In press: special issue of Advanced Drug Delivery Reviews

Whole-body and microscopic autoradiography to determine tissue distribution of biopharmaceuticals - Target discoveries with receptor micro-autoradiography engendered new concepts and therapies for vitamin D

Walter E. Stumpf, University of North Carolina, Chapel Hill, NC2612 Damascus Church Rd., Chapel Hill, NC 27616Tel.: 919 942 8646E-mail: stumpfwe@email.unc.edu

Key Words: Imaging; Drug localization; Drug homunculus; Vitamin D; Estradiol; Receptor microscopic autoradiography

Abstract

Information about the distribution of biopharmaceuticals is basic for understanding their actions. Tissue and cellular localization is a key to function. Autoradiography with radiolabeled compounds has provided valuable information with both low resolution whole-body macro-autoradiography and high resolution microscopic autoradiography (micro-autoradiography). Whole-body macro-autoradiography is a uniform and expedient single method approach, providing convenient dose- and time-related overviews with data similar to those obtained with conventional bioassays – and therefore widely used. However, whole-body macro-autoradiography, like common bioassays, has limitations. High specificity-low capacity sites of binding and deposition frequently remain unrecognized. Lack of cellular resolution can cause false negatives and provide misleading results (e.g., false blood-brain barrier). For micro-autoradiography, different methods are advertised in the literature. Most of them are, however, unsuited for drug localization because of inadequate resolution and frequent artifacts. Most drugs

interact with their receptors non-covalently by weak electrostatic forces. Therefore, translocation and loss can occur during tissue preparation. This has complicated the use of micro-autoradiography. Receptor micro-autoradiography has overcome these complications and is the method of choice. It has been validated through several diffusible compounds with known localization, extensively applied. It has contributed numerous discoveries, followed by new concepts and therapies. Pictorial evidence in this review indicates that cellular information is essential, a ‘sine qua non’ for meaningful drug distribution studies. High resolution cellular microscopic information requires tissue dissection and the necessary precautions for preserving pristine in vivo drug deposition. Receptor micro-autoradiography fulfils these requirements. It reveals crucial information that cannot be obtained with any other type of imaging.

Footnote to page 1This article is dedicated to L.J. Roth who supported pioneering research in this field. He came up with this bon mot: ‘Don’t homogenize the brain; the brain you homogenize may be your own’. Putting it on the back of his name card, he liked to present it to speakers who disregarded cellular resolution.

Contents

1. Introduction2. Whole-body macro-autoradiography 3. Micro-autoradiography multiple methods 3.1 Current problems4. Receptor micro-autoradiography 4.1Receptor microscopic autoradiography – sequence of steps 4.2 Quantification 5. Examples of micro-autoradiograms 6. Comparison between whole-body macro-autoradiography and receptor micro-autoradiography7. Cellular localization a key to function - special discoveries and concepts 7.1 Drug Homunculus 7.2 Vitamin D and central nervous system 7.3 Vitamin D regulation of the digestive system 8. ConclusionsReferences

1. Introduction

Tissue distribution of biopharmaceuticals requires identification of dose- and time-related sites of deposition regarding specific receptor binding, metabolism, storage, delivery and excretion – with both tissue overviews and cellar details. There is no single method that can inform about all simultaneously. The right choice of methods is crucial for sound results and cost effective processing. Autoradiography with radiolabeled compounds has been useful in the past and promises to be so in the future, as long as certain requirements are observed. Autoradiography is not a single method, as the term may suggest. but consists of a range of methods from whole body macro-autoradiography to multiple different micro-

autoradiography methods. This has caused confusion and failures, in particular hampered the application of high resolution micro-autoradiography [1].

The overriding concern has to be the authentic representation of in vivo distribution of labile tissue constituents and drugs. This is a challenge and includes issues pertaining to experimental design, execution of technique, pictorial resolution, and interpretation of data, with an overarching concern for authentic representation of in vivo distribution of labile tissue constituents and drugs. This topic has been reviewed repeatedly, but the necessary precision has been pervasively disregarded in careless and superficial studies with untested methods and questionable claims published in the literature. It is important to recognize when imaging methods have limitations.

