Not All Ivermectin Is Created Equal: Comparing The Quality of 11 Different Ivermectin Sources

Note from Alexandros: This is the first guest post on DYOR, one of hopefully many in the future. Tim Williams reached out to tell me about an experiment he ran on ivermectin sourced from many different manufacturers. I was blown away by the relative simplicity of the experiment, his attention to detail, as well as the striking results. I asked him if he would write up his results in longer form so I could publish them here. He obliged, and here we are. Truly an example of doing your own research if I’ve ever seen one. Without further ado, here’s Tim’s writeup:

Abstract

In this small-scale study, it was determined that various sources of ivermectin—within the United States, as well as from overseas—differ significantly in the rate at which they effectively kill parasitic worms. It is possible that these differences represent variability in the drug’s purity—depending on the manufacturing location—but this matter needs to be investigated further. Relative to the US-made Edenbridge brand of ivermectin, certain manufacturing or compounding sources demonstrated a lower rate of lethality for the parasites, whereas other sources demonstrated a faster rate of lethality—in terms of days required to kill all six parasitic worms treated—with the same final concentration (5uM) of each ivermectin source.

Introduction

Due to the pressure to stop prescribing or fulfilling prescriptions for ivermectin in the United States, many people ordered the medicine from places such as India, the Philippines, and Mexico. Others obtained ivermectin from compounding pharmacies within the US. The purity, quality, and efficacy of the drug from these sources is uncertain.

As such, this small investigational study, using the antiparasitic properties of ivermectin as a proxy for its antiviral properties—as well as its purity—tested the agent at a 5uM final concentration in petri dishes for lethality against planarian flatworms. It is acknowledged that this is a limited investigation. A more optimal look at the purity of ivermectin from different sources would consist of high performance liquid chromatography (HPLC), or some other empirical method.

Materials and Methods

Many of the materials and methods were derived from those used in the paper “Effects of Ivermectin Exposure on Regeneration of D. dorotocephala Planaria: Exploiting Human-Approved Ion Channel Drugs as Morphoceuticals,” by Ferenc and Levin.

• Ivermectin, gathered by personal online order or in-person purchase—or from orders or purchases made by personal contacts—with the country of manufacture, brand names, and other notes shown below:

• Planaria brown flatworms, live; ordered from Carolina Biological Supply Company (carolina.com Item#: 132950; L210-PLANARIA)—two jars of classroom-sized set, with plastic pipettes used to move the flatworms

• Hand pipettes with corresponding LTS tips; manufactured by Rainin

• 1000uL volume single-channel pipette

• 200uL volume singe-channel pipette

• DMSO (100%); manufactured by Roche

• 2mL tubes; manufactured by Sarstedt

• 15mL and 50mL tubes; both manufactured by Falcon

• 250mL bottles; manufactured by Corning

• Natural spring water; bottled by Crystal Geyser

• Digital pocket scale (100g by 0.01g); manufactured by Weigh Gram

• Plastic spatula tools, for scraping crushed pill tablets onto foil, scale

• 90 x 15mm Plastic Petri Dishes; manufactured by Hamiggaa

• Brushed stainless steel mortar and pestle; Manufactured by Bekith

• Microscope with 25X magnification lens; manufactured by National Geographic

• iPhone XR, for capturing images of preparation process, as well as images under magnification of planaria behavior, degradation, and confirmation of death

In preparation for the receipt of the order of live planaria flatworms, the ivermectin stock tubes were created in the following manner:

• It was determined that a starting mass of 6mg would be used from each ivermectin source in the ivermectin Table above.

• Tablets or capsules from each source were assumed to represent their effective mass (e.g. 6mg of ivermectin). Tablets that were a smaller labeled dose than 6mg (e.g. 3mg), were combined as a pair, whereas tablets or capsules of a labeled dose greater than 6mg (e.g. 12mg, or 18mg) were split proportionally. All tablets were crushed and ground using the mortar and pestle and all capsules (from compounding pharmacies) were opened and their powders used.

• First, tablets or powdered contents of capsules were weighed on the digital scaled (referenced above) to measure their total effective mass. Then, their corresponding fraction of effective mass was calculated based on the desired dosing. For example, the powdered contents of a capsule labeled as an 18mg dose was measured for its total effective mass, and one third of this total effective mass was measured out (into a 2mL Sarstedt capped tube, with scale tared) and stored in the tube.

• Pairs of 3mg tablets or single 6mg tablets were weighed before crushing/pulverizing and after scraping into tubes (with scale tared) for accuracy. This was performed for all 11 sources of ivermectin listed in the Ivermectin Table above.

• As per the Ferenc and Levin paper (above), the powdered products were dissolved and reconstituted by pipette-mixing in 100% DMSO for pipetting accuracy with single channel hand pipettes and tips by Rainin.

• This ivermectin and DMSO slurry was then taken through intermediate step-wise dilutions (using the 250mL bottles, 50mL Falcon tubes, and then 15mL Falcon tubes mentioned above) using the spring water referenced above. Ultimately, the final effective concentration of ivermectin in 10mL spring water (and 0.01% final DMSO by volume) was 5uM, also per Ferenc and Levin.

