First published on Pharma Focus Europe on Oct 9th, 2024
Ritonavir is an antiretroviral drug discovered by Abbott Laboratories to treat acquired immunodeficiency syndrome (AIDS). It was approved by the Food and Drug Administration (FDA) in 1996, and was marketed in semi-solid capsule form under the brand name Norvir.
Ritonavir works by inhibiting the growth of the causative organism of AIDS called human immunodeficiency virus (HIV) and preventing damage to the immune system. The discovery of ritonavir, together with another contemporaneous protease inhibitor, saquinavir, contributed to drop the AIDS-related death rate in the US remarkably.
In the beginning, ritonavir was thought to exist as only one crystal form. However, almost two years after reaching market authorization, multiple drug lots started failing the dissolution test. Further investigation showed the existence of a more stable, crystalline form, which created a challenge for a drug that was already on the market.
This article discusses the impact of the discovery of the new crystalline form of ritonavir after the drug had been commercialized, and how it changed the perception of polymorphism in the pharmaceutical industry forever.
Emergence of AIDS
AIDS is a chronic illness that gained global attention in the 1980s. It weakens the immune system of the body, making it hard to fight infections. When HIV enters the body, it targets CD4 T-cells, a type of white blood cell that helps defend against diseases. It uses the machinery of T-cells to make new copies of the virus, due to which the immune cells are destroyed. As the virus spreads and the number of healthy T-cells falls below 200 cells/mm3, it leads to the more advanced stage of HIV infection called AIDS. People who develop AIDS are vulnerable to opportunistic infections (infections from a weakened immune system) and certain types of cancer.
The exact origin of HIV is unknown. However, researchers believe it originally came from a virus found in chimpanzees in central Africa. The virus, called simian immunodeficiency virus (SIV), is believed to be transmitted to humans through direct contact with chimpanzee blood while hunting. Later, the SIV would mutate to HIV within the human host.
Although HIV/AIDS surfaced as far back as the 1930s, the devastating impact of this virus gripped the world in the 1980s. In the early 1980s, medical experts began to report an alarming rise in cases causing widespread concern. In mid-1981, the Centre for Disease Control and Prevention (CDC) published its initial reports describing rare types of pneumonia and cancer affecting young, previously healthy homosexual men in the US. The disease was associated with immune suppression, causing the deaths of hundreds of people within a year of diagnosis. Primarily, the cases of AIDS were reported in the gay community. However, the ongoing investigation concluded that AIDS was not specific to sexual orientation. Rather, it spread through blood transfusions, sexual contact, and breast milk.
The limited knowledge about the virus and the lack of effective treatment options exacerbated the crisis. In 1983, researchers were able to identify the virus responsible for causing deaths, marking a critical turning point in the fight against the epidemic. With this groundbreaking discovery, researchers were able to develop diagnostic tests and paved the way for the development of antiretroviral therapies.
The growing public health crisis required an urgent approach, which led to the use of experimental treatment in its early stages. One of the first drugs used against HIV infection was a Nucleoside reverse transcriptase inhibitor (NRTI), zidovudine (also called Retrovir, azidothymidine, or AZT). This drug was previously developed to treat cancer, but it failed in doing so. Marketed in 1987, zidovudine showed some promise against HIV and was effective in decreasing death rates and opportunistic infections. A year after its use, the experts found that AZT didn’t work well on its own and had toxic side effects at higher doses.
When AZT therapy didn’t prove to be as promising as the scientists hoped, they developed additional NRTIs, including didanosine, zalcitabine, stavudine, and lamivudine. These drugs gained FDA approvals in the early 1990s, expanding treatment options. Unfortunately, patients quickly developed resistance to NRTIs, rendering the treatment ineffective.
After the short-lived benefits of NRTIs, there was a critical urgency to find new treatment options as the death toll was rising. At that time, the number of AIDS-related deaths exceeded 700,000 globally. Experts were continuously working to develop a drug that could transform this fatal disease into a manageable chronic condition. With the ongoing clinical trials, scientists soon discovered a new class of HIV drugs called protease inhibitors, which suppressed the replication of the virus. In 1995, the FDA approved saquinavir as the first protease inhibitor (PI). A few months later, ritonavir, another drug of this class, was developed by Abbott Laboratories.
Figure 1. Deaths caused by AIDS by year and age group.
