favorable targeted tissue distribution properties.

A comparison of the pharmacokinetics of perfluorobutanesulfonate (PFBS) in rats, monkeys, and humans.

Materials derived from perfluorobutanesulfonyl fluoride (PBSF, C(4)F(9)SO(2)F) have been introduced as replacements for eight-carbon homolog products that were manufactured from perfluorooctanesulfonyl fluoride (POSF, C(8)F(17)SO(2)F). Perfluorobutanesulfonate (PFBS, C(4)F(9)SO(3)(-)) is a surfactant and potential degradation product of PBSF-derived materials. The purpose of this series of studies was to evaluate the pharmacokinetics of PFBS in rats, monkeys, and humans, thereby providing critical information for human health risk assessment. Studies included: (1) intravenous (i.v.) elimination studies in rats and monkeys; (2) oral uptake and elimination studies in rats; and (3) human serum PFBS elimination in a group of workers with occupational exposure to potassium PFBS (K(+)PFBS). PFBS concentrations were determined in serum (all species), liver (rats), urine (all species), and feces (rats). In rats, the mean terminal serum PFBS elimination half-lives, after i.v. administration of 30mg/kg PFBS, were: males 4.51+/-2.22h (standard error) and females 3.96+/-0.21h. In monkeys, the mean terminal serum PFBS elimination half-lives, after i.v. administration of 10mg/kg PFBS, were: males 95.2+/-27.1h and females 83.2+/-41.9h. Although terminal serum half-lives in male and female rats were similar, without statistical significance, clearance (CL) was significantly greater in female rats (469+/-40mL/h) than male rats (119+/-34mL/h) with the area under the curve (AUC) significantly larger in male rats (294+/-77microg.h/mL) than female rats (65+/-5microg.h/mL). These differences were not observed in male and female monkeys. Volume of distribution estimates suggested distribution was primarily extracellular in both rats and monkeys, regardless of sex, and urine appeared to be a major route of elimination. Among 6 human subjects (5 male, 1 female) followed up to 180 days, the geometric mean serum elimination half-life for PFBS was 25.8 days (95% confidence interval 16.6-40.2). Urine was observed to be a pathway of elimination in the human. Although species-specific differences exist, these findings demonstrate that PFBS is eliminated at a greater rate from human serum than the higher chain homologs of perfluorooctanesulfonate (PFOS) and perfluorohexanesulfonate (PFHxS). Thus, compared to PFOS and PFHxS, PFBS has a much lower potential for accumulation in human serum after repeated occupational, non-occupational (e.g., consumer), or environmental exposures.

Interspecies pharmacokinetics and in vitro metabolism of SQ109.

This study aimed at characterizing the interspecies absorption, distribution, metabolism and elimination (ADME) profile of N‐geranyl‐N′‐(2‐adamantyl)ethane‐1,2‐diamine (SQ109), a new diamine‐based antitubercular drug. Single doses of SQ109 were administered (intravenously (i.v.) and per os (p.o.)) to rodents and dogs and blood samples were analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). Based on i.v. equivalent body surface area dose, the terminal half‐life (t1/2) of SQ109 in dogs was longer than that in rodents, reflected by a larger volume of distribution (Vss) and a higher clearance rate of SQ109 in dogs, compared to that in rodents. The oral bioavailability of SQ109 in dogs, rats and mice were 2.4–5, 12 and 3.8%, respectively. After oral administration of [14C]SQ109 to rats, the highest level of radioactivity was in the liver, followed by the lung, spleen and kidney. Tissue‐to‐blood ratios of [14C]SQ109 were greater than 1. Fecal elimination of [14C]SQ109 accounted for 22.2% of the total dose of [14C]SQ109, while urinary excretion accounted for only 5.6%. The binding of [14C]SQ109 (0.1–2.5 μg ml−1) to plasma proteins varied from 6 to 23% depending on the species (human, mouse, rat and dog). SQ109 was metabolized by rat, mouse, dog and human liver microsomes, resulting in 22.8, 48.4, 50.8 or 58.3%, respectively, of SQ109 remaining after a 10‐min incubation at 37°C. The predominant metabolites in the human liver microsomes gave intense ion signals at 195, 347 and 363m/z, suggesting the oxidation, epoxidation and N‐dealkylation of SQ109. P450 reaction phenotyping using recombinant cDNA‐expressed human CYPs in conjunction with specific CYP inhibitors indicated that CYP2D6 and CYP2C19 were the predominant CYPs involved in SQ109 metabolism.

