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Drug-binding Cavities in Long-Lived Biologics: Cause for Concern but Also Potential Benefit

From Nikos Panayotatos Consulting, Orangeburg, New York.

Address for reprints: Nikos Panayotatos, PhD, Nikos Panayotatos Consulting, 95 Monmouth Court, Orangeburg, NY 10962; e-mail: nikospan{at}optonline.net.

	ABSTRACT

TOP ABSTRACT CAVITIES BIND CHEMICALS WITH... CHANCE ENCOUNTER: CAUSE FOR... DETECTION, PREVENTION, AND... CONCLUSIONS REFERENCES

 
Universally present but overlooked cavities or pockets in long-lived biopharmaceuticals, such as monoclonal antibodies (mAbs), are capable of binding small drugs. Such direct interactions can alter the pharmacokinetics of drugs and potentially affect clinical outcome. The extreme differences in the pharmacokinetic properties of these 2 classes of drugs largely account for such effects. This overlooked mechanism of biologic-chemical drug interaction should be considered before approval of new long-lived entities.

Key Words: Protein cavity • protein pocket • direct biologic–drug interactions • side effects • targeted drug delivery • drug binding • pharmacokinetics • slow release • monoclonal antibody (mAb)

	CAVITIES BIND CHEMICALS WITH HIGH AFFINITY AND SPECIFICITY

TOP ABSTRACT CAVITIES BIND CHEMICALS WITH... CHANCE ENCOUNTER: CAUSE FOR... DETECTION, PREVENTION, AND... CONCLUSIONS REFERENCES

 
The sizes and conformations of protein cavities vary extensively. As such, they bind small organic molecules and even nanoparticles strongly and specifically. One mechanism that contributes to tight binding is conformational trapping. For example, an 4400 ų cavity engineered in a protein pore was found to retain organic molecules for nearly 1 second,⁴ whereas a cavity in the chaperone GroEL trapped diffused nanoparticles for at least 400 days but released them by the conformational change imposed by adenosine triphosphate binding.⁵

With the realization that most proteins possess noncatalytic pockets or cavities that bind chemicals came the realization that such features could be identified in 3-dimensional structures and exploited for practical purposes.⁷ Following this approach, review of published X-ray structures identified a 72-ų cavity in the constant region of mAbs of the IgG type that does not exist in other immunoglobulin G (IgG) types. By limited screening, compounds that bind IgG (but not IgG mAbs) with high affinity were obtained. Subsequently, such compounds were chemically extended, immobilized on chromatography beads, and used to successfully affinity-purify IgG mAbs from other mAbs and proteins.⁶

But what is the nature of the compounds that bind at the IgG cavity? A quick search in PubChem showed that several of these compounds have been tested for inhibitory activity against various enzymes and as anticancer agents. One, VSH4 (2-[(2S)-2, 3-dihydro-1,4-benzodioxin-2-ylmethyl]-1-ethyl-1H-imidazole), is the compound imiloxan, a highly selective 2B-adrenoceptor antagonist of known human and animal pharmacology. Further comparison of the chemical structures of the compounds that bind tightly to the IgG cavity suggests that common drugs such as the chemotherapeutics 5-FU and the recently approved vorinostat (N-hydroxy-N'-phenyloctanediamide) may also bind. Thus, the IgG cavity has the potential to bind tightly and selectively not only imiloxan but also other common drugs. Such binding occurring by chance during treatment with a mAb could lead to unpredictable effects.

A crucial factor in favor of such interactions is the unusually long half-life of mAbs both in circulation (up to 3 weeks) and in a target-cell–bound state (15 days or more; Abciximab Prescribing Information-Pharmacokinetics, http://www.reopro.com). These features contrast sharply with the half-life of most other biologics and chemical drugs, which are cleared from the body in a few minutes to a few hours. In principle, binding at the IgG or other pocket of a circulating mAb could significantly prolong a drug's clearance time. And binding at the cavity of an mAb already bound at its target could repeatedly trap and release a drug, increasing its concentration near the target tissue and altering its biodistribution (Figure 1). In principle, the IgG or other pocket could be modified by genetic engineering and used for directed delivery of desired drugs to an mAb target.⁷ The problem at hand, however, is the possibility that direct binding of chemical drugs through the IgG or other pocket, such as the haptenbinding site, might alter the pharmacokinetics and distribution of the mAb or the drug. Significantly, nearly all approved therapeutic mAbs are of the IgG type, and binding could occur not only with drugs intended for combined treatment but also with unrelated drugs prescribed for other indications and administered days later.

	CHANCE ENCOUNTER: CAUSE FOR CONCERN

TOP ABSTRACT CAVITIES BIND CHEMICALS WITH... CHANCE ENCOUNTER: CAUSE FOR... DETECTION, PREVENTION, AND... CONCLUSIONS REFERENCES

 
Can such direct biologic–drug interactions actually occur in vivo and, if so, what would their effect be on treatment?