In March 1996, ritonavir was approved for the treatment of HIV/AIDS. This powerful antiviral drug works by binding to the active site of HIV protease, which is essential for the replication of the virus. Once the active site is inhibited by the drug, the virus cannot mature and produce functional proteins. As a result, the amount of HIV virus is reduced in the body, leading to slow progression of the disease. Studies have found that ritonavir also inhibits cytochrome P450-34A enzyme in the liver and intestine, which is involved in the metabolism of protease inhibitors.
Ritonavir was one of the first protease inhibitors that became a part of Highly active antiretroviral therapy (HAART), involving the use of combination drugs to treat HIV. Within two years of HAART, the AIDS diagnosis was dropped by 45%, whereas the death rate declined by 63% in the US.
Withdrawal from the market
Abbott Laboratories marketed Ritonavir (Norvir) in 1996, as a semisolid capsule formulation. But then, the nightmare began. In 1998, researchers found that several batches of ritonavir were failing the quality control tests. The drug substance was not dissolving properly, and a solid was precipitating out of the semisolid capsules. The commercial lots were depleting rapidly, and the drug was severely diminishing its efficacy.
To understand the issue, the content of capsules was examined using microscopy and X-ray powder diffraction. The investigation revealed the existence of a new crystalline form (polymorph) denoted form II. This newly discovered form was more stable and much less soluble than the existing form I.
During the drug development stage, ritonavir was known to exist in only one crystalline form, form I, which was not easily absorbed by the body in a solid state due to which it was formulated in semi-solid capsule form. However, after the appearance of form II, the oral bioavailability of the drug was compromised in the semisolid formulation. The semisolid formulation of Norvir consisted of a hydro-alcoholic solution of ritonavir which was not saturated with respect to form I, but was very supersaturated with respect to form II.
Soon after the appearance of form II, Abbott sent a team of scientists to their manufacturing facility in Italy. The goal was to investigate whether any changes had occurred during the manufacturing process that led to the development of form II. At that time, form II did not appear in the drug in detectable quantities. However, shortly after the visit, the new polymorph began appearing in manufacturing lots and in lab formulations. The exact reason for the emergence of form II remains debatable but this dominance caused the failure to formulate semisolid capsules.
Norvir was also marketed in an oral liquid dosage form which was recommended to be stored at a temperature range of 2-8°C to maintain its stability. However, after the appearance of form II, the oral solution was prone to crystallization at this temperature.
Due to the stability crisis, Abbott withdrew the drug from the market, leading to disruption in the treatment of AIDS. In October 1998, Abbott Laboratories held a press conference, explaining why they could no longer supply ritonavir capsules. It is reported that the appearance of a late polymorph caused Abbott laboratories to lose more than 250 million US dollars.
Characterization of form I and form II
The crystalline forms were analyzed using solid-state spectroscopy and microscopy techniques such as solid-state nuclear magnetic resonance (NMR), Nuclear infrared (NIR) spectroscopy, powder X-ray diffraction and single crystal X-ray. These techniques confirmed that ritonavir existed in two forms and both forms had significant differences.
The solubility of ritonavir’s polymorphs in ethanol:water solvent mixtures, which was the system used for the original formulation, showed that form II had lower solubility throughout the whole range of mixtures.
The determination of the structure of both forms by single crystal X-ray showed that the two forms had differences in three specific torsion angles, which affected the shape and stability of each form. Form II exhibits an unusual torsion angle, adopting “cis” conformation for its carbamate group. This conformation should make the crystal less stable and more soluble because it requires energy to adopt and grow as a crystal. Despite this, form II crystals were far more stable because of the presence of strong hydrogen bonds.
Figure 2. Hydrogen-bond motifs of ritonavir’s form I and form II.
Both polymorphs of ritonavir exhibit hydrogen bonds, but these differ in their bonding patterns. The difference in the bonding network influences its crystallization and dissolution. Form I has a large surface area of exposed hydrogen bond donors and acceptors, which is why it can easily interact with the solvent (like water and alcohol) to dissolve quickly. In contrast, form II shows a uniform hydrogen bonding pattern where all the hydrogen bond donors and acceptors are satisfied internally. As a result, the stability of form II is increased. Here, the ritonavir solution follows Ostwald’s rule, which states that, in a supersaturated solution, the first crystals to form are usually the ones having the lowest energy barrier. So, even though form II is more stable, form I is likely to form first.
