Cyclophosphamide Use in Multiple Sclerosis: Levels Detected in Human Milk

Introduction

Multiple sclerosis (MS) is an autoimmune disorder that affects the myelinated axons of the central nervous system. ¹ The disease is characterized by a relapsing and remitting pattern in which patients experience a vast array of neurologic symptoms, including muscle weakness, cognitive impairments, and sensory deficits. ² Approximately 400,000 people in the United States and 2.1 million people worldwide suffer from MS, with women being 3.1 times more likely to develop MS than men. ³ Although there is no definitive cure for MS, recent advances in stem cell therapy have significantly improved the condition.

Autologous hematopoietic stem cell transplantation is specifically used to treat younger patients with ongoing aggressive MS. ¹ Cyclophosphamide, a chemotherapeutic and immunosuppressant agent, is used before stem cell transplantation for its immunosuppressant effects. Antithymocyte globulin and steroids are often used in combination with cyclophosphamide to further facilitate immunosuppression. ⁴

Cyclophosphamide is a prodrug, requiring bioactivation to its active form. Its mechanism of action involves the formation of DNA cross-links, which consequently inhibit DNA replication and induce cellular apoptosis. ⁵ Liver enzymes, specifically the cytochrome P450 enzyme system, primarily metabolize cyclophosphamide. ⁶ The metabolism of cyclophosphamide is unique in two main ways. First, the drug is broken down into several active and inactive metabolites. ⁷ Second, for several days the drug exhibits autoinduction, in which repeated sequential doses of the drug autoinduce hepatic metabolism. ⁸ Hence, plasma pharmacokinetics such as maximum concentration (C_max) is reduced in each subsequent dose. Clinical dosages of cyclophosphamide vary widely, ranging from 2–6 mg/kg to as high as >6,000 mg/m ² , depending on the condition being treated. ⁷ Many studies have quantitatively analyzed cyclophosphamide in urine and plasma, but none thus far have quantified levels of the drug in breast milk. ⁷ Since cyclophosphamide is occasionally used to prepare patients for hematopoietic stem cell transplantation in breastfeeding mothers, we felt it necessary to quantify levels of the drug in human milk for 4 days of therapy.

Case Presentation

A 33-year-old woman first complained of visual loss in August 2011. A year later she was officially diagnosed with MS. Her symptoms included extreme fatigue, numbness in extremities and face, burning sensation in legs, balance issues, and heat intolerance. To treat her MS, stem cell transplantation protocol required preparation with multiple doses of cyclophosphamide. She had been exclusively breastfeeding for 6 months before undertaking this regimen, and had stored sufficient breast milk to feed her infant, whereas cyclophosphamide administration necessitated discarding the pumped milk. She received 2.8 g of cyclophosphamide daily intravenously (IV) for four consecutive days. In addition she was taking mesna, rabbit antithymocyte globulin (rATG), methylprednisolone, and furosemide during the time of the cyclophosphamide infusions. The patient did provide her consent and release form to use the data obtained from analysis. With the help of the medical team, she collected milk samples at critical time points after each IV cyclophosphamide dose.

Methods

Cyclophosphamide quantification was determined using an ABSciex QTRAP 5500 UPLC MS/MS mass spectrometer. A Phenomenex Luna C-18 column, 50 × 2 mm, 3 μ, was used. Gradient elution mode was followed using water and acetonitrile, at a flow rate of 0.5 mL/min. Milk samples were processed for protein precipitation and internal standard (final concentration 50 ng/mL) was added to each sample and standards. Data were acquired by multiple reaction-monitoring modes with mass transitions; m/z 261.1 to m/z 140.1 for cyclophosphamide and m/z 395.1 to m/z 213 for internal standard. The range for the curve constructed was 0.24–5,000 ng/mL with a correlation coefficient of 0.99.

