Abstract
Informational molecules regulate numerous processes in the body. However, most of these – if used therapeutically – are difficult to deliver safely and effectively to patients. Our work in this area started in 1974 with our studies on isolating the first angiogenesis inhibitors and has continued to this day and includes developing approaches to deliver substances that can regulate stem cell behavior. Applications range from cancer treatments, to Covid vaccines, to regenerative medicine, including possibly restoring hearing.
Introduction
I started my postdoctoral career working with the late Judah Folkman, attempting to isolate the first inhibitor of angiogenesis (new blood vessel growth). To do this, it was critical to develop a bioassay for angiogenesis inhibitors, nearly all of which were macromolecules. We conceived of using a rabbit cornea assay where we could directly visualize blood vessel growth (Langer et al., 1976) through an ophthalmic microscope. However, that assay could take up to several months, so it was critical to develop a very small biocompatible controlled release system that would not cause inflammation in the cornea, and that could slowly and continuously release macromolecules (e.g., peptides, proteins, and nucleic acids). When I started my investigations, it was widely believed that only low-molecular weight lipophilic compounds – but certainly not ionic molecules, peptides, proteins, or nucleic acids – could be controllably delivered from biocompatible materials (e.g., polymers, lipids) (Langer, 2019).
Developing a method to control the movement of macromolecules and ionic species from biocompatible materials
Nonetheless, it was crucial to create such systems if we were going to isolate angiogenesis inhibitors. Thus, I began studying this problem by examining different materials with a known safety record in humans (Langer et al., 1981) as well as different formulation approaches (Langer and Folkman, 1976; Langer, 2019).
After several hundred failures, I discovered an approach that enabled the continuous release of different molecules including nucleic acids and proteins for up to 100 days (Figure 1). This discovery was initially ridiculed by the scientific community. My first nine research grant applications were rejected. No chemical engineering department in the country would hire me as a faculty member. So I ended up joining the Nutrition and Food Science Department at MIT. But the year after I joined, the department head who had hired me left, so a number of members of the senior faculty told me I should leave, too. As my colleague, Michael Marletta recalled:
“One evening, I went to a faculty dinner with Bob Langer and some senior MIT professors. A senior scientist sat quizzing us while smoking a cigar. When the older scientist heard Langer’s concept for … drug delivery, he blew a cloud of smoke in Langer’s face and said, ‘You better start looking for another job…’”
Although much later, the National Academy of Sciences would cite this work as being “responsible for much of today’s drug delivery technology” and Nature would cite this work for the “founding the field of controlled release drug delivery” these repeated rejections at this early stage in my career were devastating to me (Langer, 2019).
I recall Dr. Folkman suggesting we file a patent. In the 1970s, Boston Children’s Hospital, where I started this research, had not filed a patent before. However, they agreed to let us file one. However, for five years in a row, the patent examiner rejected the patent application. Then the head of the Hospital’s Technology Transfer Office told me the patent would never be allowed and that I should stop trying to convince the examiner, since explaining the science was not working. However, I don’t like to give up. As discussed earlier, when we started our research, many people told us that delivery of macromolecules from small particles was impossible – that it could never work. I wondered if anyone had written that down. So, in 1982, I did a citation search of our 1976 Nature paper, and I found many papers citing us. One of them, written by five of the leading materials scientists in the world, described the drug delivery field as follows:
“Generally, the agent to be released is a relatively small molecule with a molecular weight of no larger than a few hundred. One would not expect that macromolecules, e.g. proteins, could be released in by such a technique because of their extremely small permeation rates … However, Folkman and Langer have reported some surprising results that clearly demonstrate the opposite” (Stannett et al. 1979).
“Surprising” was a critical word for the patent examiner. When the examiner saw that, he said if I could get written affidavits from all five scientists that they really wrote that, he would allow the patent. So I wrote them and they were all nice enough to write back that they really wrote it, and so the examiner agreed to allow the patent (U.S. Patent 4391797). Over time, this discovery enabled the practical use of many peptides, charged low-molecular pharmaceuticals, proteins, and nucleic acids. Since such molecules have extremely short half-lives in the body (minutes in some cases), a controlled release system must often be used (Langer, 2019).
These controlled release systems enabled us to isolate the first substances that could inhibit the vascularization of tumors (Langer et al., 1976). Using the rabbit cornea and the controlled release pellets as a bioassay for tumor-induced vascularization, we assessed the inhibitory effect of many partially purified macromolecule fractions. Pellets of drug delivery system and pieces of tumor (V2 carcinoma) were placed in the corneal pockets in over 1000 corneas (Figure 2a). Normally, the tumors grew as thin plaques, inducing vessels to sprout from the edge of the cornea 4-6 days after implantation. Vessel length and tumor diameter were measured every few days.
When drug delivery pellets were empty or if a macromolecule fraction was inactive (as was almost always the case), vessels appeared as a dense carpet sweeping over the pellets toward the tumor (Figure 2b). When vessels penetrated the tumor, it grew rapidly, into a large protruding mass occupying nearly the entire cornea. Very similar results were obtained when pellets containing substances without inhibitory activity were tested. By contrast, when an inhibitor was present, vessels were sparse, grew slowly, and failed to grow in a zone surrounding the drug delivery pellet (Figure 2c). By the 4th week, many vessels were regressing. It was remarkable to see that the blood vessels were stopped in their tracks or even regressing with my own eyes every day.
