Gary R. Erdmann

Azathioprine Metabolism: Pharmacokinetics of 6‐Mercaptopurine, 6‐Thiouric Acid and 6‐Thioguanine Nucleotides in Renal Transplant Patients

Despite extensive clinical experience with azathioprine (AZA), the disposition of various AZA metabolites remains obscure. We therefore evaluated the pharmacokinetics of three AZA metabolites: 6‐mercaptopurine (6‐MP), the immediate metabolite; 6‐thiouric acid (6‐TU), the final end product; and 6‐thioguanine nucleotides (TGN), the active moiety; in eight renal transplant patients after oral administration of AZA. The low peak plasma 6‐MP level of 73.7 ± 23.7 ng/mL (mean ± SD) and the short half‐life (t1/2) of 1.9 ± 0.6 hours suggest rapid conversion of 6‐MP to other metabolites. A peak plasma 6‐TU concentration of 1210 ± 785 ng/mL was observed at 3.5 ± 0.6 hours after the AZA dose. The strong correlation between 6‐TU t1/2 and serum creatinine (r = 0.98, P = .0008) supported our previous work showing that 6‐TU is primarily excreted by the kidneys. The total TGN levels in red blood cells (RBCs) in each patient remained largely unchanged over 24 hours with the intraindividual coefficient of variation ranging from 4.4% to 29.8%. In comparison, the mean TGN level varied considerably between patients, and ranged from undetectable to 413 pmol per 8 × 108 RBCs. However, there was no apparent correlation between white cell counts on day 0 (P > .5), day 7 (P > .5), or day 14 (P > .5) and RBC TGN level. The persistence of TGN in body tissues thus provides a pharmacokinetic rationale for the conventional once or twice daily AZA regimen. Unfortunately, the long elimination t1/2 may also predispose patients to excessive accumulation of the active metabolite and gradual development of TGN‐induced myelosuppression. Further studies including more patients are required to clarify the clinical significance of the interindividual variability in RBC TGN levels.

Experimental animal models for studying antimicrobial pharmacokinetics in otitis media

Antimicrobial treatment of otitis media, especially drug dosing considerations, is largely empiric, with few reported pharmacologic studies of drug distribution into the middle ear. A chinchilla animal model of serous and purulent otitis media has been used for some time to investigate mechanisms of disease pathogenesis. This model was adapted to investigate the penetration of amoxicillin, trimethoprim, and sulfamethoxazole into middle ear effusion. Purulent otitis media was produced by direct middle ear inoculation with type 7F Streptococcus pneumoniae. Serous otitis media was produced by eustachian tube obstruction using silastic sponge or Coeflex cement, but the Coeflex caused an undesirable local inflammatory response. The three antibiotics were administered to chinchillas with serous and purulent middle ear effusion. Plasma and ear fluid drug concentrations were measured by liquid chromatography and demonstrated the value of this model in assessing antibiotic penetration.

Systemic methotrexate exposure is greater after intrathecal than after oral administration.

Purpose To compare systemic exposure after intrathecal and oral methotrexate administration. Patients and Methods We analyzed red cell methotrexate polyglutamate concentrations with a sensitive radioligand-binding assay in 80 patients enrolled in the Childrens Cancer Group (CCG) trial 1922 for acute lymphoblastic leukemia. Methotrexate concentrations were measured 7 days after the last doses of intrathecal and oral routes, using patients as their own controls. Intrathecal methotrexate was given on an age-adjusted schedule. Data was normalized to the actual dose received per body surface area. Results The mean red cell methotrexate concentration 7 days after the last of four weekly intrathecal doses of methotrexate was 178 pmol/mL red blood cells, which was significantly greater than the result 7 days after subsequent weekly oral methotrexate of 122 pmol/mL (P = 0.00001). Intrathecal dosing resulted in an average systemic exposure ratio of 1.7 to 1 compared with oral administration. Conclusion Intrathecal methotrexate administration results in significantly greater systemic exposure than oral administration. Our data support the hypothesis that the systemic effect of intrathecal methotrexate affects ALL therapy.