A Novel Sulindac Derivative That Does Not Inhibit Cyclooxygenases but Potently Inhibits Colon Tumor Cell Growth and Induces Apoptosis with Antitumor Activity

Nonsteroidal anti-inflammatory drugs such as sulindac have shown promising antineoplastic activity, although toxicity from cyclooxygenase (COX) inhibition and the suppression of prostaglandin synthesis limits their use for chemoprevention. Previous studies have concluded that the mechanism responsible for their antineoplastic activity may be COX independent. To selectively design out the COX inhibitory activity of sulindac sulfide (SS), in silico modeling studies were done that revealed the crucial role of the carboxylate moiety for COX-1 and COX-2 binding. These studies prompted the synthesis of a series of SS derivatives with carboxylate modifications that were screened for tumor cell growth and COX inhibitory activity. A SS amide (SSA) with a N,N-dimethylethyl amine substitution was found to lack COX-1 and COX-2 inhibitory activity, yet potently inhibit the growth of human colon tumor cell lines, HT-29, SW480, and HCT116 with IC50 values of 2 to 5 μmol/L compared with 73 to 85 μmol/L for SS. The mechanism of growth inhibition involved the suppression of DNA synthesis and apoptosis induction. Oral administration of SSA was well-tolerated in mice and generated plasma levels that exceeded its in vitro IC50 for tumor growth inhibition. In the human HT-29 colon tumor xenograft mouse model, SSA significantly inhibited tumor growth at a dosage of 250 mg/kg. Combined treatment of SSA with the chemotherapeutic drug, Camptosar, caused a more sustained suppression of tumor growth compared with Camptosar treatment alone. These results indicate that SSA has potential safety and efficacy advantages for colon cancer chemoprevention as well as utility for treating malignant disease if combined with chemotherapy.

Comparative Pharmacokinetics of Perfluorobutyrate in Rats, Mice, Monkeys, and Humans and Relevance to Human Exposure via Drinking Water

Perfluorobutyrate (PFBA) has been detected in precipitation, surface waters, water treatment effluent, and in public and private wells in Minnesota at up to low microg/l concentrations. We evaluated the pharmacokinetics of PFBA in rats, mice, monkeys, and humans to provide a rational basis for dose selection in toxicological studies and to aid in human-health-risk assessment. Studies included (1) rats--iv and oral; (2) mice--oral; (3) monkeys--iv; and (4) humans--occupationally exposed volunteers. PFBA was determined in serum (all species), liver (rats and mice), urine (rats, mice, and monkeys), and feces (rats and mice). In addition, we characterized serum PFBA concentrations in 177 individuals with potential exposure to PFBA through drinking water. Mean terminal serum PFBA elimination half-lives for males (M) and females (F), respectively, in h were (1) for rats given 30 mg/kg, 9.22 and 1.76 (oral), and 6.38 and 1.03 (iv); (2) for mice given oral doses of 10, 30, or 100 mg/kg ammonium PFBA, 13.34 and 2.87 at 10 mg/kg, 16.25 and 3.08 at 30 mg/kg; and 5.22 and 2.79 at 100 mg/kg; (3) for monkeys given 10 mg/kg iv, 40.32 and 41.04; and (4) for humans, 72.16 and 87.00 (74.63 combined). Volume of distribution estimates indicated primarily extracellular distribution. Among individuals with plausible exposure via drinking water, 96% of serum PFBA concentrations were < 2 ng/ml (maximum 6 ng/ml). These findings demonstrate that PFBA is eliminated efficiently from serum with a low potential for accumulation from repeated exposure.

In vitro metabolic characterization, phenotyping, and kinetic studies of 9cUAB30, a retinoid X receptor-specific retinoid.

The present study was conducted to compare the in vitro phase I and phase II metabolic profiles of (2E,4E,6Z,8E)-8-(3′,4′-dihydro-1′(2′H)-naphthalen-1′-ylidene)-3,7-dimethyl-2,4,6-octatrienoic acid (9cUAB30) in human, rat, and dog microsomes and to characterize and identify the associated metabolic kinetics and specific isozymes from human liver microsomes (HLM) responsible for metabolism, respectively. Data from these experiments revealed that nine (M1–M9) phase I metabolites along with a single glucuronide conjugate were observed across the species investigated. With the exception of glucuronidation, no evidence of metabolism was detected for phase II enzymes (data not shown). Significant differences between species with regard to metabolic profile, stability, and gender were noted. For the eight phase I metabolites detected in HLM, the specific isozymes responsible for the biotransformations were CYP2C8, CYP2C9, and CYP2C19, with minor contributions from CYP1A2 and CYP2B6. For the glucuronide conjugate, UGT1A9 was the major catalyzing enzyme, with a minor contribution from UGT1A3. Kinetic analysis of eight of the detected metabolites indicated that four seemed to follow classical hyperbolic kinetics, whereas the remaining four showed evidence of either autoactivation or substrate inhibition.

