Treatment of a long-acting anticoagulant rodenticide poisoning cohort with vitamin K1 during the maintenance period

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Introduction
Long-acting anticoagulant rodenticides (LAARs) have been widely used in agriculture, forestry, and animal husbandry, resulting in an increase in anticoagulant rodenticide poisonings, suicides, and misuse. [1][2][3] According to the domestic reports of Wang and Jiang, [4] patients with LAAR poisoning accounted for 18% of all poison cases (772/4289) in Jingdezhen City, Jiangxi Province, China from 1996 to 2005. Internationally, similar poisoning cases [5] registered by American Poison Control Centers in 2012 reached 9555 persons. Aside from the fecal-oral route, the most common route of exposure to anticoagulant rodenticide toxicants is absorption through the skin. [6] LAARs can influence the vitamin K cycle by inhibiting vitamin K epoxide reductase (VKOR), resulting in decreased synthesis of hepatic blood coagulation factors II, VII, IX, and X. [7] Clinical examination [8,9] showed that LAARs can significantly prolong prothrombin time (PT) and activated partial thromboplastin time (APTT). The treatment for anticoagulant rodenticide poisoning [5] primarily includes administration of vitamin K1, fresh frozen plasma (FFP), prothrombin complex, and recombinant coagulation factor VIIa. Lubetsky et al [10] proposed that the curative effect of 5 mg vitamin K orally administered is equivalent to 1 mg vitamin K1 intravenously administered. However, there are no oral vitamin K1 preparations available in China. Therefore, an intermittent, long-term, large-dose vitamin K1 intravenous drip [11] is currently used as the main therapeutic schedule for treating LAAR poisoning, with an intramuscular injection as an adjuvant therapy. The highest oral dose of vitamin K1 has been reported to be 800 mg/d. [12] Because LAARs are highly lipid soluble, [13] the measured half-life of rodenticides in vivo tends to be extremely long, [14,15] with an average treatment time of approximately 168 days. [7] There are no commonly accepted methods for detecting anticoagulant rodenticides, [16] including methods using high-performance liquid phase chromatography (HPLC), and no reliable evidence exists describing how to adjust vitamin K dosages. [17] Therefore, there are no accepted standards for the use of vitamin K as a therapeutic agent. In this paper, we analyzed factors that affect the therapeutic dose of vitamin K1 in the treatment of LAAR poisoning in order to provide improved guidance.

Patients and methods
Patients diagnosed with LAAR poisoning (n = 56) by blood and urine analyses in the emergency department of our hospital from January 2013 to May 2016 were considered for inclusion in this study. Ultimately, 24 cases were included (9 female and 15 male), with an average age of 40.42 ± 19.19 years as shown in Fig. 1. All human participants signed a written informed consent.
Patients who had not received vitamin K1 therapy within 24 h of admission to the hospital, had brodifacoum or bromadiolone detected in their blood or urine, and were treated with a sustained intravenous vitamin K1 drip upon admission to the hospital were included in this study. In addition, included patients had no history of liver disease, had an abnormal international normalized ratio (INR) during the treatment period, and were not currently taking any selective serotonin reuptake inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDs), metronidazole, or cimetidine. [5] Patients who consumed alcohol or had hypoproteinemia during the treatment process were excluded from the study.
Upon admission to the hospital, an initial pulse treatment with vitamin K1 was given to normalize coagulation (INR < 1.5). A maintenance treatment was then initiated, and the type of anticoagulant rodenticide was determined. In addition, the dosage of toxicant, as well as patients' sex, age, bleeding function, blood coagulation function, INR (reference value 1-1.5), PT activity (PTA, reference value 80%-150%), PT (reference value 8.8-12.8 s), APTT (reference value 24.9-36.8 s), vitamin K1 dosage, prehospital time, and vitamin K1 sustained treatment time (VKSTT) were determined.
For statistical analyses, the vitamin K1 dosage was considered to be a dependent variable. Patient age, coagulation function, brodifacoum exposure, bromadiolone exposure, VKSTT, and prehospital time were considered to be independent variables. Continuous variables were investigated for departure from normality by use of the Shapiro-Wilk W test with a = 0.10. For normally distributed outcomes, we conducted a Pearson correlation analysis. For skewed continuous outcomes, we conducted a Spearman correlation analysis. For binary outcomes, we conducted a nonparametric Wilcoxon rank test. Based on the above results, a robust multifactor regression analysis was continuously performed on the selected independent variables (P < 0.05). Results were considered to be significant when P < 0.05.