An Experimental Model for Measuring Middle Ear Antimicrobial Drug Penetration in Otitis Media

Bacteria are an important cause of acute otitis media and successful treatment depends on achieving inhibitory or bacteriacidal antimicrobial drug concentrations in the middle ear. To evaluate further otitis media treatment success and failure, we developed a chinchilla model to study antimicrobial drug penetration through the middle ear mucosa. Using quantitative histomorphometry, we measured the middle ear space in 10 chinchillas and found a mean ±SD volume of 2.09 ± 0.08 ml and a mean SD surface area of 14.41 ± 1.48 cm2. To measure the apparent rate constant (Ke) of antibiotic elimination from the middle ear, through the middle ear mucosa, an antibiotic solution was inoculated into the middle ear cavity, and samples were aspirated between 1 and 8 hr later. In normal ears, the mean Ke ±SD for amoxicillin was 0.118 ± 0.013 hr−1, that for a trimethoprim 0.461 ± 0.090 hr−1, and that for sulfamethoxazole 0.265 ± 0.062 hr−1. In ears inoculated with type 7F Streptococcus pneumoniae to induce acute otitis media, the Ke ±SD increased for all three drugs (P < 0.05): amoxicillin to 0.286 ± 0.089 hr−1, trimethoprim to 0.662 ± 0.118 hr−1, and sulfamethoxazole to 0.411 ± 0.056 hr−1. These values demonstrate that amoxicillin had the lowest apparent penetration rate constant of the three antibiotics but the greatest increase from normal to infected mucosa (142%). Trimethoprim had the highest apparent penetration rate constant of the three antibiotics but the smallest increase from normal to infected mucosa (44%), while the sulfamethoxazone apparent penetration rate constant increased from normal to infected mucosa by 55%. The Ke for amoxicillin was the same for inoculation volumes of 0.8 and 1.6 ml (P = 0.557) and the same for sampling intervals of 4 and 8 hr (P = 0.054). All three antimicrobial drug concentration–time curves were log-linear, as predicted by Ficks first law of diffusion. In conclusion, this model overcomes the technical limitations of previous models and permits investigation of the many factors that can influence antibiotic penetration into the middle ear and reduce otitis media treatment efficacy.

Mizoribine pharmacokinetics and pharmacodynamics in a canine renal allograft model of local immunosuppression

We utilized a canine renal transplant model to estimate the first-pass extraction of mizoribine (MZB) during renal artery infusion and to compare the efficacy and toxicity of continuous intraarterial (i.a.) versus intravenous (i.v.) MZB delivery, with and without a background of oral cyclosporine. Five autotransplanted mongrel dogs with programmable, implantable pump/catheter systems were given MZB by both i.v. bolus (5 mg/ kg) and i.a. infusion (5.0 mg/kg/day). Mean ± SD elimination half-life was 3.02±0.81 hr, and the transplanted kidney removed as much as 47–59% (mean 56%) of locally infused MZB. With increasing local and systemic MZB delivery in a single autografted dog undergoing both i.a. and i.v. pump/catheter placement, renal extraction decreased from at least 47% (5.0 mg/kg/day) to 33% (7.5 mg/kg/day), and finally to 18% (10.0 mg/kg/day). A dose of 3.0 mg/kg/day MZB did not significantly prolong survival of renal allograft recipients over that of untreated controls (median survival time [MST]=8 days) when administered either locally (MST=9 days) or systemically (MST=12 days). All dogs receiving 4.0 mg/kg/ day MZB i.a. died from rejection, and a survival advantage was still not realized (MST=7 days). In contrast, 4.0 mg/kg/day i.v. prolonged survival over controls (MST=14 days; P=0.03) but not when directly compared with the i.a. group (P=0.30), and produced death from severe debility in five of seven animals with significantly higher mean systemic MZB levels (P=0.02). Four of six dogs receiving 5.0 mg/kg/day MZB i.a. (MST=14 days) and two of four dogs receiving 5.0 mg/kg/day i.v. (MST=14 days) died from severe debility, though survival in both groups was prolonged over control values (P=0.01 and P=0.05, respectively). Coad-ministration of a subtherapeutic dose of oral CsA (5 mg/ kg/day) significantly prolonged the overall survival of dogs receiving MZB 4.0 mg/kg/day i.a. (MST=23; P=0.01) but not i.v. (MST=11; P=1.00), so that a significant difference in overall survival between the combined MZB i.a. + CSA and MZB i.v. + CSA groups was now realized in favor of the former (P=0.04). We conclude that at local doses required to achieve immunosuppression, the transplanted kidney was not able to extract enough MZB to prevent death from systemic toxicity, presumably as a result of saturation of renal elimination mechanisms, so that an overall survival benefit was not realized. However, combination of oral CsA with i.a., but not i.v., MZB infusion conferred a survival advantage with lower systemic MZB concentrations, suggesting mediation via a local immunosuppressive effect.

