Two clinical trials monitored methemoglobin after 3-AP infusion (). In the first Phase I dose-escalation clinical trial in patients with advanced solid cancers [20
], 3-AP was administered intravenously over 2–4 h at dose levels of 105, 140 or 185 mg/m2
on days 1, 8 and 15 of each 28-day cycle. In this study, gemcitabine was also given over a 30-min intravenous infusion 1–4 h after 3-AP infusion at a dose level of 0–1000 mg/m2
. Of 26 patients, a total of six manifested dyspnea at approximately the time of 3-AP infusion. In three patients, methemoglobin levels were determined and were 12, 12 and 11%. A suggested association of 3-AP-related dyspnea was claimed.
Because of this, a second Phase I dose-escalation clinical trial in patients with advanced-stage cervical cancer tracked serum levels of 3-AP and methemoglobin [15
]. In this trial, 3-AP (25 or 50 mg/m2
) was given as a three-times weekly intravenous 2-h infusion during once-weekly cisplatin (40 mg/m2
) and daily radiation. Here, 3-AP serum concentrations were measured in heparinized blood samples by high-pressure liquid chromatography tandem mass spectrometry with parallel online turbulent flow extraction and positive ion selected reaction monitoring [15
]. Samples were taken on day 1 and day 10 of treatment before, and at 2, 4, 6 and 24 h after the start of the 2-h 3-AP infusion (). For procedural detail, serum concentrations of 3-AP were determined according to a modified internally standardized high-pressure liquid chromatography UV procedure provided by the manufacturer [JD Talton
., FL, USA, Pers
.]. Linear calibration curves of 3-AP:internal standard peak height ratios versus 3-AP concentration were established over a concentration range of 0.02–10 μg/ml of 3-AP in serum for each analyzed batch of serum specimens. Quality-control specimens at low (0.05 μg/ml), medium (0.8 μg/ml), and high (8.5 μg/ml) concentrations of 3-AP were determined concurrently with each analytical specimen batch. Permissible control ranges were ± 20, ± 15 and ± 15% of nominal concentration for the low-, medium- and high-concentration quality-control specimens, respectively. Typical within-day precision for replicate determinations of the low-, medium- and high-concentration quality-control specimens was 6–21, 5–8 and 2–6%, respectively (relative standard deviation [SD], n = 6 within-day replicates, 3 days of results compared). Interday precision for six replicate determinations of the low-, medium-and high-quality-control specimens during method validation studies was 21, 7 and 5%, respectively (relative SD, n = 3). Corresponding time-point serum methemoglobin concentrations were determined by direct spectrophotometry with a verified detection range of 0–100% [27
]. Methemoglobin specimens were drawn into heparinized arterial blood-gas syringes on wet ice and measured within 10 min of procurement.
3-aminopyridine-2-carboxaldehyde thiosemicarbazone pharmacokinetics and methemoglobin pharmacodynamics
Patient 3-AP and methemoglobinemia levels were measured in blood samples collected for pharmacokinetic evaluation from ten patients with advanced-stage cervical cancer. A total of 20 treatment courses (ten on day 1 and ten on day 10) were evaluated for each of two dose levels of 3-AP (25 mg/m2 [n = 6] or 50 mg/m2 [n = 4]) studied. Maximal plasma concentrations (Cmax) of 3-AP were measured at the termination of the 2-h intravenous 3-AP infusion. The mean 3-AP peak plasma concentration was 262 ng/ml (SD = 51 ng/ml) at the 25 mg/m2 dose level and 560 ng/ml (SD = 59 ng/ml) at the 50 mg/m2 dose level. The mean elimination half-life was 2 h, with no change in elimination half-life between day 1 and day 10 for either the 25 mg/m2 (p = 0.18) or 50 mg/m2 (p = 0.35) dose levels. The plasma concentration of 3-AP 6 h after the start of the 2-h infusion fell to 2% Cmax (25 mg/m2) and 13% Cmax (50 mg/m2). The corresponding peak methemoglobin level was 1% (SD = 0.6%) at the 3-AP 25 mg/m2 dose level and was 6% (SD = 2.8%) at the 3-AP 50 mg/m2 dose level.
Although patient numbers were small in the two clinical trials performed, these two trials suggest that higher doses of 3-AP lead to decreased mean oxygen saturation and higher peak methemoglobin levels (). For example, the peak methemoglobin levels recorded in one of these studies follow peak serum 3-AP levels by a 2 h delay (). The elevated methemoglobin levels usually resolved within 2 h of the peak (). Repeated 3-AP dosing had no substantial additive effect (3-AP at 25 mg/m2: day 1 mean [SD] = 1% [0.6% ], day 10 mean [SD] = 0.9% [0.4% ]; 3-AP at 50 mg/m2: day 1 mean [SD] = 6% [2.8%] day 10 mean [SD] = 4.7% [2.2% ]), suggesting that mechanisms responsible for recycling methemoglobin were not irreversibly impaired.
For clinical and interpretative context, chemically-induced methemoglobinemia occurs most commonly from inhalation or tactile exposure to oxidizing chemical agents. Agents known to induce methemoglobinemia up to 15% after prolonged exposure, include acetonitrile (e.g., nail varnish remover), anesthetics (e.g., lidocaine, prilocaine), aniline dyes, chlorates (e.g., matches, explosives, weed killers), naphthalene (e.g., moth balls), volatile nitrites, phenazopyridine (e.g., pyridium), quinones (e.g., chloroquine and primaquine), sulfonamides (e.g., sulfamethoxazole) and dapsone. Mechanisms of methemoglobin induction involve direct oxidizing effects on the hemoglobin or generation of oxygen and peroxide free-radicals capable of oxidizing hemoglobin.