The lack of rigor in this area is at least partly due to the failure of regulatory agencies to demand sufficient identification of targets in new drug applications [1]. Against this backdrop, it has been easier to stick with simpler methods, even though current in vivo imaging lacks resolution and sensitivity. Perceived technical difficulties and exposure times of weeks and months, have likely also contributed to the reluctant uptake of ‘micro-autoradiography’ despite its superior potential. In general, not enough is known about drug target tissues and target kinetics. Micro-screening methods for drug target identification in tissues, other than receptor micro-autoradiography and, to a degree, topographical biochemistry, are non existent. Current in vivo imaging methods lack resolution and sensitivity. Whether or to what degree new spectrometric methods can replace or complement existing autoradiographic methods remains to be seen [2].

Labeling with radioisotopes has been applied successfully for tracing biopharmaceuticals in the body. As soon as artificial radioisotopes became available during the middle of the 20 th century, methods for autoradiography were developed at National Laboratories, university departments of pharmacology, and pharmaceutical companies [3,4]. These methods include the liquid emulsion technique [5] and the stripping film technique [6]. Soon, however, concern was expressed about the validity of autoradiograms in the study of diffusible substances when liquid fixatives, solvent dehydration, embedding, and liquid emulsion were used. Adequate preservation of cell and tissue morphology, while at the same time retaining diffusible compounds in situ, appeared very difficult or impossible [7]. Therefore, as a convenient alternative, whole-body macro-autoradiography with thick frozen sections was introduced [8]. Efforts toward cellular micro-autoradiography for drugs continued and ultimately proved successful with histological preparations that went beyond the traditional fixation and embedding procedures and produced the necessary cellular and tissue quality. Freeze-drying of frozen sections and thaw-mounting of thin unfixed frozen sections provided visible proof of adequate preservation, while, at the same time, retaining radiolabeled compounds at their authentic in vivo sites [9-11].The resulting and simplified receptor micro-autoradiography has since proven superior to other micro-autoradiography methods. Receptor micro-autoradiography has established itself as the only acceptable and safe general method for the cellular and tissue localization of biopharmaceuticals [1,2,12,13].

2. Whole-body macro-autoradiography

Whole-body macro-autoradiography with thick frozen sections was introduced by Ullberg at the Veterinary University in Stockholm [8]. Recognizing that information at different levels of resolution would not only be desirable but also necessary, Ullberg made continued efforts to obtain information [14]. This even included electron microscopy, although that was later abandoned. Attempts toward light microscopic autoradiography remained few and scattered. Despite several efforts [15], no generally applicable method for light-microscope autoradiography for drug localization was developed in the Ullberg laboratory. Instead, the whole body method was advanced from the initial cold-room condition to automated cryostat sectioning. Replacement of X-ray exposure with digitized imaging further facilitated its use [16-18]. Whole-body autoradiography now provides convenient qualitative and quantitative overviews of dose- and time-related distributions of radiolabeled compounds administered to experimental animals (Fig. 1). This renders it more expedient for routine use than the conventional biochemical dissection method [19,20].

Fig. 1: Example of whole-body autoradiography that shows a sequence of changing distribution of radiolabeled compound at different time intervals after injection of 3H-Oxa-Calcitriol, an analogue of vitamin D, at a dose of 10 μg/kg (male rat), sacrificed at 30 minutes, two, eight and 24 hours after the injection [13].

The information gained with whole-body autoradiography is, however, limited. If these limitations are not considered, false conclusions may lead to erroneous decisions and costly failures in drug

development. With whole-body autoradiography, the distribution of the radiolabel does not distinguish between specific target binding and unspecific deposition. A distinguishing competition with excess unlabeled compound is not possible, primarily because the diagnostic dose required for signal recognition is too high. For the same reason, distribution studies of low-dose therapeutics are readily compromised. In routine experiments, high capacity-low specificity sites of deposition can easily be demonstrated. However, high specificity-low capacity sites – most frequent in brain, spinal cord, and pituitary[2] – often are not recognized, for example, when they remain undetected or are overshadowed by signals from strong unspecific depositions. Interpretation of results may be difficult without complementary support from more sensitive high resolution receptor micro-autoradiography [21]. Without cellular resolution, over-interpretation of data with both false negatives (e.g., surmised blood-organ barriers) and false positives can result, with costly consequences.

The 40 μm or 20 μm thick sections used in whole body autoradiography cause superposition of structure and absorption of radiation from layers remote from the detector. During the freezing of whole animals, tissue and cell structures are disrupted. If such sections are viewed with a microscope, the destruction through intracellular and extracellular hexagonal ice formation is so severe that cell types can no longer be identified. These and other factors contribute to low resolution pitfalls and the need to complement whole-body autoradiography with receptor micro-autoradiography.