The Effect of Rho-Associated Kinase Inhibition on the Ocular Penetration of Timolol Maleate

PURPOSE To assess the effects of Rho-associated kinase (ROCK) inhibition on the intraocular penetration of timolol maleate. METHODS Ex vivo porcine corneal penetration of timolol maleate, sotalol hydrochloride, or brinzolamide incubated with or without Y-27632 was determined in vertical Franz diffusion cells. The effect of ROCK inhibition on the vasodilation of porcine conjunctival vasculature was assessed by scanning electron microscopy (SEM) and immunohistochemical staining with subsequent laser-scanning confocal microscopy (LSCM). Experiments were conducted in New Zealand White (NZW) rabbits to assess the effect of ROCK inhibition on the intraocular distribution of timolol maleate. RESULTS ROCK inhibition resulted in minimal alteration of ex vivo porcine corneal drug penetration of timolol, sotalol, or brinzolamide. SEM and LSCM experiments conducted with conjunctiva and sclera tissue in Franz diffusion cells suggested vasodilation in the conjunctival vasculature in the presence of Y-27632. Pretreatment of the eyes of NZW rabbits with Y-27632 resulted in aggregate fold reductions (1 hour, 0.25-fold; 4 hours, 0.45-fold) of timolol maleate drug concentrations in intraocular tissues (aqueous humor, lens, and iris) versus eyes not receiving Y-27632 pretreatment. Pretreatment with a vasoconstrictor, phenylephrine, resulted in a reversal of the effect of Y-27632 on diminished timolol maleate intraocular penetration in NZW rabbits. CONCLUSIONS ROCK inhibition reduced the intraocular penetration of administered timolol maleate presumably due to increased systemic elimination through the conjunctival vasculature. It is anticipated that care in order and timing of ROCK inhibitor administration will be warranted for those patients who may be on a multiple topical drug regimen for primary open-angle glaucoma.

Mass spectrometric method for detecting carbon 13 enrichment introduced by folate coenzymes in uric acid

Using [2-13C]uric acid as a test material, we developed a mass spectrometric procedure that detects and estimates the difference in 13C enrichment at the positions of carbons 2 and 8 of the purine ring. This method could replace radiochemical methods and could trace the incorporation of carbon fragments into the purine ring from 13C-labeled metabolites in humans.

Evaluation of FR901464 analogues in vitro and in vivo

This study was designed to determine the in vitro and in vivo antitumor behaviour of three analogues of the natural product FR901464. All analogues demonstrated effective inhibition of cell proliferation. Minimal in vivo antitumor activity was observed, warranting further PK and antitumor efficacy studies.

Pharmacokinetics and pharmacodynamics of Phor21-βCG(ala), a lytic peptide conjugate

Phor21‐βCG(ala), a 36‐amino acid peptide comprised of a lytic peptide (Phor21) conjugated to a modified 15‐amino acid segment of the β‐chain of chorionic gonadotropin (βCG(ala)), selectively kills cancer cells that over‐express luteinizing hormone/chorionic gonadotropin (LH/CG) receptors by disrupting cellular membrane structure. These studies were designed to further characterize its in‐vitro inhibition and in‐vivo destruction of prostate cancer cells, biostability and pharmacokinetics to determine its pharmacokinetic and pharmacodynamic profile. Inhibitory effects of Phor21‐βCG(ala) were tested in PC‐3 and Caco‐2 cells as well as in nude mice bearing PC‐3 cells transfected with the luciferase gene (PC‐3.luc). Plasma stability, protease hydrolysis and pharmacokinetics of Phor21‐βCG(ala) were measured by using liquid chromatography mass spectrometry (LC/MS/MS). Phor21‐βCG(ala) selectively inhibited proliferation in‐vitro and in‐vivo metastases of PC‐3 cells. Phor21‐βCG(ala) was relatively stable in mouse, rat, dog and human plasma. Its degradation was partially due to protease hydrolysis and thermodynamic catalysis. Intravenous administration of Phor21‐βCG(ala) showed its blood Cmax and AUC0→∞ around the in‐vitro effective levels. In the tested rodents, Phor21‐βCG(ala) displayed a moderate volume of distribution at steady state (VdSS) and slow clearance (Cl) in the rodents. In conclusion, Phor21‐βCG(ala) displayed promising in‐vitro and in‐vivo anti‐cancer activity with favourable pharmacokinetics, and may offer a novel approach to metastatic cancer chemotherapy.