Substitution of oral and intravenous thioguanine for mercaptopurine in a treatment regimen for children with standard risk acute lymphoblastic leukemia: A collaborative Children's Oncology Group/National Cancer Institute pilot trial (CCG‐1942)

Although mercaptopurine (MP) is conventionally used to treat childhood acute lymphoblastic leukemia (ALL), thioguanine (TG) is a more potent thiopurine in vitro and, when administered orally to patients, achieves cytotoxic drug concentrations in the cerebrospinal fluid (CSF). We performed a pilot study incorporating oral and 24‐hr continuous IV infusion (CIVI) TG in children with newly diagnosed standard‐risk ALL.

Reversed-phase high-performance liquid chromatographic approach to determine total lymphocyte concentrations of 6-thioguanine, methylmercaptopurine and methylthioguanine in humans.

A reversed-phase high-performance liquid chromatographic (HPLC) procedure was developed to quantify intracellular lymphocyte 6-thioguanine, methylmercaptopurine and methylthioguanine. The free base of each metabolite was obtained by acid hydrolysis, which allowed for a total determination of thiopurine metabolites. 6-Thioguanine was analyzed on an octadecylsilane column using acetonitrile-10 mM sodium phosphate (11:89), pH 7, containing 0.06% tetrabutylammonium chloride. 6-Thioguanine was oxidized with potassium permanganate, and fluorescence was measured at 330 nm excitation and 410 nm emission. Methylmercaptopurine and methylthioguanine were separated on a cyanopropylsilane column using methanol-40 mM sodium phosphate (22:78), pH 2.7, and detected by ultraviolet absorbance at 314 and 290 nm, respectively.

Local immunosuppression with reduced systemic toxicity in a canine renal allograft model

We compared the efficacy of continuous intraarterial versus intravenous 6-mercaptopurine (6-MP) infusion in a mongrel canine renal allograft model with regard to overall survival, incidence of systemic and renal toxicity, and systemic drug exposure. Arterial anastomoses were done end-to-end, and infusion catheters were placed in the iliac artery or vena cava and connected to a subcutaneously placed programmable pump. A dose of 0.5 mg/kg/day 6-MP did not prolong survival over heparin-treated or untreated controls (MST = 7 days for both groups) when administered either locally or systemically. However, 0.75 mg/kg/day 6-MP i.a. (MST = 20 days) significantly prolonged survival over both untreated (P = 0.007) and heparin-treated controls (P = 0.02), with all dogs eventually dying of rejection. In contrast, 0.75 mg/kg/day i.v. (MST = 7 days) failed to prolong survival over controls (P greater than 0.1) and produced death from systemic toxicity in 3 of 7 animals. Six of 7 dogs receiving 2.0 mg/kg/day 6-MP i.a. (MST = 12 days) developed azotemia secondary to drug-induced nephrotoxicity. Identical renal histologic changes occurred in the same time frame in autotransplants treated similarly. Of 7 animals receiving 2.0 mg/kg/day i.v. (MST = 12 days), 5 died from early, severe systemic drug toxicity and 2 from early rejection. During 6-MP infusion at 0.5 mg/kg/day, systemic exposure was significantly less in the locally treated than in the systemically treated dogs when Cr concentrations were normal or moderately elevated (P less than 0.0005 and P = 0.01, respectively) but not when renal function became severely impaired (P = 0.34). In contrast to i.v. infusion, i.a. 6-MP delivery dissociated immunosuppressive efficacy from systemic toxicity, supporting previous work demonstrating high first-pass renal elimination of 6-MP. We conclude that tightly controlled local delivery of an immunosuppressive agent can effectively prolong graft survival with reduced systemic toxicity in a large animal model employing a pump/catheter system applicable to man.

