Efficacy of continuous ascorbate infusion on lymphopenia and neutrophil counts in the supportive care of cancer patients

Abnormal blood counts and, in particular, lymphopenia commonly occur in cancer patients and predict poor prognosis. In this study data from a previous Phase 1 clinical trial of continuous vitamin C infusions were analyzed. The trends in the absolute lymphocyte counts (ALC), absolute neutrophil counts (ANC) and neutrophil to lymphocyte ratio (NLR) were evaluated. In addition, pharmacokinetic data on IVC in patients who were treated by continuous infusion were determined for different dosages. During continuous infusion treatment, data for all patients, who completed 6-8 weeks of the treatment, demonstrated improvement of lymphocyte counts in patients with lymphopenia. On average the percentage of improvement in ALC was 22% for ALC <1300 / µl and 69% for ALC <1000 cells/µl. Our data also indicate that lower doses (less than 20 g/70 kg) were more favorable for the improvement of ALC. In addition, the data suggest a normalizing effect of continuous infusions of ANC levels and changes in NLR. In summary, the present analysis demonstrated the normalizing effect of continuous IVC on lymphopenia, ANC and NLR.


Introduction
The use of high dose ascorbate in cancer therapy remains an area of controversy.
Currently, there are no clinical data that show the importance of several factors in the treatment schedule using high dose vitamin C, such as dose, frequency and duration of administration on the effectiveness of the cancer patients' treatment [1]. Most practitioners administer IV ascorbate to cancer patients by bolus infusions 2-3 times per week [2]. Bolus infusion of high dose ascorbate (1g/kg) can reach very high concentrations in blood based on the data of pharmacokinetics, but is very quickly eliminated from the body with a half-life of about two hours [3,4]. Such treatment is generally well tolerated 3 and safe, with few adverse events reported, and there are anecdotal case reports of an anticancer effect of such a regimen [5][6][7].
There have been two clinical trials that used continuous IVC infusions. Cameron and Pauling performed a clinical trial in 100 terminal cancer patients [8,9]. The protocol included an initial 10 day course of IV ascorbate, at a relatively low daily dose of 10 g/day given by continuous infusions, followed by daily oral intakes of 10-30 g/day, in divided doses. Their results showed increased survival time and improved the quality of life of the patients compared with patients who had not received IVC.
The ideas of Linus Pauling were extended in the "The dynamic flow model" developed by Dr. Hickey et.al [10,11].
The second trial of the treatment of cancer patients by continuous infusions was conducted by Dr. Riordan [12]. In this Phase I clinical trial, patients were administered continuous infusions using an infusion pump. The first data analysis published in 2005 was focused on the safety of the treatment. In the next publication [13], we analyzed previously unpublished parameters from the Riordan clinical study, including blood chemistry and blood count parameters that are reportedly related to patient prognosis and degree of inflammation. This included: absolute neutrophil and lymphocyte counts and the neutrophil-to-lymphocyte ratio; lactate dehydrogenase, an enzyme involved in tumor initiation, metastasis, and recurrence; creatinine, the depletion of which is associated with cachexia; and glucose, as hyperglycemia is common in cancer patients. The analysis demonstrated the regulatory effect of continuous IVC on lactate dehydrogenase enzyme, neutrophil-to-lymphocyte ratios, lymphopenia, neutrophil count and hyperglycemia.
In the present study, we further analyzed the pharmacokinetics of the blood ascorbate levels, effect of the dosages of continuous IVC infusion on absolute lymphocyte counts, and improvement of neutrophil counts and neutrophil to lymphocyte ratios as the 4 result of continuous IVC treatment.

Materials And Methods
A description of how the Phase 1 IVC continuous infusion clinical trial was conducted was given previously [12]. A total of 24 patients with late stage cancer were included in the study. All patients had several rounds of chemotherapy or radiation prior to entering the study. Seventy nine percent of the patients had a metastatic tumor; 17  Blood cell counts, blood chemistry parameters, progression or disease, and adverse events were monitored in these patients. Samples were collected one week prior to therapy and at weekly intervals during intervention. White blood cell counts, hemoglobin and hematocrit, red blood cell counts, glucose, lactate dehydrogenase and blood chemistry parameters related to renal function (creatinine, BUN, and uric acid) were determined using standard procedures at the Eppley Institute for Research in Cancer and Allied Diseases at the University of Nebraska Medical Center (Omaha, NE) [12]. Plasma ascorbate concentrations were measured as the Riordan Clinic laboratory by colorimetric method.
The clinical trial was approved by the ethics committee of the Eppley lnstitute for Research in Cancer and the Institutional Review Board of the Riordan Clinic. Written informed consent was obtained from all patients.

