Lomustine, also known as 1-(2-chloroethyl)-3-cyclo-hexyl-1-nitrosourea, is used to treat human malignancies, especially brain tumors and lymphoma.1 Lomustine is metabolized in the body into compounds that alkylate and crosslink DNA and thereby block cell proliferation. In veterinary medicine, lomustine is used alone or in combination with other drugs to treat resistant lymphoma,2 mast cell tumors,3 intracranial meningioma,4 epitheliotropic lymphoma,5 and histiocytic sarcoma.6
Lomustine is commercially available as US FDA–approved capsules or as compounded capsules, and its use in veterinary patients is extralabel. The purity, potency, and efficacy of compounded drugs for veterinary use have been ongoing concerns for veterinarians and the FDA Center for Veterinary Medicine. The FDA defines compounding in general as a practice in which a licensed pharmacist, a licensed physician, or, in the case of an outsourcing facility, a person under the supervision of a licensed pharmacist combines, mixes, or alters ingredients of a drug to create a medication tailored to the needs of an individual patient.7 The Center for Veterinary Medicine recognizes the need for compounding in veterinary medicine; however, compounding from bulk drug substance (APIs) for use in animals is illegal. In addition, compounded drugs are not evaluated by the FDA for compliance with Good Manufacturing Practice guidelines, they may not meet regulatory standards for safety and effectiveness, and they may not be labeled or marketed appropriately.
Compounded veterinary drugs are receiving renewed attention from the FDA. In May 2014, 10 horses died as a result of a formulation error.8 Over the last 5 years, many other cases of compounding formulation errors resulting in toxicosis, lack of efficacy, or lack of equivalence with reference formulations have been reported.9–14 These problems can arise not only from an incorrect amount or purity of the API but also from changes in bioavailability, absorption, or stability resulting from the use of different carrier agents, excipients, or formulations.15
Compounded chemotherapeutic drugs are becoming more widely used for veterinary patients, and lomustine is currently available from many commercial compounders. The purpose of the study reported here was to measure the lomustine content (ie, potency) in compounded and FDA-approved lomustine capsules.
Materials and Methods
Lomustine
Compounded lomustine was obtained from 3 commercial compounders (Wedgewood Compounding, Swedesboro, NJ; Diamondback Drugs, Scottsdale, Ariz; and Global Technology, Boca Raton, Fla), and the FDA-approved druga was obtained from the commercial manufacturer. The compounding pharmacies were selected from pharmacies that advertised lomustine compounding on websites, at national veterinary meetings, or through direct email marketing and were arbitrarily assigned number designations to maintain anonymity for reporting purposes. The capsules (6/source/dose) were requested for office use only, and 2 capsule formulations (termed low dose and high dose) were purchased from each source. Low-dose capsules obtained from compounding sources were labeled (by the source) as containing 7, 10, or 11 mg of lomustine (the API), and high-dose capsules were labeled as containing 48, 42, or 40 mg of lomustine. Advertised or available strengths close to those of the FDA-approved product were selected from compounding sources. The low-dose and high-dose FDA-approved capsules were labeled as containing 10 and 40 mg of lomustine, respectively. All capsules of a given lomustine dose from each source were of the same lot.
All capsules were stored at room temperature until analysis and were analyzed before the expiration or beyond-use date on the product label. Five capsules of each dose from each supplier were tested. Analysis was conducted over 2 days. The high-dose FDA-approved capsules were analyzed on the first day, and the low-dose capsules were analyzed on the second day. The compounded capsules were divided and analyzed on the first (4/6 formulations) and second days (2/6 formulations). Capsules were blocked by compounder and strength to ensure dilutions were consistent. Quality control samples were analyzed on each day before, during, and after capsule analysis.
A lomustine standardb was dissolved in methanol to a concentration of 1 mg/mL, vortexed, and sonicated for 10 minutes to ensure complete solvation. The standard solution was stored protected from light at 4°C for ≤ 72 hours prior to use. Standard curve samples were prepared daily in 50% methanol at 250, 100, 50, 25, 10, 5, and 2.5 μg/mL concentrations.
