Two-hundred and ninety-nine NVP-filled pouches were submitted to the Southern Testing and Research Division of Microbac Laboratories, Inc. (Wilson, NC) for twelve months of controlled storage and testing. Samples were stored in walk in stability chambers at 25ºC/60% relative humidity (RH) and 40ºC/75% RH and in a reach in chamber at 30ºC/65% RH. The selected temperature and humidity ranges are expected to represent extremes of African home storage and match the US Food and Drug Administration (FDA) recommended testing conditions [6, 8]. Storage conditions did not fall outside of +/- 2ºC and +/- 5% RH of marked values. Each pouch was stored at 5ºC after being pulled until it was tested.

Microbiological, weight change, and chemical testing was performed on the pouches. Microbiological testing was performed at time zero and after 12 months. Flora and fauna challenges (total microbial aerobic, yeast and mold) were attempted on twenty pouches at each storage condition at the start of the experiment and on another twenty pouches after twelve months of storage following US Pharmacopeia part 61 (USP61) using USP33-NF28 methodology. (The United States Pharmacopeia (USP) and The National Formulary (NF) is a book of public pharmacopeia standards for the testing of chemical and biological drug substances.) Ten pouches at each storage condition were tested for the presence of E. Coli (USP62) at the start of the experiment and retested on another ten pouches after twelve months of storage also following USP33-NF28 methodology.

Samples were weighed prior to stability testing and again when pulled at months 2, 4, 6, 8, 10, and 12, and weight loss or gain was used to estimate moisture loss during the stability storage.

Lastly, chemical testing was performed at months 2, 4, 6, 8, 10, and 12 following a modified USP32-NF-27 methodology. The modifications to the standard procedures were 1) use of the bulk NVP pulled from the original bottles (stored at 5ºC) for the preparation of method standards at time points 2, 4, 6, 8 and 10 (time points zero and twelve months were completed with USP NVP reference standard as per USP32-NF27 without modification), and 2) a reduced sample volume. Specifically, the three pouches were combined, and approximately 0.4 g-aliquots of test samples were volumetrically diluted to 10.0 mL (refer to monograph procedure) to yield working solutions at a NVP concentration of about 400 µg/mL (nominal). For each instrumental set-up, the system suitability was established prior to samples analysis, and continued system suitability was monitored following the USP method. All samples were analyzed by high performance liquid chromatography (HPLC) with duplicate injections for NVP assay, related compounds and impurities determination. Duplicates were combined to form single data points according to USP32.

In addition to determination of the NVP assay, levels of Methyl paraben and Propyl paraben (preservatives) in the samples were estimated using independently prepared standards. This was performed at all measured time points. The standards were prepared at 50 µg/mL for Methyl paraben and at 10 µg/mL for Propyl paraben.

The chromatographs were analyzed to determine if impurities were induced in the medication or extracted from the plastic [9, 10]. To determine the origin of the impurities, two additional analyses were made: preservatives by themselves and NVP by itself. Methyl and Propyl paraben standard solutions were prepared in water/methanol mixed-solvent and were stored in clear glass containers at ambient temperature for four months. USP NVP reference standard was prepared according the USP 32-NF27 methodology and analyzed.

Three pouches were combined for each measurement of NVP assay. Twenty pouches were used for each USP61 measurement and ten for each USP62 measurement. Because the assay testing was completed at each of seven time points and USP61 and USP62 were each completed twice, the total number of pouches required for testing was 243. The remaining 56 were prepared but never used.

For the NVP assay and related compound data, each reported data point represents the analysis of three sample pouches and multiple data points combined, as required by USP testing. Since, only one data point results, no statistical analysis can be performed on any one individual time point and test condition.

As a comparison, thirty syringes were filled with water then placed in pouches of identical material to that described above. After sealing with double-sided adhesive tape, the pouches were weighed and moved to a storage compartment at room temperature. Pouches were removed and weighed approximately every other day for twenty days.

To determine the acceptability of the pouch among healthcare workers and end users in Sub-Saharan Africa, ten nurses in six hospitals in northern Tanzania were interviewed. All participating nurses interviewed worked directly with HIV+ mothers in their hospital’s maternity ward and were familiar with NVP and prevention of mother-to-child transmission (PMTCT) national protocols. Three questions were asked, as shown in Table 1.

As a more objective measure of the volunteers’ ability to open the pouches, tear time was measured by asking ten volunteers in the United States to open the pouches. The tear time was measured as the time from when the pouch was handed to the volunteer until the volunteer felt they had opened the pouch. Participants were provided with two pouches filled with placebo doses of the same viscosity as NVP and with picture instructions with minimal text. No verbal instructions were provided. Participants were asked to empty the pouches into a small cup (to simulate giving the infant liquid medication). If the participant indicated that the picture instructions were unclear, the participant was provided with the same picture instructions with the addition of one to three word instructional captions in English (for example, “tear”).