Once ritonavir is dissolved, both crystalline forms become identical, so either polymorph of ritonavir can be used in the production of Norvir soft gelatin capsules, as long as a new formulation is developed to maintain the drug fully dissolved.
However, ritonavir form II has certain drawbacks due to which it is not the most desirable form for manufacturing. Extensive studies on form II determined that lots prepared with the new polymorph had failure rates of up to 50%. The reason for this failure lies in the fact that it easily co-crystallizes with impurities, making it difficult to purify during the crystallization process. It also requires extended drying time to get rid of residual solvents, affecting the quality of the final product. Finally, it dissolves much slower than form I, which compromises the bioavailability of the product.
Since the new polymorphic form was not the form of choice for manufacturing, the next goal was to identify how the desired polymorph (form I) could be obtained from form II.
First, it was key to understanding if the supersaturated solution of form II kept its crystal memory. Using sonication (a technique that applies sound energy to agitate particles in a liquid), the scientists created a super-saturated solution of ritonavir form II which was kept in a closed system to prevent any contamination. After this, it was seeded with crystals of form I to induce crystallization. The final product showed that form I had crystallized, and no memory of form II was retained. This observation confirmed that ritonavir form I can be selectively generated from form II under a controlled environment.
To obtain seed crystals of form I, a reverse crystallization technique was used. This technique involved adding a small amount of form I seed crystals in a liquid solvent in which ritonavir is less soluble. Then a small amount of ritonavir solution was slowly added to the anti-solvent containing the seeds of form I. Since the solution is added in a small amount, the product immediately begins to crystallize and yields a large amount of seed crystals.
This approach proved successful as it led to the generation of form I using a minimal amount of seed crystals. Moreover, this process ensured the production of form I crystals starting with 100% form II. Scientists quickly put this technique into practice, which contributed to the reformulation of ritonavir. Finally, in 1999, ritonavir was re-marketed with a new formulation, which included butylated hydroxytoluene, ethanol, gelatin, iron oxide, oleic acid, polyoxyl 35 castor oil, and titanium dioxide, thus, once again, providing relief to patients.
Why did form II initially happen?
After the new polymorph was fully characterized, the researchers found the reason for the occurrence of form II. They established that crystallization of form II can only happen in a highly supersaturated solution if it was seeded with form II crystals. Since such seeds didn’t exist initially, this crystallization might have been due to heterogeneous nucleation by an impurity. When ritonavir is exposed to a base, it undergoes a degradation reaction that produces a cyclic carbamate linkage, structurally similar to form II. It is likely that this degradation product acted as a seed for the nucleation of ritonavir form II.
Coincidental discovery of ritonavir form III
The sudden stability crisis of Norvir highlighted the importance of comprehensive identification of all solid forms of an active ingredient. A thorough polymorph screening, and the identification of new forms are fundamental for effective drug development. It also helps in understanding solubility, stability, bioavailability, and other important physical properties of the different polymorphs, and how these can affect the development and manufacture of the drugs.
To screen polymorphs, multiple techniques have been developed to date including direct solid-solid conversion (i.e. grinding, thermal and moisture stress, etc.) and solvent mediated techniques (i.e. anti-solvent addition, cooling, evaporation, Ostwald ripening, etc.) and other techniques which expose the samples to non-ambient conditions (i.e. high-pressure crystallization, crystallization from the melt, sublimation, etc.). So, even after the drug was re-marketed, the researchers continued their efforts to understand the complex polymorphic behavior of ritonavir. In 2003, scientists from Cambridge, MA, performed an estimated 2000 experiments using high-through crystallization platforms to study ritonavir’s polymorphs. These experiments led to the discovery of two new solvated forms (form III and form V) and one metastable, anhydrous form (form IV).
A few years later, in 2014, scientists in Japan discovered a new crystalline form by crystallizing ritonavir from its melt after heating it in the oven at 60°C for several days. This form was initially believed to be similar to the anhydrous form IV found earlier in 2003. However, when the crystalline structure was observed under X-ray powder diffraction (XRPD), it did not match the published data. Despite this, it was referred to as form IV.
Almost twenty years after the appearance of form II, in 2022, a report was published describing the discovery of a new true polymorphic form of ritonavir, denoted form III[1]. The XRPD of the new crystal form was compared with the published data on the form IV pattern found in 2014, which revealed that the crystalline form discovered by Kawakami was not form IV; instead, it was a mixture of both form III and amorphous material. Therefore, although form III was, in fact, discovered in 2014, it was not recognized as a new form until 2022.