Sample preparation and reverse-phase HPLC
All samples were analyzed at the Analytical Pharmacology Laboratory of Kansas State University by 1 investigator (MW). All sample capsules were opened and emptied into 50-mL culture tubes. Capsule exteriors were then dropped into the tubes to ensure complete transfer of the entire capsule content. Samples were diluted to a nominal concentration of 1 mg/mL in methanol, according to the labeled content. Samples were sealedc tightly to prevent leakage, capped, gently vortexed for 30 seconds, and sonicated for 10 minutes. The samples were then allowed to stand for 10 minutes to allow inert capsule filler to settle. Next, 100 μL of sample was diluted to a 100 μg/mL concentration in 50% methanol in glass culture tubes and vortexed for 10 seconds. Samples (100 μL) were then diluted in microcentrifuge tubes to a final nominal concentration of 50 μg/mL by mixing with an equal volume of 50% methanol, vortexed for 10 seconds, and centrifuged for 5 minutes at 15,000 × g to remove insoluble filler. A 100-μL aliquot of each sample was then transferred to an injection vial for testing by HPLC.
Lomustine content was assessed using a validated reverse-phase HPLC method,16 performed through use of an HPLC system with a thermostat-controlled column oven,d a C18 column,e and UV detection at 254 nm. Prior to the first injection, the column was conditioned for 5 minutes and equilibrated for 5 minutes in 0.1% formic acid-0.01% trifluoroacetic acid in water. Injections (25 μL; 1 injection/sample) were made when the baseline fluctuation was stable. The HPLC was performed at 40°C with a flow rate of 1 mL/min, 0.1% formic acid-0.01% trifluoroacetic acid in water as mobile phase A, and acetonitrile as mobile phase B. The gradient program was as follows: 90% phase A from 0 to 0.10 minutes, followed by a decrease to 50% phase A from 0.10 to 2.00 minutes; after holding at 50% phase A from 2.00 to 2.79 minutes, phase A was increased to 90% at 2.80 minutes and held at that percentage for 1 minute.
Calculation of lomustine concentration
The concentration of lomustine was determined by comparing the peak height for the sample with the peak heights of the linear lomustine standard curve (from 2.5 to 250 μg of lomustine/mL). Standard curves included a blank (0 μg/mL). Standard curves were fit with linear regression and accepted if R2 was ≥ 0.99 and measured concentrations were within 15% of the actual concentration. Lomustine quality control standards at 10, 50, 100 and 250 μg/mL were also included in each run. The accuracy of the assay (measured content divided by the actual content) and CV of the quality samples were determined. The lomustine content in capsules was determined as 100% × (measured concentration [μg/mL]/50 μg/mL). All calculations were performed with a data-processing spreadsheet.f An a priori acceptance range of 90% to 110% of the stated API content was used according to US Pharmacopeia standards17 for compounded drugs and for lomustine capsules.
Results
All lomustine quality control samples measured within 91% to 99% of the actual concentration (mean ± SD, 96 ± 2%) for concentrations of 10, 50, 100, and 250 μg/mL (n = 27 replicates). The accuracies of the assay were 97%, 96%, 96%, and 97% at 10, 50, 100, and 250 μg/mL, respectively (n = 5 at each concentration). The CVs were 3%, 1%, 1%, and 1% at 10, 50, 100, and 250 μg/mL, respectively (n = 5 at each concentration). In all sample analyses, the only or main HPLC peak eluted at the same time as the reference standard (2.6 minutes), although for all capsules obtained from compounder 3, a small secondary peak eluting at 2.9 minutes was detected (Figure 1).
Representative reverse-phase HPLC profiles for lomustine in low-dose capsules obtained from 4 sources (compounders 1 [A], 2 [B], and 3 [C] and the commercial manufacturer of the FDA-approved product [D]) and a lomustine reference standard (E). Lomustine content was assessed for 2 capsule formulations (termed low dose [n = 5] and high dose [5]) from each of the 4 sources with detection at 254 nm. The stated API content for low-dose formulations ranged from 7 to 11 mg, and that for high-dose formulations ranged from 40 to 48 mg. mAU = Milliabsorbance units.
Citation: Journal of the American Veterinary Medical Association 250, 3; 10.2460/javma.250.3.322
Representative reverse-phase HPLC profiles for lomustine in low-dose capsules obtained from 4 sources (compounders 1 [A], 2 [B], and 3 [C] and the commercial manufacturer of the FDA-approved product [D]) and a lomustine reference standard (E). Lomustine content was assessed for 2 capsule formulations (termed low dose [n = 5] and high dose [5]) from each of the 4 sources with detection at 254 nm. The stated API content for low-dose formulations ranged from 7 to 11 mg, and that for high-dose formulations ranged from 40 to 48 mg. mAU = Milliabsorbance units.