5
The data were analyzed by Systat software (Systat, Inc, San Jose, USA) and Kaleidagraph software (Synergy software, PA, USA). Statistical significance was evaluated by using a paired, two-tailed, Student's t test where p < 0.05 was considered significant.

3.1.
Ascorbate concentrations in blood after continuous infusions.
During the continuous infusion clinical trial, the levels of ascorbate were measured before intervention, every day for the first four days and then at the end of each week. Pretreatment measurements demonstrated that patients had hypovitaminosis C, with two thirds of the subjects having levels below the normal range (0.6mg/dL-2mg/dL) [12,13].
During IVC treatment, ascorbate levels increased, and reached a plateau, but average maximum values did not differ significantly between different doses and were in the range of 1.2 mM -1.5 mM. The ascorbate concentrations in blood for low (150 mg/kg/day) and high (710 mg/kg/day) dosages, averaged for all patients in these dosage groups, are presented in Figure 1. Data were extrapolated by Michaelis-Menten equation.

Effect of continuous infusion on ALC
The effect of continuous infusions on the absolute lymphocyte count (ALC) was analyzed for 22 patients from 24 terminally ill cancer patients. More than half of the patients enrolled in the study had lymphopenia or absolute lymphocyte counts less than 1300 cells/ µl (normal range 1300-4000 cells/µl). The data of the patients' diagnosis, lymphocyte and neutrophil counts one week before treatments, at the beginning and end of the treatment, and the duration and the dosages of the treatments are shown in Table 1. *Excluded from the data analysis due to lack of post treatment data.
The percentage of change in ALC was calculated based on the pre-treatment ALC (6-12 days before intervention), initial ALC values and the ALC at the end of the treatment.
According to our data, the tendency of ALC was to decrease in 56% of patients before treatment. For patients with lymphopenia (15 subjects

The effect of continuous infusion on ANC
As absolute neutrophil counts (ANC) and neutrophil-to-lymphocyte (NLR) ratios are useful prognostic factors in a variety of cancers, with higher values of NLR indicating lower survival times [14], the effect of continuous injection on these parameters was analyzed.  The levels of ANC were divided to three regions: less than the middle of the normal range (2000-4500cell/µl, n=6), higher than the middle of normal range (4500-7000cells/µl, n=10) and higher than normal range (ANC>7000 cells/µl, n=2). The normal range for neutrophil counts is 2000-7000 cells/µl.

The effect of continuous infusions on NLR
At the beginning of IVC therapy, 75 % of subjects had NLR levels higher than normal range (0.78 -3.53). An improvement in the NLR was seen in 36% of the patients. However, for continuous infusions we were able to calculate the tendency in the change of NLR before and during treatment, as the values for ALC and ANC were measured a week before treatment, at the beginning, and each week during treatment. To calculate the initial ΔNLR (prior to therapy), NLR on day zero was subtracted from NLR measured one week prior to therapy, and this difference was divided by the number of days between the two measurements. The rate of change in this ratio (ΔNLR) for each patient before and after therapy was described in our previous article [13]. The comparison of the trend in the change of NLR measured for periods one week before treatment and during treatment demonstrated that the rate of change was decreased. The average ΔNLR values for the patients who completed 6-8 weeks of treatment were 4.2%/day pre-therapy and 1.0 %/ day post-therapy ( Figure 5). According to our data, the treatment resulted in the suppression or prevention of the progression of the rate of growth of NLR. This improvement of the rate of change of NLR was found for 54% of the patients. For patients with initial NLR higher than the upper level of the normal range of 3.5, improvement was seen in 64% of patients who completed 6-8 weeks of treatment.