Appropriate validation should be required for any imaging before its experimental use. Where there are apparent limitations, validation of method is even more critical. This means comparing localization of diffusible compounds for which detailed distribution data are known, such as estradiol and vitamin D. To make comparisons with bioassay results – which themselves are lacking resolution [22-25] - would not be valid. Whole-body autoradiography has never been adequately validated by its users. Advantages and limitations of whole-body autoradiography have been reviewed [21].

3. Micro-autoradiography multiple methods

Since the inception of micro-autoradiography by Belanger and Leblond (1946) [5] and Pelc (1947) [6], many variations have been recommended in the literature, mostly related to tissue preparation, resolution and application of nuclear emulsion. For the localization of covalently bound substances and precursors that are incorporated into macro-molecules, like amino acids for protein synthesis, uridine and thymidine for RNA and DNA synthesis, traditional preparations with liquid fixation and embedding can be used [26]. For diffusible compounds, like most drugs, that are bound to their receptors not covalently but by weak van der Waals and ionic forces, tissue preparation must avoid liquid fixatives, embedding media, any liquid treatment, and also liquid emulsion, in order to avoid translocation and loss.

As a result, the scientific struggle for the development of a workable general method for drug localization at the microscopic cellular level has been formidable. It has been difficult to preserve both cellular-subcellular structure in the same preparation, with results comparable to those obtained with fixed and embedded thin sections, and without altering in vivo distributions of diffusible substances. So

difficult that this was all considered impossible [7]. Many investigators abandoned this pristine pursuit or resorted to a simpler apposition technique with two slides, i.e., a temporary assembly with the mounted tissue section on one slide and nuclear emulsion on the other slide. While disassembly after photographic exposure allows separate processing of the two slides, it undermines realignment of the tissue structure and encumbers the radiation record. With this apposition method, cellular resolution is not possible.

Among the numerous methods and modifications advertised in the literature, none matches the expectations of a generally applicable high resolution micro-imaging approach that is sufficiently devoid of artifacts and adequately tested using compounds with known localizations.

Without digressing further into the history of this complicated matter, potential users of micro-autoradiography should appreciate the importance of method details. They should also be aware of problems that can result from digressing from receptor micro-autoradiography, the only well-tested method documented by evidence [12,27].

3,1 Current problems

Despite repeated admonitions and incontrovertible evidence of artifacts, confusion continues through erroneous ‘expert’ publications and claims. Widespread endorsement of deficient methods and untested modifications of existing methods have prevented the broad and successful use of micro-autoradiography for drug localization.

Two recent publications [28,29] illustrate this situation. The technique used in these publications is purported ‘based on the methods of Appleton and Stumpf’, without specifying which steps used in the technique belong to the one and which to the other. Although both methods are ‘micro-autoradiography’, they cannot be mixed. Their differences have been discussed [13] and differing results are apparent in the literature.

In the first publication [28], one single ‘micro-autoradiogram’ is included, showing localization of a 14C-labeled test compound ‘in the sebaceous glands of rat skin after topical application’ (Fig. 2, left). While it is recognized that topical applications pose special problems, the demonstration in this review is not an acceptable example of micro-autoradiography. Demonstration of cellular localization would be a prerequisite. In this specimen, however, neither cells nor sebaceous glands can be recognized. There are dense clusters of silver grains which may or may not be related to specific cells. Artifacts cannot be excluded because of the variability of the (overexposed) clusters that cover different structures. For comparison, note a picture of a sebaceous gland from receptor micro-autoradiography studies of vitamin D with nuclear concentration of radiolabel in keratinocytes and basal cells, diminished or absent deposition in mature sebocytes (Fig.2, right).

Similarly, in the second publication, which was a follow-up review [29], the author presents two sections of the eye with disrupted morphology, claiming the use of micro-autoradiography. These specimens are without cellular detail, and again are not representative of micro-autoradiography. Furthermore, to substantiate the micro-autoradiography technique purportedly used in this case, the

author refers to an article by Nagata [30]. However, Nagata did not study drugs, but instead used RNA- and DNA-precursors that are chemically bound to macromolecules. Nagata’s methods are used in electron microscopic studies and have not been applied to light-microscopic autoradiography of drugs. The reader is misled, both as to the specific results and the general application of micro-autoradiography. This kind of contemporary review article thus compromises the potential successful use of ‘micro-autoradiography ‘, which if properly conducted is capable of producing meaningful results.