Citation: Journal of the American Veterinary Medical Association 250, 3; 10.2460/javma.250.3.322
Representative reverse-phase HPLC profiles for lomustine in low-dose capsules obtained from 4 sources (compounders 1 [A], 2 [B], and 3 [C] and the commercial manufacturer of the FDA-approved product [D]) and a lomustine reference standard (E). Lomustine content was assessed for 2 capsule formulations (termed low dose [n = 5] and high dose [5]) from each of the 4 sources with detection at 254 nm. The stated API content for low-dose formulations ranged from 7 to 11 mg, and that for high-dose formulations ranged from 40 to 48 mg. mAU = Milliabsorbance units.
Citation: Journal of the American Veterinary Medical Association 250, 3; 10.2460/javma.250.3.322
Compounded capsules
For all compounded capsules, the measured lomustine content was less than the stated amount of API. For compounder 1, low-dose capsules contained 78% to 93% and high-dose capsules contained 77% to 80% of the stated amount. For compounder 2, low-dose capsules contained 59% to 90% and high-dose capsules contained 70% to 74% of the stated amount. For compounder 3, low-dose capsules contained 85% to 95% and high-dose capsules contained 66% to 83% of the stated amount (Table 1). For low-dose capsules, 2, 4, and 2 of 5 capsules tested from compounders 1, 2, and 3, respectively, had results outside of the acceptable range (90% to 110% of the stated API content). None of the compounded high-dose capsules had results within the acceptable range.
Lomustine content (ie, potency) as measured by a validated HPLC method in capsules obtained from 3 compounding sources (compounders 1, 2, and 3) and from the commercial manufacturer of the FDA-approved product.
| Measured lomustine content (% of stated API) | |||||
|---|---|---|---|---|---|
| Source | Dose formulation | Range | Mean ± SD | Proportion of capsules outside of acceptance limits* | CV (%) |
| Compounder 1 | Low | 78–93 | 86.2 ± 7.6 | 2/5 | 8.8 |
| High | 77–80 | 79.0 ± 1.4 | 5/5 | 1.1 | |
| Compounder 2 | Low | 59–90 | 70.2 ± 11.7 | 4/5 | 16.7 |
| High | 70–74 | 71.0 ± 1.7 | 5/5 | 2.4 | |
| Compounder 3 | Low | 85–95 | 89.8 ± 73.2 | 2/5 | 4.1 |
| High | 66–83 | 73.2 ± 7.9 | 5/5 | 10.8 | |
| Commercial | Low | 107–108 | 107.4 ± 0.5 | 0/5 | 0.5 |
| manufacturer | High | 104–110 | 106.6 ± 2.4 | 0/5 | 2.3 |
Capsules of 2 formulations (termed low dose and high dose) were purchased from each source. Compounded low-dose capsules were labeled as containing 7, 10, or 11 mg of lomustine; compounded high-dose capsules were labeled as containing 48, 42, or 40 mg of lomustine. The low-dose and high-dose FDA-approved capsules were labeled as containing 10 and 40 mg of lomustine, respectively. Lomustine content in each of 5 capsules/dose was determined by comparing the absorbance at 254 nm with a standard curve prepared by use of a lomustine reference standard.
An acceptance range of 90% to 110% of the stated drug content was selected a priori on the basis of US Pharmacopeia standards.17
Content variability as measured by the CV was between 1.1% and 16.7% (Table 1). For compounders 1 and 2, variability among capsule contents was higher for low-dose than for high-dose capsules, whereas for compounder 3, variability was lower for low-dose than for high-dose capsules.
FDA-approved capsules
The measured amount of lomustine in low-dose capsules from the commercial manufacturer was 107% to 108% of the stated API content, and that for high-dose capsules was 104% to 110% of the stated amount (Table 1). All capsules tested from this source had results within acceptable limits. Variability among FDA-approved capsule contents was between 0.5% and 2.3%.
Discussion
Results of the present study revealed that lomustine capsules of 2 formulations (termed low dose and high dose) from 3 different compounding sources frequently contained < 90% of the stated API (lomustine) content as measured by HPLC. With an acceptable content range of 90% to 110% of the stated amount of API (selected a priori on the basis of US Pharmacopeia standards17), the product failure rate of the compounded products ranged from 2 of 5 to 5 of 5 capsules tested. Furthermore, content of the compounded capsules was often highly variable, even though all capsules tested of a given lomustine dose from each source were from a single lot. In contrast, all of the FDA-approved capsules were within the acceptable range, and lomustine content was fairly consistent.