Discussion
Vitamin C contributes to immune defense by supporting various cellular functions of both the innate and adaptive immune systems [15]. The role of vitamin C in leukocyte function and its multiple beneficial effects on immune function was summarized in a previous review [15]. In the present study we analyzed the effect of continuous infusions on two immune cell populations, lymphocytes and neutrophils. leveling off of the ascorbate concentration can be explained by observations that high doses will saturate renal tubular ascorbate reabsorption, leading to increased ascorbate excretion. Recently, the kinetic curves of the continuous infusions for two dosages 2 g/d and 10 g/day were presented in the study [16]. According to this study, the steady-state concentrations of ascorbate were 40-210 µM for 2 g/d and 230 µM -1.470 mM for 10 g/d at 48 h, and vitamin C excretion and renal vitamin C clearance increased with the dosage.
IVC high dose bolus infusions can reach much higher concentrations [17], but these peaks are only transient.
In the Riordan Clinic trial [12,13], patients were treated by continuous infusion, which was administered over much longer periods of time than bolus intermittent treatments.
For most patients the duration of the continuous infusion was at least 20 hours, as the duration of bolus infusion is from one hour to three hours depending on the dosage. The present analysis demonstrated the regulatory and normalizing effect of continuous IVC infusions on lymphopenia, neutrophil-to-lymphocyte ratios, and absolute neutrophil counts. In addition, our data also demonstrated the relationship between the survival of patients and the rate of growth of NLR in continuous infusion group [13].
The analyzed data also suggested a benefit of using lower IVC doses in continuous infusions for improvements in immune cell counts, as raising the dose above 20 g/70 kg body weight showed a tendency to decrease improvements in lymphocyte counts and exhibited increased frequency of side effects, the latter of which were discussed in detail in the previous articles [12,13].
Most practitioners administer IVC to cancer patients by bolus infusions of pharmacological doses of ascorbate several times per week [2]. According to the prevailing hypothesis, pharmacological bolus dosages of 50 -100 grams have an anti-cancer effect by increasing hydrogen peroxide in the tumor environment [18,19]. However, ascorbate has multifactorial mechanisms of action. In addition to the proposed mechanism of the pro-13 oxidant effect of high dose ascorbate, studies have demonstrated that the mechanisms of anti-tumor activity of ascorbate include changes in metabolic activity, stimulation of the 2-oxoglutarate dependent dioxygenase family of enzymes, which regulate the hypoxic response, collagen stabilization, and epigenetic histone and DNA demethylation [20,21].
There are studies supporting the anti-tumor effect of relatively lower concentrations of vitamin C than pharmacological doses given by bolus IVC. For example, ascorbate can inhibit hypoxia-inducible factor-1 (HIF-1) activation in vitro at intracellular concentrations between 150 and 300 µM [22], and pharmacokinetic data on ascorbate in tumor tissues following vitamin C administration determined an optimal dose for HIF-hydroxylase activity of ~1-3 mM [23]. The in vitro study of the optimal concentration of ascorbate as a cofactor for hydroxylases that regulate gene transcription and cell signaling pathways shows that ascorbate concentration less than 1,000µM dose-dependently increases the 5-hmC signal [24].
The frequency of IVC treatment has an effect on mechanisms of tumor suppression.
Campbell et al. [25] examined the effects of treatment schedule on the ability of intravenous ascorbate to inhibit HIF-1 expression (and the expression of its target proteins) in tumor bearing mice. It was found that a single bolus injection inhibited expression temporarily while daily injections maintained the inhibition. Increased tumor ascorbate was associated with slowed tumor growth, but alternate day administration of ascorbate resulted in lower tumor inhibition and did not consistently decrease HIF-1 pathway activity compared with daily injections [25]. Our retrospective analysis of prostate cancer patients treated with bolus IVC at the Riordan Clinic (1994-2015) showed that PSA levels increased more slowly in subjects given more frequent IVC treatments [26].
14 Thus, the paradigm of maximum tolerated dose, or the "more must be better" philosophy,  The dosage effect of vitamin C on the change in lymphocyte counts.
22 Figure 4 Dependence of the ANC change on the initial cell counts for continuous treatment. The levels of ANC were divided to three regions: less than the middle of the normal range (2000-4500cell/µl, n=6), higher than the middle of normal range (4500-7000cells/µl, n=10) and higher than normal range (ANC>7000 cells/ µl, n=2). The normal range for neutrophil counts is 2000-7000 cells/µl. 23 Figure 5 The rate of NLR changes (percentage per day) before and after treatment for patients who completed 6-8 weeks of treatment.