Results
During the study period from January 2013 to May 2016, 56 patients with LAAR poisoning were admitted to the Affiliated Hospital of Military Medical Sciences. Among them, 24 patients were included in this study ( Fig. 1) and 32 patients were excluded (10 patients received vitamin K1 24 h before hospital admission; 8 patients had an abnormal INR during the treatment period; and 14 patients were discharged from the hospital ahead of schedule because of economic or family conflicts, resulting in no follow-up examinations). Table 1 shows baseline characteristics of patients, and Table 2 shows toxicant concentration, hemostasis, coagulation indices, and dosage of vitamin K1 (all of which correspond to the time when blood and urine samples were taken), as well as the patients' VKSTT (the time from the first day after vitamin K1 was administered until the time of toxicant detection in blood and urine) and prehospital time (the exact time of poisoning or 3 days before the onset of first symptoms [17] ). This study included 24 patients with an average age of 40.42 years (median, 39 years;   (Table 3). Correlations between vitamin K1 and parameters such as PTA, PT, and APTT were analyzed by the Pearson correlation test. Correlations between vitamin K1 and variables such as age, brodifacoum exposure, bromadiolone exposure, VKSTT, and prehospital time were analyzed by the Spearman rank correlation test. Significant correlations were observed among VKSTT, prehospital time, and vitamin K1 (P < 0.05).
Because the studentized residuals distribution of the multiple linear regression model did not conform to the residual normality or homogeneity requirements (refer to Supplemental figure, http://links.lww.com/MD/B425, which shows the studentized residuals distribution of the multiple linear regression model), the authors chose a robust regression analysis method to continue the study. As shown in Table 4, only VKSTT (partial regression coefficient À1.133, 0.59, P = 0.035) showed statistical significance after the robust regression analysis (Prob > F = 0.0415 < 0.05). The regression equation was as follows: y VK1 = 81.435 À 1.133X VKSTT . P values for other factors were >0.05 and were not statistically significant.

