Abstract
Pyridorin®, a naturally occurring metabolite of vitamin B6 that inhibits and scavenges reactive oxygen species, is being developed as a potential therapeutic for acute kidney injury. An investigational new drug application (IND) was opened for Pyridorin in support of its ongoing oral drug clinical development program. Currently, a Pyridorin intravenous (IV) formulation is being developed for use in surgical patients. To support the IND for Pyridorin, a full battery of nonclinical Good Laboratory Practice compliant studies was performed with no neurological or behavioral signs of toxicity seen following oral or IV administration of pyridoxine dihydrochloride (the active ingredient in Pyridorin). However, excessive ingestion of vitamin B6 has been reported to cause neurotoxic syndrome in humans. Therefore, under Food and Drug Administration recommendation, a 7-day IV study in rats was conducted to further evaluate the drug’s potential to cause neurotoxicity. Blood plasma samples indicated that exposure to pyridoxamine dihydrochloride and its metabolites, pyridoxal, pyridoxine, and 4-pyridoxic acid was linearly dose proportional and independent of gender. At doses of up to 200 mg/kg/day pyridoxine dihydrochloride, no treatment-related effects were seen in rats, providing further evidence for the absence of pyridoxine dihydrochloride-related changes in the nervous system. A no observed adverse effect level of 200 mg/kg/day was identified for this study.
Introduction
Acute kidney injury (AKI) commonly occurs in hospitalized patients, with its true prevalence being difficult to enumerate because of the lack of a universal definition. 1,2 However, new definitions which allow a more standardized classification have been proposed. 1,3 –5 These new definitions are based on an increase in the concentration of creatinine in serum or reduction in urine output. These definitions reflect the fact that even a relatively small increase in concentration of creatinine in serum is associated with adverse patient outcomes, 1,6,7 including increases in length of hospital stay, mortality, and costs. Currently, there is no therapeutic intervention for chronic outcomes such as chronic kidney disease (CKD).
Incomplete recovery from a severe episode of AKI is a well-recognized pathway to persistent and progressive CKD. 8 Interestingly, more recent studies have demonstrated that even apparent complete recovery from AKI is associated with a subsequent risk for CKD development. 9 –11 Thus, an intervention that would result in a reduction in a chronic outcome such as CKD development would be an important advance over current therapies.
The estimated incidence of AKI in patients undergoing cardiac surgery is 11–30%. 11 Severe AKI requiring renal replacement therapy occurs in an estimated 1–2% of these patients. Therefore, patients undergoing elective cardiac surgery represent a population worthy of investigation for a prophylactic treatment, and a trial is planned with an intravenously administered form of Pyridorin®, a drug originally designed by BioStratum, Inc. (Durham, NC), to prevent the progression of diabetic nephropathy. The active ingredient in Pyridorin is pyridoxamine dihydrochloride. Pyridoxamine is a naturally occurring and structurally distinct metabolite of vitamin B6 pyridoxal phosphate. It is present in the body at extremely low concentrations. At pharmacological doses, it inhibits and scavenges a broad range of pathogenic oxidative chemistries that develop in patients with type 2 diabetes and cause irreversible kidney damage.
Pyridorin is being developed as an oral therapeutic agent by NephroGenex, Inc. (US IND Number: 58,684, Raleigh, NC). Additionally, NephroGenex, Inc. is also studying the application of an injectable formulation of Pyridorin for the treatment of specific types of AKI where pathogenic oxidative chemistries have been identified as a possible contributing factor to the severity of this condition.
Pyridorin intravenous (IV) formulation is being developed to be administered as part of the patient’s preoperative, operative, and postoperative care. Given the limited half-life of the orally administered Pyridorin, an IV formulation is preferable in order to carefully control and maintain constant plasma levels before, during, and after surgery.