Pharmacokinetics of 6-thiouric acid and 6-mercaptopurine in renal allograft recipients after oral administration of azathioprine

SummaryThe immunosuppressive activity of azathioprine (AZA) is unpredictable and depends on the formation of intracellular thiopurine ribonucleotides. However, the quantification of these active thiopurines presents difficult analytical problems. It has recently been postulated that plasma concentrations of 6-thiouric acid (6-TU) and 6-mercaptopurine (6-MP), metabolites of AZA, may provide more readily measurable indices of the pharmacologic activity of AZA. In order to evaluate the utility of 6-TU and 6-MP plasma concentrations in monitoring AZA therapy, we studied their pharmacokinetics in 6 renal transplant patients, and their in vitro immunosuppressive potency in a mixed lymphocyte proliferation assay.A peak plasma 6-TU concentration of 710.7 ng/ml was observed at 3.8 h after oral dosing. Good correlation was observed between the elimination t1/2 of 6-TU and serum creatinine, and between AUC over 24 h and serum creatinine. However, we did not observe a second peak in plasma 6-TU concentration that could be attributed to the degradation of active AZA metabolites. 6-MP plasma concentrations in the patients were low (mean peak concentration 36.0 ng/ml) and rapidly disappeared within 8 h. In vitro immunosuppressive activity could not be demonstrated for 6-TU over a concentration range of 1.25 ng/ml to 0.25 mg/ml.We conclude that 6-TU is pharmacologically inert and is primarily eliminated by the kidneys. Our findings currently do not support the use of plasma concentrations of 6-TU or 6-MP to monitor AZA therapy. In order to optimize AZA therapy, analytical techniques that are technically feasible and that can directly quantify the active intracellular thiopurines are being explored.

An Implantable Pump for Intrarenal Infusion of Immunosuppressants in a Canine Autotransplant Model

We developed a canine renal allograft model utilizing implantable infusion pumps and biocompatible catheters to investigate the pharmacokinetics of local immunosuppressive drug administration. Seven mongrel dogs underwent bilateral nephrectomy and autotransplantation of one kidney to the iliac vessels. The proximal end of an infusion catheter directed into the iliac artery was tunneled to a subcutaneously placed programmable pump. A second, sampling catheter was placed with its tip in the iliac vein. Simultaneous regional (iliac vein) and systemic (jugular vein) venous concentrations of 6-mercaptopurine (6-MP), the immunosuppressive metabolite of azathioprine, were determined during a continuous 24-h intraarterial infusion (10 mg/kg/24 hr). The gradient between regional and systemic 6-MP concentrations was maximal initially when the pump was turned on, continuously decreased until steady state was reached, and disappeared immediately after the pump was turned off. The mean ratio of steady-state iliac vein to systemic 6-MP concentrations was 5.0 ± 1.4, demonstrating a pharmacokinetic advantage of continuous intraarterial 6-MP infusion to the autotransplanted kidney. The novel canine renal allograft model described herein overcomes the technical limitations of earlier models and represents a foundational step in the design of intrarenal infusion patterns of immunosuppressive agents which we expect to prolong survival of the allotransplanted kidney with minimal systemic drug exposure and side effects.