The most critical function of the pouch is to preserve the active ingredient for later delivery to the infant. Fig. (2) shows that the NVP concentrations remained nearly unchanged for all time points at all storage conditions. The average change of the NVP was observed to be a 1.36% drop (maximum 1.40%) in the concentration of NVP when compared to the bottle stored at 5°C during the same time period. This is well within the 10% USP acceptability standard. In other words, the pouch preserves the medication adequately for up to twelve months.

Because we used the USP reference standard at time points zero and twelve, we can also determine how well the manufacturer’s bottle can store NVP. The original material was 106.8% of the specified 10 mg/mL marked on the bottle. After twelve months of storage, the bottle dropped to 102.9% of the specified 10 mg/mL. Again, these values are well within the USP standard (+/-10%).

At time zero and twelve months later, the pouches passed all microbial challenges. E. coli was absent. The aerobic and yeast counts were both below 10 cfu/mL, which is well below the acceptance criteria for the tests. In summary, the testing demonstrated a complete absence of any sort of biological or microbial contamination despite having sealed the pouches in conditions typical of a pharmacist, not a sterile medical processing facility.

Infant NVP is held in an aqueous suspension by emulsification agents. As mentioned earlier, we suspected that, when stored in a syringe or a syringe inside a foil pouch, moisture loss quickly concentrated the NVP beyond usability. As shown in Fig. (3), when NVP is stored in the pouch, it loses less than 0.7% of its weight over twelve months. In the syringe, moisture is lost well beyond tolerable limits in less than two months (Fig. 3). The pouch nearly eradicated moisture loss.

Handling the medication, including exposure to light, oxygen and mechanical treatment can damage the medication. As already presented above, we did not see any evidence of this in the concentrations of NVP. However, there are other ways to determine if material handling affected the medication, specifically preservatives and impurities. Fig. (4) does show that some preservatives were lost during the filling and sealing of the pouches (time point zero). Propyl paraben dropped 38%, and Methyl paraben dropped 12% initially. However, after the initial time point, preservative levels were stable in the package over twelve months under all storage conditions (4.1% average concentration drop after initial drop). The initial drop could be due to the medicines exposure to heat, oxygen or light when it is drawn into the syringe but probably occurred within minutes or hours of repackaging. The bag itself can not be the cause, as Fig. (4) shows no loss of preservatives after the initial stabilization period. The lack of significant change in NVP levels and lack of microbial growth suggest that the preservatives did act as sacrificial oxidants, leaving sufficient preservatives to protect the medication for twelve months.

There were five impurities noted in the samples from the bottle upon opening (time point zero), the stored bulk samples from the bottle (5°C, time point twelve months) and the stored samples in the pouch (all storage conditions and time points). The same five impurities were found in all three: the freshly opened bottle, the stored bottle and the stored pouches. All impurities were present at concentrations below 1% in all samples stored under all conditions. Storage in the pouch did not introduce any impurities that were not already present in the original material analyzed when the bottles were first opened. This strongly suggests that the pouch does not induce impurities in the medication and the medication does not extract impurities from the pouch (or at least none that are not also extracted from the manufacturer’s bottle).

For this discussion purposes we arbitrarily number the impurities from the highest to the lowest relative retention times. Of the five impurities, USP reference standard NVP shows impurity four. Therefore, we conclude that this is a breakdown product of NVP. Aged samples of the preservatives show impurities two and five. Therefore, we conclude that these are breakdown products of the preservatives. The breakdown products of these chemicals were studied and their safety documented in the original manufacturers submission to the FDA. This leaves impurities one and three.

Impurities one and three were present in very small quantities (<0.3%) in all samples including recently opened bottles, stored bottles and samples stored in pouches. Therefore, we conclude that storage in the pouch poses no additional risk over storage in the bottle. However, we can gain some additional insight from our data. Impurities one and three seem to be most dramatically affected by handling, as they increased from <0.02% to 0.04 and 0.06% at time point zero. However, storage and storage conditions did affect their concentrations (0.05 and 0.08% at four months for 25C/60% and 0.11 and 0.18% at four months for 40C/75%). Nevertheless, storage for twelve months at extremes of temperature and humidity created only as much of impurities one and three as would handling the medication approximately four times (0.19% and 0.29% at twelve months 40C/75%). The bag itself is probably not involved in the creation of impurities one and three as storage for twelve months at 25C/60% created just barely measurable additional concentrations of the impurities over those present at time point zero (0.06% and 0.12% at 12 months)

Nearly all (90%) of the ten healthcare workers (nurses) thought mothers could empty the pouch contents into their infants mouths with pictures alone and no training (Table 1). All (100%) nurses felt that with proper training mothers could empty the pouch properly at home. About half of the nurses expressed concerns about using the device, mostly relating to the HIV stigma (the risk of revealing one’s status by using the pouch) and to a sound understanding of pouch use. For example, one nurse was worried that despite an instructional consultation, a mother may squeeze the drug into a contaminated cup or spoon before giving to the infant, which may cause diarrhea.

The engineering study in the US indicated that end users can easily tear open the pouch without a cutting device. No failures to open the pouch were observed during the tear time study. The overall average time to tear the pouch open was 1.98 s with a standard deviation of 1.28 s.