Discussion
In this clinical cohort study, the patients were initially treated with large-dose vitamin K1 pulse therapy in order to stabilize the bleeding and coagulation functions. Then, an appropriate dosage of vitamin K1 was adopted as a maintenance therapy as shown in Fig. 2. The concentration of LAAR in patient V was very low; however, a high dose of vitamin K1 (40 mg/d) was still needed. In contrast, the concentration of LAAR in patient V and XXII was high, yet the required dosage of vitamin K1 was similar to that of patient V (40-50 mg/d as static drops), suggesting that there is not a significant dose-effect relationship between the LAAR concentration and vitamin K1 requirements during the maintenance period. These results are consistent with the dosing requirements for vitamin K1 seen in clinical practice. Patient prognoses were good in this cohort, as they all survived. Moreover, the required daily dosage of vitamin K1 (10-120 mg/d, intravenous drip) showed a downward trend that was related to the VKSTT (i.e., vitamin K1 maintenance therapy), but not significantly related to the toxicant concentration.
In patients receiving sustained vitamin K1 treatment, the maintenance dose of vitamin K1 gradually decreased over time as shown by the equation y VK1 = 81.435 À 1.133X VKSTT (effective range: 10-120 mg/d), an effect that was not related to the concentration of LAAR. These results might be attributable to the anticoagulant rodenticide combining with the target of VKOR, [5,7] in which the sustained stimulation of vitamin K1 could increase the expression of VKOR analogs that did not combine with the anticoagulant rodenticides. This would result in a further decrease in vitamin K1 requirements. We did not determine the final concentration of vitamin K1 required by these patients.
Treatments for anticoagulant rodenticide poisonings that have been reported at home and abroad are summarized in Table 5. Gunja et al [18] analyzed the relationship between brodifacoum poisoning and vitamin K1 treatment and reported the INR, poisoning time, brodifacoum concentration, and multidimensional tendency chart of vitamin K1 for 2 cases. Case 1 was treated with a large dose of vitamin K1 (100 mg/d orally administered) sustained over 6 months. When the concentration of brodifacoum reached 5 ng/mL, the vitamin K1 was stopped.    provide little information for establishing guidelines, with the primary outcome being a threshold value of 10 ng/mL brodifacoum; once the concentration of brodifacoum reached less than the 10 ng/mL threshold, vitamin K1 treatment could be stopped.
In this study, we did not evaluate threshold values because of the low incidence of patients exposed to brodifacoum only. There is no oral vitamin K1 preparation available in China; therefore, we adopted a protocol requiring intramuscular injections of vitamin K1 (10 mg/d) plus follow-up treatments instead of infusion therapy during the maintenance period. King et al [7] summarized the treatment of 41 cases of LAAR poisoning. The treatments for each case were similar in the early stages, consisting primarily of FFP (31 cases, 2-6 U, with the majority receiving 2 U) plus vitamin K1 (100 mg/d as an intravenous drip) combined with supportive treatment. During maintenance therapy, vitamin K1 was primarily orally administered at a dosage of 15 to 600 mg/d, with the majority of patients receiving 100 mg/d. Two patients received vitamin K long term by intravenous drip (100 mg/d). A mixed therapy was used in 1 case, in which the patient received 200 mg vitamin K1 orally plus 10 mg subcutaneously twice a day in order to maintain PT at a normal level. The median treatment period was 140 days (average, 168 days; range, 28-730 days).
A large number of studies suggest that anticoagulant rodenticide poisonings require a longer maintenance period; however, the suggested maintenance treatments using vitamin K1 present potential risks to patients, and there are no standards of care. For example, intramuscular injections [23] easily cause hematomas, while intravenous administrations [24,25] can result in anaphylactic shock. Further, patient compliance is poor and the poisoning easily relapses, resulting in an increased risk. [12] The present study provides certain guidance for the maintenance treatment of LAAR poisonings and promotes the formation of a standard of care. A treatment curve is shown in Fig. 3. Table 5 Clinical and treatment data of long-acting anticoagulant rodenticide poisonings. This study has certain limitations. Genetic differences among patients were not considered in these analyses. Specifically, differences in suppression of the CYP2C9 and VKORC1 genes [26] may have resulted in a weakening of LAAR metabolism and an increase in the toxic effects, contributing to an increased risk of bleeding and coagulation. Further, the therapeutic dose of vitamin K1 was limited to 10 to 120 mg/d (intravenous dose q.d.), and the lowest concentration of monotherapy for brodifacoum was 5 ng/mL. There is no further research available on higher or lower therapeutic doses. Finally, only some clinical phenomena were explained, and the therapeutic strategies were investigated by a multifactor regression analysis in this study; therefore, the mechanisms behind these phenomena remain unclear. These results were interpreted based on inferences through published reports, clinical experience, and regression analysis of the data. The results of this study may be attributable to a lack of competitive inhibition between the LAARs and vitamin K1. After successive administration, the distribution of vitamin K1 reached a steady state, and only a small amount of vitamin K1 was required for maintenance treatment. However, successive administrations greater than the minimum dosage resulted in interference (Gunja et al [18] and this study).

Conclusion
Standardized methods (including HPLC methods) for detecting anticoagulant rodenticides in the blood and urine have not been accepted, and LAAR poisoning occurs mostly in underdeveloped areas. [27,28] Further, the dose of vitamin K1 injections should not exceed 40 mg according to the manufacturer's instructions. The above limitations make the rescue of LAAR poisoning patients a national problem. The results from our robust multifactor regression analysis provide a standardized treatment strategy for anticoagulant rodenticide poisoning. Specifically, successive vitamin K1 treatment was conducted after the bleeding, and coagulation functions were initially stabilized. The vitamin K1 maintenance dosage (10-120 mg/d, intravenous drip q.d.) was gradually decreased over time in a manner that was not related to the poisoning type or concentration of toxicant.