Fig. 2 (left) is offered as representative of light-microscopic autoradiography with ‘labeled sebaceous glands’ [28]. However, in this low-power picture, cellular morphology is not recognizable, nor can the clusters of silver grains (arrowed and non-arrowed) be attributed to defined structures. The claim that the underlying structures are sebaceous glands is conjectural. In this disrupted tissue section (pressure) artifacts cannot be excluded. Such a picture is misleading and should not be offered as an example of micro-autoradiography which by definition includes cellular resolution and recognition of labeled cells. Compare Fig. 2 (right), a 4 μm thin section prepared with receptor microscopic autoradiography after injection of a 3H-labeled vitamin D analogue [31]. This shows a cut through lanugo hairs with attached sebaceous gland together with outer hair sheaths’ strongly labeled keratinocytes. Individual cells and nuclei can be recognized in the associated sebaceous gland with weakly labeled basal cells and unlabeled sebocytes.

4. Receptor micro-autoradiography

For localizing drugs at the light-microscopic cellular-subcellular level with simultaneous low power tissue overview, receptor microscopic autoradiography is documented as the best and most reliable general method available. This method has evolved after multiple tests during the early and mid 1960s, and subsequent facilitation, followed by extensive applications with numerous discoveries and development of new concepts. Design and development was aimed at recognizing ‘where it goes’ [32], to obtain microscopic in vivo information that was authentic, pristine, and undistorted. The goal was ‘to study the unmolested tissue’. This drove the elimination of steps in the preparation of tissue and autoradiogram that could pose a potential threat of translocating labile tissue constituents. Any unnecessary treatment was to be avoided. Liquid fixation, embedding, and liquid emulsion needed to be excluded. In test experiments with two diffusible compounds, 3H-estradiol and 3H-mesobilirubinogen, with known localization, it became apparent that each of the six tested methods provided different method-specific,

reproducible results [10]. The least deviations from the known and assumed localization was achieved by ‘dry-mount’ and ‘thaw-mount’ procedures, then still under elaborate tests [11].

In developing this method, some erroneous scientific concepts had to be overcome and a number of discoveries emerged. These related, for example, to the exploration of optimal cooling temperatures and freezing speed, ice crystal formation, and the feasibility of cutting thin frozen sections. At the beginning, it was not yet clear whether temperature of minus 40°C or even minus 80°C would be required, or whether frozen sections had to be cut at such low temperatures in order to minimize disruptive hexagonal ice grows. Along the way, fixation was shown to be unnecessary - cellular structures could be preserved without it. It turned out that freeze-dried sections were useful not only for biochemical analyses as with the Lowry method, but, if well prepared, could also provide high subcellular resolution, such as kidney brush border and mitochondrial basal striation [11]. Furthermore, the notion that sections cannot be cut below -45°C was disproved [33]. Cutting sections at much lower temperatures succeeded [34], giving rise to utra-cryomicrotomy.

These and other trial-and-error experiments with their many controls led to two acceptable methods: ‘dry-mounting of freeze-dried frozen sections’ and ‘thaw-mounting of thin frozen sections’. Since the results obtained with the thaw-mounting procedure resembled those obtained with the more pristine but more elaborate and demanding freeze-dried section dry-mounting procedure, thaw-mounting of thin 4 μm sections became the standard approach for routine experiments. The method was named ‘Receptor Microscopic Autoradiography’ to distinguish it from similar but less reliable methods. Among the important features of receptor micro-autoradiography is the restricted use of frozen sections of 4 μm or less (depending on the type of tissue). Thawing of frozen sections 5 μm or more can lead to chemical interaction of wet tissue constituents with nuclear emulsion to create either positive or negative chemography artifacts. Dry-mounting of frozen sections on dry emulsion coated slides without thawing, while desirable, incurs section loss during photographic development, rinsing and staining.