Evidence for direct biologic–drug interactions resulting in pronounced pharmacokinetic effects has been obtained in animal models. When an mAb and its small chemical hapten were coadministered, the half-life of the hapten in vivo increased 17 times relative to an mAb control.⁹ Furthermore, the mAb could be recharged in situ with fresh hapten, even days after the initial antibody infusion.¹⁰ In other animal studies, endogenous antibodies against a small chemical molecule were found to serve as carriers and result in substantial decreases in clearance relative to mock controls.¹¹ Although such in vivo evidence is not available for drug binding through the IgG or other mAb pocket, the fact that compounds binding at the IgG cavity are sufficiently avid and specific so as to trap IgG mAbs out of a mixture of Escherichia coli protein contaminants⁶ speaks strongly for such a possibility. This evidence reinforces the concerns that direct mAb–drug binding can alter the pharmacokinetics of a drug and that the consequences of direct interactions could manifest even when an mAb and a small molecule are administered days and possibly weeks apart.

View larger version (14K): [in this window] [in a new window]   Figure 1. Effect of complex formation on drug half-life. Schematics of a typical antibody structure (adapted from Silverton et al8) and 2 compounds are shown, along with their complexes. Binding through a cavity could prolong half-life, alter distribution, and deliver a drug to the target of the mAb. Binding through the antigenbinding site increases the half-life of a drug 17-fold.9 Note that a typical antibody/drug size difference is 20-fold greater than shown.

	DETECTION, PREVENTION, AND POTENTIAL BENEFITS

TOP ABSTRACT CAVITIES BIND CHEMICALS WITH... CHANCE ENCOUNTER: CAUSE FOR... DETECTION, PREVENTION, AND... CONCLUSIONS REFERENCES

 
Undoubtedly, a prerequisite for pharmacologically effective direct biologic–drug interactions is a strong binding affinity, which may be possessed by very few drugs. But the difference in the half-lives of mAbs and drugs mitigates this requirement to a great extent.

In cases where strong interactions are involved, their effect should be evident in common pharmacokinetic studies, as in the mAb–hapten direct interactions mentioned above, and should be addressed. In contrast, if only weak direct biologic–drug interactions are involved, their effect may not be detectable by pharmacokinetic studies, because of the high molar excess at which drugs are used relative to biologics. In such cases, however, bound mAb could still redirect to its target a small but pharmacologically significant amount of free drug, prolonging its half-life in situ. In cancer patients, where the intratumoral half-life of the chemotherapeutic drug 5-FU increases from 10 minutes to 30 minutes, there is correlation with positive clinical outcome.^15,16 If increased intratumoral half-life can be achieved at lower dose as a result of half-life prolongation and targeted delivery of the drug, the potentially life-threatening side effects of chemotherapy could also be alleviated. But a beneficial effect seen under a specific protocol where a cytotoxic drug is coadministered with a cytotoxic mAb may not be reproducible if chemotherapy is initiated at a later time, after mAb clearance. The same could be true with adverse reactions. Ultimately, the nature of biologic and drug, the strength of their binding, their dose, and timing of administration will affect the outcome.

To prevent direct biologic–drug interactions, biologics could be tested prior to approval for binding drugs, particularly those that are likely to be administered during the course of treatment and a few weeks thereafter. One parameter to consider is that the conformation of the binding pocket may be different in the free and membrane-bound forms of the biologic, but appropriate experiments could be carried out to address this issue. Bioinformatics may be of help. Computational approaches to protein–chemical binding appear now capable of predicting novel protein–drug interactions at the genome scale.^17,18 And because most approved mAbs and their derivatives share the same structure in their constant region, only the variable regions would need to be modeled in order to study drug binding in silico. The complementary approach should also be instituted, namely, testing newly approved drugs for binding to the relatively small number of approved biologics—a much easier task at present.

Chance encounters between mAb cavities and drugs may be undesirable, but encounters by design could turn mAb cavities into unique drug-delivery systems. Protein cavity redesign for the purpose of binding desired molecules can now be achieved with structure-based computational methods without the need for high-throughput screening.¹⁹ Furthermore, the IgG pocket can tolerate modifications that do not impair the function of the mAb. Accordingly, it would be straightforward to identify drugs that bind an IgG mAb and, if necessary, modify the pocket to optimize binding. Treatment with such mAb–drug combinations would have considerable clinical guidance from the broad experience with its parent components.

	CONCLUSIONS

TOP ABSTRACT CAVITIES BIND CHEMICALS WITH... CHANCE ENCOUNTER: CAUSE FOR... DETECTION, PREVENTION, AND... CONCLUSIONS REFERENCES

 
The extended half-life of mAbs and other long-lived proteins combined with their capacity to bind common chemicals can result in direct biologic–chemical drug–drug interactions occurring by happenstance. If not recognized, such interactions could spuriously improve or confound results of clinical trials or subsequent treatment. Existing biologics, particularly mAbs and those engineered for increased half-life, such as erythropoietin and interferon derivatives modified by glycosylation or pegylation, should be evaluated for such effects. Development of future entities, particularly mAbs "humanized" by amino acid substitutions, should include the possibility that such manipulations may inadvertently create novel binding sites for drugs. Theoretically, such sites could be designed and engineered for targeted drug delivery.

Financial disclosure: None declared.

	REFERENCES

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Request Reprints Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Panayotatos, N. Search for Related Content PubMed PubMed Citation Articles by Panayotatos, N. Social Bookmarking                   What's this?