An extensive nonclinical safety program was conducted by BioStratum (the predecessor to NephroGenex) to support the opening of IND 58,684 under which there is an ongoing oral drug clinical development program, to monitor plasma levels of drug well above those achieved in oral studies or that might potentially be achieved in proposed oral studies. Additionally, IV administration studies were also conducted to confirm route-specific safety as well as a single IV dose study. No neurologic or behavior signs have been seen with the drug by oral or single IV dose administration. However, some years ago, excessive ingestion of vitamin B6 (pyridoxine) when used as a dietary supplement at doses up to 2 g/day was reported to cause neurotoxic syndrome in humans characterized (clinically) by paresthesia, muscle weakness, and numbness most pronounced in the extremities. 12 In Beagle dogs, 150 mg/kg/day of orally administered pyridoxine for 100 days has been linked to a neurologic syndrome characterized clinically by ataxia, which correlated to degeneration of nerve fibers (axons) from sensory ganglia neurons. 13,14
Accordingly, with feedback from the Food and Drug Administration, a 7-day IV administration study of pyridoxamine dihydrochloride in male and female Sprague-Dawley rats was designed to specifically evaluate the potential for the drug to cause neurotoxicity. Neurologic examination of animals during in-life and histopathological evaluation of the peripheral nervous system was conducted. The resulting study and its outcome are described here.
Materials and methods
Test article
Pyridoxamine dihydrochloride was supplied by NephroGenex, Inc. (Raleigh, North Carolina, USA) as a white powder. Formulations were prepared daily by dissolving pyridoxamine in saline solution. The pH value was initially adjusted with 1 N sodium hydroxide (NaOH) and 0.1 N NaOH to a pH of 6 ± 0.1. On the first day of dosing, the pH for the preparation of the low- and high-dose formulations went above 6 ± 0.1. So, 1 N hydrochloric acid (HCl) was used to adjust the pH. The dosing formulations were filter sterilized using 0.2 μM acetate membrane filters and were put in sterile amber glass vials protected from light.
Chemicals and materials
Saline solution (0.9% NaCl in sterile water for injection) was supplied by Abbott Laboratories (Lake Bluff, Illinois, USA). One (1) N NaOH and 0.1 N NaOH were supplied by Ricca (Arlington, Texas, USA) and Calvert Laboratories (Scott Township, Pennsylvania, USA), respectively. One (1) N HCl was supplied by Sigma-Aldrich (St Louis, Missouri, USA). Filtration sterilization filters (0.2 μM acetate membrane) were supplied by Thermo Fisher Scientific (West Palm Beach, Florida, USA).
Animals
A total of 162 (81 males and 81 females) cannulated Sprague-Dawley (Hsd:SD) rats obtained from Harlan (Indianapolis, Indiana, USA) were individually housed in suspended wire cages upon receipt and assignment to study in compliance with National Research Council Guide for the Care and Use of Laboratory Animals. 15 Study animals were acclimated to their housing for a minimum of 1 day prior to their first day of dosing. Room temperature was maintained at 20.7–23.8°C with a 12-h light and 12-h dark cycle, except for when room lights were turned on during the dark cycle to accommodate blood sampling or other procedures. Drinking water and commercial rodent diet (Harlan Teklad certified rodent diet) were provided ad libitum. All animals were assessed as to their general health prior to study initiation. During the acclimation period, each animal was observed at least once daily for any abnormalities or for development of infectious disease. Only animals that were suitable for use were assigned to the study.
Experimental design
One hundred sixty-two (162) experimentally naïve SD rats were used. The study had two portions: the toxicity study (main study/80 rats) and the toxicokinetic study (TK study/82 rats). Main (toxicity) study: Following acclimation, 80 SD rats (40 males and 40 females) were randomly assigned to four groups, which were intravenously dosed (30-min infusion) for seven consecutive days with either vehicle or one of the three pyridoxamine dose solutions (12.5, 25, and 50 mg/mL). The daily dose volume was set at 4 mL/kg for all four groups. The daily pyridoxamine dose levels were 50, 100, and 200 mg/kg/day, respectively.