Figure 3. Hydrogen bonding motifs in ritonavir polymorphs.
The detailed analysis of form III found that it is the least stable and dense form when compared to form I and form II. The previously described metastable form IV was not considered for this comparison, owing to its obvious lack of stability. Form III presents a needle-like morphology and a lower melting point at approximately 114°C, while the melting point for form I and form II was around 120 and 121°C respectively.
When the solubility of the ritonavir polymorphs was determined under different pH conditions, the order of solubility was form III > form I > form II, which also confirms that form II is the most stable of this trio, while form III would be the most labile.
The hydrogen bonding patterns of form III are even more complex, forming a two-dimensional structure as compared to the other two forms that exhibit one-dimensional bonds. Similarly, the difference of conformations in various functional groups also affects the behavior and stability of this crystalline form. For instance, the N-methyl urea group of form III adopts trans conformation just like form II while the carbamate group forms trans conformation the same as form I.
Kaletra, a solution to solubility challenges
Following the challenges with ritonavir, Abbott Laboratories leveraged the knowledge gained to create Kaletra, a combination of two protease inhibitors: lopinavir and ritonavir. Approved in 2000 for HIV treatment, Kaletra incorporated ritonavir as a booster, enhancing lopinavir’s bioavailability through cytochrome P450 inhibition.
However, Kaletra’s formulation presented solubility issues. The solution was an amorphous solid dispersion (ASD), a critical technology to increase the bioavailability of poorly soluble drugs. Unlike crystalline forms, amorphous dispersions lack the orderly molecular structure, improving solubility and absorption.
The choice of ASD for Kaletra reflects a broader trend in addressing drug solubility challenges, particularly in protease inhibitors like ritonavir, where polymorphism can destabilize formulations. Amorphous dispersions continue to serve as a powerful tool for poorly soluble drugs, offering flexibility in API selection and increased therapeutic efficacy. Kaletra’s success with this technology emphasizes its potential to enhance other formulations struggling with solubility.
Conclusions
The commotion caused by ritonavir’s polymorphism emphasizes the need for comprehensive solid state investigation during the early stages of drug development. When a new, less soluble crystalline form of ritonavir appeared, it changed the stability and efficacy of the drug, leading to a costly market withdrawal. This sudden polymorphic transformation taught researchers that new crystalline forms can appear even years after rigorous testing and approval of the drug. The ritonavir case also caused a shift in the attitude towards the control of the physical properties of the APIs, such that the ability to demonstrate that these properties are well understood and will undergo no changes during development, manufacturing or storage, became a regulatory requirement.
The unpredictability of polymorphism highlights the need for pharmaceutical companies to adopt a proactive approach to understand how different solid forms of active pharmaceutical ingredients (API) relate to each other and to environmental changes, minimizing the risks in later stages. With adequate knowledge of the solid state behavior of the drug, it becomes easier to select the most suitable form that will remain stable throughout development and manufacturing. For precise characterization and ongoing monitoring, the companies should implement strategies to reduce risks and increase success rate, thus saving time and money.
Dr. Eugene Sun, from Abbott Laboratories, speaking in 1998 at a press conference on the ritonavir crisis, explained it very eloquently: “There are many mysteries of nature that we have not solved. Hurricanes, for example, continue to occur and often cause massive devastation. Meteorologists cannot predict months in advance when and with what velocity a hurricane will strike a specific community. Polymorphism is a parallel phenomenon. We know that it will probably happen. But not why or when. Unfortunately, there is nothing that we can do today to prevent a hurricane from striking any community or polymorphism from striking any drug.”
But we can, at least, learn from the past and minimize the impact on the drugs of the future. I am hoping.
References
- HIV-AIDS: A global epidemic and the leading cause of death in some countries, https://ourworldindata.org/hiv-aids
- Morbidity and Mortality Weekly Report, 2011, 60, 689-93
- Pharm. Res., 2001, 18, 859-866
- Org. Proc. Res. & Dev., 2000, 4, 413-417
- Cryst. Growth Des., 2023, 23, 320−325
- J. Pharm. Sci., 2023, 112, 237−242
- PNAS, 2003, 100, 2180-2184
- Mol. Pharmaceutics, 2023, 20, 3854–3863
[1] This form III is a true new polymorph of ritonavir, not to be mistaken by the solvated form discovered in 2003, also denoted form III.
