A long-standing drug for ancient diseases found itself at the center of a modern pandemic. What did the early laboratory science actually show?
In the frantic early days of the COVID-19 pandemic, with no approved treatments in sight, scientists embarked on a global hunt for existing drugs that could be repurposed to fight the novel coronavirus. One of the first to emerge from the shadows was a decades-old medication: hydroxychloroquine (HCQ). Familiar to many as a treatment for malaria and autoimmune conditions like lupus and rheumatoid arthritis, HCQ suddenly found itself in the international spotlight. The burning question was simple: could this old, widely available, and relatively inexpensive drug become a weapon against a new global threat? The first clues didn't come from hospital wards, but from laboratory petri dishes. This is the story of that initial, crucial in vitro discovery .
Before we dive into the experiment, let's clarify two key ideas.
The term in vitro (Latin for "in glass") refers to experiments conducted in a controlled laboratory environment outside of a living organism—think cells in a petri dish. This is the first crucial step to see if an idea has merit. In vivo ("within the living") studies are conducted in whole living organisms, like mice or humans. A positive in vitro result is a promising starting point, but it is far from a guarantee that a treatment will work in the complex system of the human body.
Viruses are cellular hijackers; they can't replicate on their own. SARS-CoV-2, the virus that causes COVID-19, typically enters a human cell by latching onto the ACE2 receptor on the cell's surface. It then gets engulfed by the cell in a tiny bubble called an endosome. For the virus to release its genetic material, the endosome needs to become acidic. Scientists hypothesized that HCQ, which is known to interfere with cellular acidification, could raise the pH of the endosome. Think of it as neutralizing the acid that the virus needs to escape its bubble. A trapped virus is a harmless virus .
Hydroxychloroquine is thought to work by increasing the pH of intracellular compartments like endosomes and lysosomes. This alkalization disrupts the acidic environment needed for the fusion of the viral envelope with the endosomal membrane, a critical step for SARS-CoV-2 to release its RNA into the host cell cytoplasm.
One of the earliest and most cited studies came from a team led by Dr. Michel Viénot in France, published in March 2020 . Their goal was straightforward: to test whether hydroxychloroquine, and its parent drug chloroquine, could stop SARS-CoV-2 from infecting cells in a lab setting.
The researchers designed a clean, controlled experiment to isolate the drug's effect.
They used a line of monkey kidney cells (Vero E6), which are known to be easily infected by SARS-CoV-2 and are a standard model for this type of research.
The cells were exposed to a live sample of the SARS-CoV-2 virus.
The crucial step. The team treated the infected cells with different concentrations of hydroxychloroquine and chloroquine.
After a set period (48 hours), they measured the viral load in the cells—essentially, how much of the virus had successfully replicated.
The results were striking. Both chloroquine and hydroxychloroquine demonstrated a powerful, dose-dependent ability to inhibit the virus.
The higher the concentration of the drug, the more effectively it blocked viral infection.
Notably, hydroxychloroquine was found to be even more potent than chloroquine in this cellular model.
The combination with azithromycin showed enhanced antiviral activity.
The data below illustrates these findings clearly.
| Drug | EC90 (the concentration for 90% virus inhibition) |
|---|---|
| Chloroquine | 4.51 µM |
| Hydroxychloroquine | 0.72 µM |
| Treatment | Viral Load Reduction (after 48 hours) |
|---|---|
| Untreated Cells | 0% (Baseline) |
| Hydroxychloroquine (HCQ) alone | ~90% |
| HCQ + Azithromycin | ~99% |
| Drug | Effective Concentration (EC50) | Cytotoxic Concentration (CC50) | Therapeutic Index (CC50/EC50) |
|---|---|---|---|
| Chloroquine | 2.71 µM | 261.3 µM | ~96 |
| Hydroxychloroquine | 0.72 µM | 249.5 µM | ~346 |
Interpretation: The therapeutic index for hydroxychloroquine was significantly higher, meaning there was a much wider gap between the dose that kills the virus and the dose that starts to harm the host cells. This suggested it could be a safer candidate.
To conduct an experiment like this, scientists rely on a specific set of tools and materials. Here's a look at the essential "toolkit" used in this in vitro study.
| Research Reagent | Function in the Experiment |
|---|---|
| Vero E6 Cell Line | A standardized line of monkey kidney cells that acts as a model for how the virus might behave in a living host. |
| SARS-CoV-2 Virus Isolate | The specific strain of the live virus, cultured and used to infect the cells. |
| Hydroxychloroquine Sulfate | The active pharmaceutical ingredient being tested, dissolved in a solution so it can be applied to the cells. |
| Cell Culture Plates | Plastic plates with multiple small wells, allowing scientists to test many different conditions (e.g., different drug concentrations) simultaneously. |
| qRT-PCR Machine | A sophisticated device that measures the amount of viral RNA present in the cells, providing a precise count of the viral load. |
The in vitro data was clear, compelling, and provided a solid scientific rationale for further investigation. It showed that, in a petri dish, hydroxychloroquine could effectively block the SARS-CoV-2 virus. This discovery ignited a wave of hope and prompted immediate clinical trials around the world.
Subsequent large-scale, randomized controlled trials—the gold standard in medicine—ultimately found that hydroxychloroquine did not provide significant benefit for treating COVID-19 in hospitalized patients and could cause serious heart-related side effects .
The story of hydroxychloroquine and COVID-19 is a powerful lesson in the scientific process. It highlights how a promising laboratory discovery is not an end point, but rather the first step in a long, rigorous, and often unpredictable journey to find a cure.