This study (Langer et al., 1976) established that angiogenesis inhibitors did, in fact, exist. Over many decades, the above controlled release systems have proven fundamental to the isolation and study in vivo of nearly all angiogenesis stimulators and inhibitors, as well as numerous other informational molecules in developmental biology studies (Langer, 2019).
There are also many controlled release systems used by patients worldwide that continuously release peptides for up to six months from a single injection (e.g. Lupron Depot©, Zoladex©, and Decapeptyl©). Similar or related microspheres or nanospheres, or other systems containing bioactive molecules have led to treatments of schizophrenia (Risperdal Consta©), alcoholism, opioid addiction (Vivitrol©), arthritis (Zilretta©), controlling bleeding (Floseal©, Surgiflo©), pituitary dwarfism (Nutropin Depot©), Type 2 Diabetes (Bydureon©), all drug eluting stents, and many other diseases. Nucleic acids like RNA are also protected and delivered from small particles. Examples are OnPattroã and all Covid mRNA vaccines (e.g., SpikeVax©). (Langer, 2019; Dong et al, 2021).
One additional area where drug delivery may be useful is in tissue engineering/regenerative medicine. Every year millions of patients suffer tissue loss or end-stage organ failure. For the most part, physicians treat organ or tissue loss by transplanting organs from one individual into another, performing surgical reconstruction, or using mechanical devices such as kidney dialyzers. Although these therapies have saved and improved countless lives, they are imperfect solutions. In the early 1980s, my colleague, Jay Vacanti, who was head of the liver transplantation program at Boston Children’s Hospital asked me if we could create a new liver. Prior to this, several research groups had worked to try creating two-dimensional systems to form certain tissues. We started to use two-dimensional cell/material systems as well. However, after much work in trying to grow liver cells in two-dimensional structures (e.g., Discs, Petri dishes) to test our prototypes, we realized that we could not get enough cells per unit volume to create tissue with enough liver function. One day, Jay was in Cape Cod and saw some seaweed. So, he called me and said, “Bob, could you make a polymer system that was three-dimensional, like seaweed, and if so, could that solve the surface-to-volume problem?” So, we did (see Figure 3) and these eventually led to new ways to create cartilage, spinal cords, blood vessels, and many other tissues and organs. It also helped lead to organs and tissues on a chip, which may reduce drug testing in animals and people (Langer, R., 2019., Molecular Frontiers Journal).
Regenerative Medicine, Hearing Loss, and Drug Delivery
One area of regenerative medicine where we combined novel drug delivery systems with discoveries in stem cell biology involves hearing loss. An estimated 1.1. billion people are at risk of disabling hearing loss worldwide for which there is currently no pharmacologic treatment. Chronic sensorineural hearing loss (SNHL) accounts for roughly 90% of this sensory deficit and is likely caused by noise, chemical, viral, and aging insults with potentially debilitating effects. In people with SNHL, audibility (loudness of sound) and intelligibility (clarity of words) deteriorate due to the aforementioned auditory insults. Except for “retrocochlear” hearing loss, more than 80% of chronic SNHL is due at least in part to loss of cochlear hair cells. While many vertebrates such as birds and reptiles generate hair cells spontaneously to restore hearing after various insults, mammals do not. Mammalian progenitor cells that produce hair cells during embryonic development persist into adulthood but are quiescent. (McLean, W., 2021) It occurred to us that Lgr5+ stem cells exist in other parts of the body and are precursors to hair cells. Although Lgr5+ intestinal stem cells have been expanded in vitro as organoids, homogenous culture of these cells has not been possible thus far. Building on the work of Hans Clevers, who collaborated with us in our initial study (Yin et al, 2014), we discovered that two small molecules (CHIR99021 and valproic acid (VPA), synergistically maintain self-renewal of mouse Lgr5+ intestinal stem cells resulting in nearly homogenous cultures. We found that the colony forming efficiency of cells from these cultures is ~100-fold greater than that of cells cultured in the absence of CHIR99021 and valproic acid (CV), and multilineage differentiation ability is preserved. We used these homogenous cultures to identify conditions employing simultaneous modulation of Wnt and Notch signaling to direct lineage differentiation into mature enterocytes, globet cells, and Paneth cells. We then showed that the combination of CHIRS99021 and valproic acid (VPA) acts synergistically to activate proliferation of quiescent mammalian cochlear progenitor cells in vitro from mice, nonhuman primates, and humans (McLean et al., 2017). Additionally, a murine ex vivo study showed the application of CHIR99021+VPA (CV) following aminoglycoside ototoxicity induced supporting cells in the organ of Corti expressing the leucine-rich repeat-combining G-protein receptor 5 (Lgr5) to divide and regenerate hair cells (McLean, W. et al., 2017).