The low API content and content variability of the compounded capsules are a medical concern because it could potentially result in subtherapeutic effects, which can lead to treatment failure or more rapid development of resistant neoplasia. All capsules were analyzed prior to the beyond-use date indicated by the supplier; thus, it was unlikely that the contents had degraded. The low drug content and variability could have resulted from formulation errors. One possibility is that the API used for the compounded capsules may not have been pure lomustine. For example, impurities such as carmustine-related compound A and lomustine-related compounds A, B, and C could have been present in the API used for compounding, resulting in a smaller amount of lomustine in these capsules. However, specific testing for impurities was not conducted in this study.
Another possible explanation was incomplete dissolution attributable to the use of various carrier agents, excipients, or chemical forms of the drug (eg, salts or esters); if this was the case, the in vivo solubility could also be altered, resulting in less complete drug absorption from the gastrointestinal tract. The measured lomustine content in the FDA-approved capsules agreed with the stated content, suggesting that poor solubility of lomustine in capsule form alone was not the reason.
Finally, if an alternate form or chemically similar form of the drug was present, it could have eluted at later times and therefore have been undetected by the HPLC method. This can happen, for example, with metronidazole, which elutes much later during reverse-phase HPLC when present in the benzoate form than when present as a free base.18 Chemically related compounds such as carmustine and semustine, which would be expected to produce pharmacological effects, could have been present but would not have been measured as lomustine on the HPLC assay. Testing for related compounds was outside the scope of the study; we did identify a small peak that eluted later than lomustine for all capsules from 1 compounding source, but whether this reflected the presence of an impurity, inactive ingredient, chemically related compound, or alternate form of the drug was un-clear. However, none of the compounded capsules were labeled as containing active compounds other than lomustine. We believe that the most plausible explanations for the findings were that incorrect amounts of the drug were incorporated into the compounded capsules during formulation and manufacturing or that the API used for the compounding was not pure and stable.
We used a validated HPLC method to detect lomustine and determined concentrations by comparing UV absorbance of samples at 254 nm to that of a reference standard. To ensure the validity of each run, we included lomustine control samples, which measured within 91% to 99% of the actual concentration in each test. In addition, we followed a standard protocol for sample preparation in which each capsule was diluted to the same nominal concentration to minimize the potential influence of solubility differences. To keep dilutions consistent and minimize errors in dilution volume, capsules were analyzed in a nonrandomized block design created on the basis of supplier and dose formulation (high or low). Unfortunately, this precluded blinding of the investigator to the capsule source, which could have resulted in bias. Such bias, however, was considered unlikely given the many controls and the similar results for each sample source. Importantly, similar laboratory findings for compounded lomustine capsules and the clinical effects of such deviations from the stated drug content have been reported elsewhere.19 In a retrospective analysis of dogs with cancer treated with lomustine, Burton et al19 found that FDA-approved lomustine capsules induced neutropenia in 21 of 21 (100%) dogs, with 15 (71%) developing ≥ grade 3 (severe) neutropenia (scale of 1 [mild] to 5 [death]), whereas compounded lomustine capsules induced neutropenia in only 4 of 16 dogs that received it. Subsequent HPLC analysis of 3 compounded capsules (labeled as containing 5 mg of lomustine) from that same compounding pharmacy revealed that they contained only 50% to 54% of the stated lomustine content.
The present study had other potential limitations that should be considered in evaluating the results. Samples from only 1 lot for each dose and source were assessed. Additionally, products from only 3 compounding pharmacies were included in the study. Although another investigation19 had findings similar to those reported here, it is possible that other lots from these compounders or compounded lomustine capsules from other sources could have contents consistently within the acceptable range according to US Pharmacopeia standards. Further studies that include multiple lots from multiple sources are needed to assess this.
Frequent product content (potency) failure was found for compounded capsules from all sources in the present study but not for the FDA-approved capsules. We therefore recommend that compounded veterinary formulations of lomustine not be used when appropriate doses can be provided with FDA-approved capsules or combinations of the FDA-approved capsules.
Acknowledgments
The study was funded by NextSource Biotechnology.
The authors thank Dr. Phillip S. Leventhal for scientific writing.
ABBREVIATIONS
| API | Active pharmaceutical ingredient |
| CV | Coefficient of variation |
| HPLC | High-performance liquid chromatography |
Footnotes
Lomustine, NextSource Biotechnology, Miami, Fla.
Sigma-Aldrich Corp, St Louis, Mo.
Parafilm M, Bemis, Neenah, Wis.
Prominence, Shimadzu Scientific Instruments, Columbia, Md.
Waters XBridge Shield RP 1C8, 50 × 2.1 mm, 5-μm pore size, Waters Corp, Milford, Mass.
Microsoft Excel 2013, Microsoft Corp, Redmond, Wash.
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