For the TK portion of the study, 82 SD rats (41 males and 41 females) were randomly assigned to four groups with 5 rats per gender assigned to the vehicle control group and 12 rats per gender for each pyridoxamine dihydrochloride dose group of 50, 100, or 200 mg/kg/day. All animals received seven consecutive daily IV infusions (over 30 min/day). Blood samples for TK analyses were collected from TK animals on days 1 and 7 at the following time points: pre-dose, and 15, 30, 60, 90, 120, 180, 240, 480, and 720 min following dose administration.
All rats were observed twice daily for signs of morbidity and mortality and once daily for clinical signs until study termination. Body weights were recorded prior to the initial dosing, on days 0 and 7, and on day 8 prior to termination. Full feeder weights and feeder weigh backs were recorded on days 1 and 7 for determination of food consumption. For all main study rats, a functional observational battery test as described elsewhere was conducted prior to dose initiation (baseline) and on day 7 at approximately 1–2 h post-dose. 16 On day 8, rats were euthanized by carbon dioxide asphyxiation. After the animals were killed, blood samples for evaluation of hematology, coagulation, and serum chemistry parameters were collected, and the organs and tissues were removed, weighed, and examined for gross and microscopic pathological anomalies.
Data collection and statistical analyses
In-life data (clinical observations, body weights, feeder weights, dose administration, and other related data) were collected using Pristima version 6.3.2 or on paper when necessary. Hematology data were collected using the Advia 120 (Siemens Medical Solutions USA, Inc., Malvern, PA) Hematology Analyzer. Coagulation data were collected using the Diagnostica STA (Diagnostica Stago, Inc. Parsippany, NJ) Compact Coagulation Analyzer. Clinical chemistry data were collected using the AU400 (Olympus, Tokyo, Japan) Chemistry Analyzer. Once collected on their respective instruments, hematology, coagulation, and clinical chemistry data were transferred to Pristima version 6.3.2 via a validated interface. Necropsy data were collected on paper. Any other data not collected online were manually tabulated for inclusion in the report.
In-life data, clinical pathology data, and necropsy data were tabulated and statistically evaluated using Pristima version 6.3.2. Pristima software was developed by Xybion Corporation (Morris Plains, New Jersey, USA).
Statistical evaluation was performed on in-life, clinical pathology, and organ weight numerical data. For in-life, clinical pathology, and organ weight parameters, the software determined statistical significance by following a decision tree. First, the homogeneity of the data was determined by Bartlett’s test. If the data were homogeneous, a one-way analysis of variance was performed to assess statistical significance. If statistically significant differences between the means were found, Dunnett’s test was used to determine the degree of significance from the control means (p < 0.05, p < 0.01, and p < 0.001). If the data was nonhomogeneous, the Kruskal–Wallis nonparametric analysis was performed to assess statistical significance. If statistically significant differences between the means are found (p < 0.05, p < 0.01, and p < 0.001), the Mann–Whitney U test was used to determine the degree of significance from the control means (p < 0.05, p < 0.01, and p < 0.001).
Results
No treatment-related mortality was seen during the study. One mid-dose male in the TK group was found dead immediately post-dose on day 3 with gross necropsy findings indicating the presence of red fluid in the thoracic and lumbar areas. All organs appeared normal, and the cannula appeared intact and was not dislodged or dislocated. It is believed that during handling of the animal while dosing the end of the cannula tore the vessel wall causing internal bleeding and subsequent death. No clinical signs were observed during the treatment period. Body weights and body weight gains are summarized in Table 1. No test article effects on body weight were seen. At day 7, the mean bodyweight gain was lower in 100 mg/kg/day females when compared to controls. However, normal mean body weight gain relative to control was seen in females administered 200 mg/kg/day Pyridorin. Therefore, there is no dose-related decrease in body weight gain and this finding is considered incidental. There were no treatment-related effects on food consumption. All animals appeared normal during the functional observation battery tests.
Summary of body weights and body weight gains.a
BW: body weight.
aAll data are presented as mean values ± standard deviations with N = 10; For BW gain, percent increase in body weight from day 1 to day 7 is included in parenthesis.
bStatistically significant from the vehicle control group (p ≤ 0.01).