Working with Jeff Karp, Will McLean, Chris Loose, David Lucchino, and others, we thought that applying compounds that regenerated hair cells could provide a novel approach to improve auditory function in subjects with chronic SNHL. CV was formulated for human use by developing a novel drug delivery system. In particular, we used a thermoreversible poloxamer named Pluronics that can be injected intratympanically as a liquid which will then transition to a gel in the middle ear to allow the prolonged diffusion into the cochlea. The combination of the gel and CV is called FX-322. It is important to note that this is the reverse of what normally occurs with gels (normally increases in temperature decreases gelation). We then conducted clinical cochlear pharmacokinetics (PK) and pharmacodynamics (PD) studies to examine FX-322 as a potential therapy for restoration of hearing in patients with SNHL. We first evaluated spatial and temporal drug distribution in guinea pig cochlea. Predicted concentrations were compared to those that showed activity in ex vivo mouse and human studies. Drug concentrations were measured for samples of middle ear contents and perilymph to calibrate the human PK model and validate measured and modeled values. A Phase 1b clinical trial was conducted to assess safety of intratympanically administered FX-322 in adult human subjects with chronic SNHL and to study its PD effect on hearing. (McLean et al., 2021)
In humans, comparison of baseline and Day 90 pure tone thresholds showed no statistically significant differences between the groups at any frequency. However, Day 90 pure-tone assessment showed that 4/15 FX-322-treated patient ears had 10 cB improvement at the highest test frequency (9kHz), whereas no placebo-treated ears showed this level of improvement. Changes in individual word recognition (WR) performance were analyzed to determine if any clinically meaningful change had occurred based on parameters set forth by Thornton and Raffin’s binomial distribution. Of the 23 participants, 10 displayed a deficit in WR performance (≤90%) before treatment so could be assessed for hearing improvement without a “ceiling effect”. Of these ten patients, six were treated with FX-322 and four with placebo. Four of the six FX-322-treated ears showed statistically significant and clinically meaningful improvements from baseline to 90 days in the prespecified WR test, exceeding expected results of test-retest variability for this measure. In contrast, no placebo-treated ears showed statistically significant changes. The four FX-322-treated ears that had clinically meaningful improvements has an absolute mean (SE) WR increase of 35.4 (5.5) percentage points. FX-322-treated subjects’ speech recognition improved over the duration of the study while placebo-treated subjects did not. In WR assessment, FX-322-treated ears showed a statistical improvement in percentage change from baseline scores versus placebo on average across all time points (p=0.029). The effects were sustained throughout the study, with the following least-square mean difference (SE)=18.3% (11.0); Day 30=14.2% (11.4); Day 60=s2.1% (11.4); and Day 90=21.9% (11.0) (McLean, W.J. et al., 2021).
Speech recognition in a noisy background using WIN also improved over time for FX-322-treated patient ears but not placebo-treated patient ears. Performance was quantified as the signal-to-noise ratio (SNR: 0-24 dB) consistent with 50% correct WR, with lower SNR values indicating better speech perception in background noise. Analyses showed a significant improvement in average SNR from baseline to Day 90 in FX-322-treated ears (-1.3 dB; P=0.012) but not placebo-treated patient ears (-0.21 dB, p=0.71) (McLean, W.J. et al., 2021).
Individual responses across intelligibility tests for four FX-322-treated patient ears showed clinically significant improvements. Absolute improvements in WR from baseline to Day 90 range from 18 to 42% in these four patients. Two of these four ears showed substantial and clinically meaningful improvements in WIN testing from baseline to Day 90, with SNR improvements that exceeded the 3.1 dB threshold representing the theoretical difference exceeding expected test-retest variabilities established by Wilson and McArdle. Subjects from both etiologies and dose volume cohorts responded to treatment (McLean, W.J. et al., 2021). A summary of clinical data is shown in Figure 5.
Conclusion
In summary, the totality of the above studies has led to the first delivery systems for administering macromolecules and has helped enable the discovery of new angiogenesis inhibitors, the development of Covid vaccines, and many other new therapies. These delivery systems and extensions thereof have also played a role in regenerative medicine and could possibly play a role in new approaches for treating hearing loss.
References
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McLean, W.J., Yin, X., Lu, L., Lenz, D.R., McLean, D., Langer, R., Karp, J.M., & Edge, A. (2017). Clonal expansion of Lgr5-positive cells from mammalian cochlea and high-purity generation of sensory hair cells. Cell Reports, 18(8), 1917-1929. https://doi.org/10.1016.
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McLean, WJ., Hinton, A.S., Herby, J.T.J., Salt, A.N., Hartsock, J.J., Wilson, S., Lucchino, D.L., Lenarz, T., Warnecke, A., Prenzler, N., Schmitt, H., King, S., Jackson, L.E., Rosenbloom, J., Atiee, G., Bear, M., Runge, C.L., Gifford, R.H., Rauch, S.D., Lee, D.J., Langer, R., Karp, J.M., Loose, C., LeBel, C. (2021). Improved speech intelligibility in subjects with stable sensorineural hearing loss following intratympanic dosing of FX-322 in a phase 1b study. Otology & Neurotology, Aug 1;42(7):e849-e857. doi: 10.1097/MAO.0000000000003120. PMID: 33617194; PMCID: PMC8279894.