4.1 Receptor microscopic autoradiography – sequence of steps

APPLICATIONof radiolabeled compound with high specific activity (includingcompetition)

EXCISIONof multiple tissues and positioning on holders

FREEZE-MOUNTINGon tissue holders

LIQUID NITROGEN STORAGEof mounted tissues

CRYO-SECTIONINGof 4 μm (or less) sections

THAW-MOUNTINGon emulsion-coated slides

PHOTOGRAPHIC EXPOSUREin desiccator boxes

PHOTOGRAPHIC PROCESSING

STAINING single step with MBBF or MGP

AIR-DRYING and COVER-SLIPPING

EVALUATIONqualitative and quantitative

Each step of the method requires special attention, which is discussed in detail [13]. To give some examples: Excision of tissue samples must be done rapidly and without damage to tissue structure. Utmost care must be applied, including not grasping with the forceps those parts of the tissue to be sectioned later. The excised tissue should be placed immediately on the ice-cooled tissue holder und anchored, gently positioned there so that the intended cut surface is exposed for later sectioning. During freeze-mounting, a two-step procedure is followed (conductive freezing from the holder immersed in the coolant fluid followed by slow direct freezing of the experimental sample). Depending on the tissue size, the procedure needs to be adapted in order to freeze as fast as possible and as slow as necessary, taking care not to lose the sample by fracturing it. While this may sound complicated, it is well explained [13] and with a few trial experiments, the necessary experience is quickly achieved. Thin cryo-sectioning requires a sharp knife. Without one, sufficiently thin sections are not possible, skipping occurs and cut sections can be double in thickness. It should be possible to cut ribbons of 4 μm sections. Several of these sections are then thaw-mounted onto an emulsion-coated slide with a gentle touch and quick removal of the section-mounted slide. Some practice with test slides can assure optimal histology without pressure artifacts.

Any method of high resolution and sensitivity requires special attention to detail and preparatory practice. Less sensitive methods are typically not as exacting, and this is one reason why those accustomed to using whole body autoradiography have difficulty practicing receptor micro-autoradiography side by side.

Investigators in micro-autoradiography should be trained in cell biology, pharmacology and nuclear medicine. Experience and skills in the execution of the various technical steps are required in order to minimize errors and artifacts. For these and other reasons, this author has advocated education in Histopharmacology for pharmacologists and pharmacists, enabling them to train and gather experience in established Laboratories of Histopharmacology in pharmaceutical institutions [35].

4.2 Quantification

Quantification of autoradiograms has been performed in two different ways: (1) counting of labeled cells or structures without counting individual silver grains, or (2) counting of individual silver grains in a defined compartment or structure. Silver grain counting is possible only without clustering of grains, i.e., when coincidence quenching, which prevents linear analysis, is excluded. It is even possible to calculate the number of molecules from the number of silver grains, if the specific silver grain yield has been assessed [38]. Precise quantification is one of the attractive possibilities of micro-autoradiography. This is not possible, for example, with immunocytochemistry.

5. Examples of micro-autoradiograms

Samples are provided below to demonstrate the potential of the receptor micro-autoradiography method. Two sections from the early freeze-dried frozen section dry-mounting procedure are included in Fig. 3. The thaw-mounting procedure is the simpler, more routine method (Figs. 4 to 6). The pictures demonstrate the importance of cellular resolution for meaningful drug distribution studies. Noteworthy are the simultaneous low and high resolution in the same microscopic slides, the different information from different exposure times, the relevance of controls, and the possibility of characterizing cellular localization by combining micro-autoradiography with immunocytochemistry. From the specific localization depicted in the autoradiograms, clues for specific functions can be derived. This is discussed elsewhere [13].

Fig. 3: Early high resolution autoradiograms with freeze-dried frozen sections. Left: hypothlamic neuron (2 μm). Right: uterine gland (1 μm). Two hours after injection of 0.63 μg of 3H-estradiol, rat. Concentration of silver grains over nuclear regions. First demonstration of histological quality sections without fixation and embedding (Stumpf and Roth, 1969)

Fig. 4: Autoradiograms of rat pituitary after injection of 3H-estradiol, demonstrating the heterogeneity of receptor binding to different cell types. Left:The strongest labeled cells have been characterized by combined autoradiography-immunocytochemistry as gonadotrophs [38]. Center: After long exposure, all pituitary cell types appear labeled; differences are less apparent (unpublished). Right: Pituitary thyrotropes after injection of 3H-1,25(OH)2 vitamin D3 and combined immunocytochemistry with antibodies to thyroglobilin [39].

Fig. 5: Autoradiograms after injection of 3H-1,25(OH)2 vitamin D3. Left: Brain, central nucleus of amygdala with strongly labeled, weakly labeled, and unlabeled neurons. Center: Cerebellum, labeled Golgi type 2 cells (arrows) with unlabeled granule cells and Purkinje cells. Right: Spinal cord ventral horn motor neurons with strong nuclear concentration of radiolabeled compound [40,41].