Statistically significant clinical pathology findings are summarized in Table 2. The mean hemoglobin and hematocrit concentrations were significantly lower in females given ≥50 mg/kg/day while the mean corpuscular hemoglobin concentration was significantly lower in females given 100 mg/kg/day when compared to control females. The mean neutrophil counts were significantly higher in females given 100 and 200 mg/kg/day, while the mean relative reticulocytes and large unstained cells were significantly higher in females given 100 and 200 mg/kg/day, respectively. These values were not considered adverse because of the small magnitude of change and the differences occurred only in the females. Rare to slight polychromasia (+1) was noted in males (including controls) while rare to moderate polychromasia (+1 to +2) was noted in all females (including controls). No changes were seen for prothrombin time and activated partial thromboplastin time or urinalysis parameters. Decreased mean sodium and creatinine were noted in 50 mg/kg/day males and 200 mg/kg/day females, respectively. Based on the small magnitude of these changes and the differences occurring only in one sex, they were not considered adverse. It is concluded that there were no adverse treatment-related effects on any clinical pathology parameters.
Summary of statistically significant clinical pathology values.a
HGB: hemoglobin; HCT: hematocrit; MCHC: mean corpuscular hemoglobin concentration; #Neut: absolute neutrophils; %Neut: percent neutrophil; %LUC: percent large unsustained cells; #LUC: absolute large unsustained cells; %Retic: percent reticulocytes; #Retic: absolute reticulocytes; Creat: creatinine; Na: sodium.
aHematology and clinical chemistry data are presented as mean values ± standard deviations (n). Red blood cell morphology data are presented as number examined/number abnormal (grade). For grade: 1 = rare slight; 2 = moderately; 3 = marked.
bStatistically significant from the vehicle control group (p ≤ 0.05).
cStatistically significant from the vehicle control group (p ≤ 0.01).
No treatment-related changes were seen for absolute or relative organ weights, gross necropsy findings or microscopic findings, including absences of nerve fiber degeneration in the spinal cord, spinal nerve roots, or peripheral nerves.
Toxicokinetics
The TK parameters for pyridoxamine and its metabolites (pyridoxal, pyridoxine, and 4-pyridoxic acid) are summarized in Tables 3 and 4, respectively. Based on observed maximum plasma concentration (C max) and partial area under then curve (AUCpartial), plasma concentrations of pyridoxamine increased roughly proportionally with increasing dose, reaching a C max of 85 µg/mL in female rats on day 1. No obvious differences were seen in gender or time on study. Half life (T ½) values ranged from 1.2 h to 3.7 h. Accumulation would not be expected with single daily dosing. Clearance values ranged from 1.0 L/(h.kg) to 1.3 L/(h.kg) and volume estimates ranged from 1.9 L/kg to 5.2 L/kg. Again, there were no obvious patterns.
Toxicokinetic parameters of pyridoxamine on day 1 following a single IV dose and on day 7 following seven consecutive daily IV infusions (30 min) of pyridoxamine.
NE: not estimable; IV: intravenous.
Toxicokinetic parameters of pyridoxamine metabolites on day 1 following a single IV dose and on day 7 following seven consecutive daily IV infusions (30 min) of pyridoxamine.
NE: not estimable; IV: intravenous.
Female and male plasma concentrations of pyridoxal remained consistent across doses and day of dosing, reaching a maximum of 2.4 µg/mL in females receiving 100 mg/kg/day on day 7. Maximum values for C max (2.4 µg/mL) and AUCpartial (20 µg.h/mL) were 3% and 9%, respectively, of maximum pyridoxamine values (85 µg/mL and 215 µg.h/mL). Although concentrations appeared slightly higher in females, there was a less than twofold difference in the C max and AUCpartial ranges at 200 mg/kg/day. T ½ values were obtained in only 5/13 profiles and ranged from 5 h to 30 h.