• These 5uM in 10mL stocks were kept until their use, respectively, in treatment of six worms in each designated petri dish, after randomization (see below).

• The Ferenc and Levin study used other concentrations and also involved cutting worms and observing the regenerative properties of the planaria (in control and ivermectin treatment conditions), but since the investigator here noted that the observations by Ferenc and Levin were that no worms had survived past Day 2 of treatment at the highest final ivermectin concentration (5uM in 10mL), this concentration alone was selected for the study presented here.

• No cutting actions were taken on the worms nor regeneration observations gathered, but rather a survival evaluation tally was made each day instead.

Controls

Negative control tubes (in 15mL Falcon tubes), of 10mL plain spring water, as well as a 0.01% DMSO by volume in 10mL spring water, were also prepared and used. The 0.01% DMSO treatment condition was used in order to demonstrate that any efficacy in lethality for the planaria was not due to the DMSO agent but rather each ivermectin source itself. The plain water negative control condition was to demonstrate the contrasted longevity of the planaria worms (which will eventually die of starvation if no food source is added to the petri dishes). This brought the total treatment conditions to 13 (11 different ivermectin sources normalized to 5uM in 10mL, plus two control conditions).

Feeding

There was no food source added to any petri dish, in order to control for variability in survival based on the worms feeding.

Planarian Worm Handling

Upon receipt, live planarian worms had their two jars opened (in which they were shipped by Carolina Biological Supply Company), for aeration and health of the worms. The worms were counted, totaling in number to sufficient to distribute six worms to each of the 13 petri dishes. They were dispensed by the plastic pipettes included in the shipment. The water in which the worms were shipped was aspirated, and 10mL of a fresh spring water pre-soak was used for the first 24 hours. Upon ensuring survival of all six worms per petri dish, the investigator pipetted off all of the spring water of the pre-soak and poured in the entire 10mL contents of each randomized and number-labeled 15mL Falcon tube, as described below.

Randomization

In order to control for any bias against any source of ivermectin, its country of manufacture, or any information gathered about one brand’s efficacy over another, the 13 tubes of corresponding solutions were randomized and the investigator blinded to their source. A key was determined by an assistant (not involved in any other preparation, lab work, or analysis) wherein each treatment condition was assigned a number, as selected using the site random.org, between 1 and 13. This key was kept by the assistant and not shared with the investigator until the conclusion of the study. Once the key was determined, the 10mL of contents of each labelled "treatment" tubes (15mL Falcon) by the assistant to new tubes labeled only with their corresponding number, per the key. As such, the investigator poured contents of each tube (labeled 1-13) into a petri dish labeled with the matching number label, and with six planarian worms. Thus, for the course of the study, the investigator was unaware of which source was in which dish, so as to not impact any observations or conclusions made.

Storage Conditions

As per the information sent by Carolina Biological Supply Company, the planaria were kept out of direct light, with each group of six worms kept in each of the 13 petri dishes. The entire set of dishes was stored in a cardboard box, taped closed to protect from light.

Daily Planaria Survival Evaluation

The time was noted during the addition of the 13 treatment conditions (on Day 0) and—as much as possible—the six worms in each dish were evaluated for survival at the same time on each subsequent test day. Worm survival tallies were kept in an Excel document and added to daily, with notes and observations inserted about worm behavioral, shape, and motility changes. These daily tallies and observations were performed until Day 16, when all the worms in 12 of the petri dishes had died (and many disintegrated). At this point, the assistant revealed the key referenced in the randomization section above to the investigator, and the below survival plot was made for all 13 of the treatment conditions after this unblinding.

Results

The results of this study are summarized in table format below (with the randomly assigned number association from the key listed), along with the observed day whereupon all planaria worms were observed to have died. The microscope used had an overhead as well as an underneath light, and the latter was used when evaluating the survival of each set of six worms for each treatment condition. This was helpful in determining death with more certainty and less subjectivity, as any slowing and crumpling worms would begin moving when agitated by the light underneath the sample area and clear petri dish.

To better and more dynamically represent the path and trending features of the data, the results are also shown in the below survival plot, with each unique color representing a different treatment condition (and, for those that were positive for ivermectin, a different source or brand).

Discussion

As mentioned earlier, there were interesting findings here regarding the variability of the antiparasitic efficacy of the 11 different ivermectin sources (compared to one another as well as to the two types of negative controls). One very interesting observation was that, at least in antiparasitic activity, there were multiple sources and brands of ivermectin that seemed to prove deadly for the worms earlier than even the US-made and mass produced Edenbridge brand: Veridex, Austro, Strive, CustomRx, and Beaker—as well as once source that killed worms equally as quickly as Edenbridge—Ivexterm.

As also discussed previously, more work needs to be done in investigating the variations in purity (and, perhaps therein, efficacy) of ivermectin in its different manufacturing sources and brands the world over. This can be done, in part, with tools such as HPLC.

This study, nevertheless, holds some value in terms of measuring the effective potency of each source of the medication in perhaps a more direct manner.