Results

In this case report we observed the levels of cyclophosphamide in the milk samples obtained from a breastfeeding mother from days 1 to 4 after daily IV dose of 2.8 g. Detected levels of milk cyclophosphamide were reduced each subsequent day as the area under the curve was 364.1 μg.hour/mL on day 1 and 74.4 μg.hour/mL on day 4 in Figure 1. The C_max in milk observed on day 1 was 40.8 μg/mL, which peaked at 4 hours. For 24 hours and before the next injection, levels of cyclophosphamide gradually receded to minimum concentrations. The pharmacokinetic parameters are described in Table 1. The relative infant dose (RID) for a period of 4 days ranged from 4.8% on day 1 to 0.9% on day 4, respectively. Although these data suggest that cyclophosphamide is transferred into milk at relatively low concentrations, the incredible toxicity of this compound would preclude its use while breastfeeding an infant. The levels might not seem to be of clinical significance; however, its active metabolism and systemic availability in infants might be hazardous.

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FIG. 1.

Mean concentration-time profile of cyclophosphamide in human milk after daily 2.8 g dose during four consecutive days.

Discussion

Recent advances in hematopoietic stem cell transplantation have proven beneficial to younger patients with severe persistent MS. ⁴ In the aforementioned case, the immunosuppressant drug cyclophosphamide was administered before institution of stem cell transplantation. Many studies have both qualitatively and quantitatively determined levels of cyclophosphamide and its inactive metabolites in plasma, urine, cerebrospinal fluid, saliva, and sweat.^7,9 Although previous studies have qualitatively detected cyclophosphamide in human milk,^9,10 no studies to date have accurately quantified levels in human milk, particularly after a 4-day IV treatment regimen.

To our knowledge, this is the first case report to quantify levels of cyclophosphamide in human milk. Levels of cyclophosphamide were found to be relatively low, with an average drug concentration time interval (C_avg) of 15.1 μg/mL on day 1 and 3.1 μg/mL on day 4. These values produced a range of RID from 4.8% on day 1 to 0.9% on day 4. As expected, C_avg, C_max, and, consequently, the RID progressively decreased with each subsequent day of treatment. This phenomenon is explained by hepatic autoinduction of metabolism, and is frequently reported in other literature reporting decreasing plasma levels. Although it is well known that hepatic autoinduction causes increased formation of the drug's active and inactive metabolites, ¹¹ it remains unclear as to whether this also implies increased exposure to the active metabolites. ⁷

Hepatic microsomal oxidases present in the liver are primarily responsible for cyclophosphamide bioactivation. It is a prodrug extensively metabolized to both active and inactive forms. The metabolites formed are highly unstable and exist in equilibrium with inactive forms. 4-hydroxycyclophosphamide is an active metabolite which diffuses into the cells but not cytotoxic by itself. It spontaneously converts to a phosphoramide mustard, which is highly toxic as a DNA alkylating agent. ¹² The determination of its metabolites has been limited due to its instability.

Although the exact exposure of cyclophosphamide's metabolites in the infant is unknown, there have been reports of immunosuppression in breastfeeding infants whose mothers were being treated with cyclophosphamide. In one case report, a mother received 800 mg/week dose of cyclophosphamide for 6 weeks of therapy and still breastfed her infant despite warnings. Her 4-month old infant developed neutropenia thereafter. ¹³ In another case report of a mother receiving 6 mg/kg of cyclophosphamide delivered IV for 3 days (total 300 mg/day), her 23-day old infant developed major leukopenia and thrombocytopenia. ¹⁴ Although case reports such as these are limited, they are informative and add to the data found in our work. In both of these cases, the mothers received significantly less total dosages (4.8 and 0.9 g) of cyclophosphamide than the mother described in this our case report, who received a total dosage of 11.2 g for 4 days. Despite the lower dosages used in the aforementioned case reports, their infants still exhibited significant immunosuppression. Although the reported levels of cyclophosphamide in milk in this study are low, the potential risk that this drug might pose to a breastfeeding infant cannot be minimized.

In conclusion, further studies on the transference of cyclophosphamide into human milk are required to fully describe the drug's risk to a breastfed infant. Although autoinduction may reduce the total amount of cyclophosphamide in human milk with repeated subsequent treatments, the transfer of the drug's metabolites into human milk remains unknown. Ultimately, great caution is recommended in using cyclophosphamide in breastfeeding mothers.