Concentrations of pyridoxine in males remained consistent across doses and day of dosing (although slightly elevated on day 7), reaching a maximum of 29 ng/mL on day 7 after receiving 100 mg/kg/day doses of drug. Concentrations in females tended to be somewhat higher, reaching 47 ng/mL on day 7 after 50 mg/kg/day. For females, AUCpartial values tended to increase with increasing dose, but not in a dose proportional manner, reaching a maximum of 485 ng.h/mL. Concentrations (as measured by C max) were, however, consistent across doses over 7 days. Maximum values for C max and AUCpartial were about 0.05% and 0.2%, respectively, of pyridoxamine values.
Exposure to 4-pyridoxic acid was uniform over dose amount, gender, and day of dosing. C max concentrations reached 445 ng/mL in male rats receiving 100 mg/kg drug on day 1. Maximum values for C max and AUCpartial were 0.5% and 1%, respectively, of pyridoxamine values.
Discussion
Sensory neurons are pseudounipolar, meaning they have one main appendage (an axon). These axons are typically relatively long, some coursing from the sensory site (peripheral skin/soft tissue/organs) through the peripheral nerves, continuing through the dorsal spinal nerve roots, entering the spinal cord, and coursing to the brain via the dorsal tracts (fasciculus gracilis and fasciculus cuneatus) to the nucleus gracilis and nucleus cuneatus in the brain stem. Some sensory axons synapse in the spinal cord gray matter, forming local reflex arcs. This study comprehensively evaluated the nervous system and included evaluation of sites pertinent to the sensory (afferent) components of the peripheral and central portions of the nervous system including (1) longitudinal paraffin sections and osmicated cross sections of peripheral nerves; (2) cervical, thoracic, and lumbar sections of the spinal nerve/spinal nerve roots and ganglia; 3) cervical, thoracic, and lumbar sections of the spinal cord; and (4) the brain and brain stem. No treatment-related morphologic changes were observed in the nervous system.
In one high-dose female, there was a focal area of nerve fiber degeneration and Schwann cell hyperplasia in a dorsal nerve root (cervical). Because these changes were unique in this animal (i.e. did not occur in other animals), occurred in only a single dorsal nerve root (i.e. was not bilateral; did not occur at the thoracic or lumbar levels in this animal), the change was interpreted to be due to a focal injury at this level without any relationship to treatment with pyridoxine dihydrochloride.
Inflammation at the catheter emptying site was variable, but this is a common occurrence at any implant site and is not indicative of a treatment-related effect. Changes in the spleen (increased germinal centers) were present in one control male and one high-dose female and were strictly secondary to the inflammatory reactions at the catheter emptying sites.
In summary, SD male and female rats were exposed to pyridoxamine dihydrochloride and its metabolites pyridoxal, pyridoxine, and 4-pyridoxic acid following single, 30-min IV infusions of pyridoxamine dihydrochloride at 0, 25, 50, 100, or 200 mg/kg/day. Exposure to pyridoxamine dihydrochloride appeared to be linearly dose proportional. Exposure to pyridoxal, pyridoxine, and 4-pyridoxic acid was a small fraction of the exposure to parent drug, comprising 9%, 0.2%, and 1%, respectively, of the AUCpartial of pyridoxamine dihydrochloride. For the most part, exposure to the metabolites was independent of dose, gender, and days of dosing, suggesting that the metabolism of pyridoxamine to pyridoxal was saturated at the lowest dose and was rate limiting for subsequent metabolic steps.
Administration of up to 200 mg/kg/day (males and females) pyridoxamine dihydrochloride via IV infusion for seven consecutive days was not associated with any treatment-related findings including gross or microscopic findings in the nervous system (brain, spinal cord, spinal nerve roots/ganglia, and peripheral nerves), liver, kidneys, heart, spleen, thymus, pancreas, or the catheter emptying site. Based on clinical observations, functional observational battery tests, changes in body weight, food consumption, and clinical pathology and histopathology findings, the no observed adverse effect level in SD rats was determined to be 200 mg/kg/day.
Footnotes
Declaration of Conflicting Interests
The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: DW Sullivan, Jr., CV Mujer, and SC Gad declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. RC Peterson was an employee of NephroGenex, Inc.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by NephroGenex, Inc.