Fig. 6: Autoradiograms after injection of 3H-1,25(OH)2 vitamin D3 (Stumpf, 1995; 2003; 2008) showing (A and B) low magnification overviews of duodenum with nuclear concentration in epithelium of villi and crypts, radioactivity in intestinal lumen with epithelial barrier; (C) transition between unlabeled cells of stomach antrum and labeled cells of duodenum absorptive epithelium; (D) duodenum at the region of Brunner gland with a few labeled pyloric muscle cells (arrow) above unlabeled smooth muscle cells of the intestinal muscularis; (E) duodenum labeled absorptive epithelium with unlabeled Goblet cell; (F) liver sinusoid litoral cells (probably Ito cells) with cytoplasmic concentration and retention of

L

radiolabeled compound (arrows), hepatocytes do not show concentration of radioactivity; (G) pancreas labeled islet beta cells stained with antibodies to insulin [43]; (H) kidney macula densa cells with nuclear concentration of labeled hormone (center); (I) vibrissa hair section with outer hair sheath labeled cells (arrows); J: parathyroid overview with strongly labeled chief cells; (K) parathyroid (high magnification, short exposure time), nuclear concentration of radioactivity; (L) parathyroid after injection of 3H-estradiol, serving as control, - compared to 3H-(OH)2 vitamin D3, with 3H-estradiol only connective tissue cells are labeled (arrows), while parathyroid chief cells are unlabeled (unpublished).

6. Comparison between whole body macro-autoradiography and receptor micro-autoradiography

Distribution data obtained with whole body autoradiography in general agree with those obtained with biochemical dissection procedures. When data from each procedure have been compared und used to validate each other, deficiencies have not become apparent. However, validation of a deficient method with another similar deficient method is suspect and should be unacceptable.

With radiolabeled vitamin D, when data with similar experimental conditions from both radioassays and whole-body autoradiography were compared with data from receptor micro-autoradiography, numerous false negatives were revealed [1,13]. With biochemical assays and macro-autoradiography, brain and spinal cord were found negative [44-47], while at the same time maps of vitamin D target neurons were being published [2]. Other false negatives with whole-body autoradiography and/or bioassays include: heart atrial myocytes, pyloric muscle, adrenal medullary cells, gastric endocrine cells, gastric gland isthmus cells, myoepithelial cells, salivary gland cells, and many others [12]. The schematic below illustrates some of the differences in the distribution of vitamin D between whole-body autoradiography and receptor micro-autoradiography, reproduced from a memorandum to the Food and Drug Administration and International Conference of Harmonization [1]. The results of such comparative studies indicate a need for adequate controls and validation. Designating whole-body autoradiography as a stand alone method for determining the localization of drugs can be misleading since distribution cannot be completely assessed. An uninformed reviewer may accept such ‘localization’ to be representative and accurate, when it is not.

Fig. 7: Comparison of Vitamin D distribution assessed by whole-body autoradiography (rat, black and white center) with negative results for brain (BRA), spinal cord (SC), stomach (STO), and skin (SK), and by receptor microscopic autoradiography (side color images) with specific nuclear receptor binding demonstrated in distinct cell populations in brain, spinal cord, stomach endocrine G-cells; and skin keratinocytes, as well as parathyroid chief cells (PTH), heart atrial myocytes (HRT) and many others not shown [1].

7. Cellular localization a key to function - special discoveries and concepts

In the early experiments with both dry-mount and thaw-mount micro-autography procedures, contributions were made toward the cellular and tissue distribution of estradiol and mesobilirubinogen [48-50]. With 3H-estradiol, it was established in collaboration with biochemists that the hormone concentrates not in or at cell membranes but in target cell nuclei, to which the hormone translocates [48, 49]. Furthermore, 3H-estradiol specific binding sites were discovered in different parts of the brain,

not confined to the hypothalamus, but at projection sites of the stria terminalis in the piriform-endorhinal cortices, amygdala, preoptic-septal region, thalamus, and midbrain [51-52], as well as in female and male reproductive and ‘non-reproductive’ organs [53], and even in atrial muscle of the heart [54]. With these and a list of other target discoveries, the thinking about estradiol actions changed. Estradiol was no longer viewed as a mere hormone of female reproduction, but one with multiple functions in both female and male organs.Similar to estradiol, the autoradiographic exploration of testosterone, dihydrotestosterone, corticosterone, aldosterone, and dexamethasone expanded knowledge about their respective distributions and related functions [55]. For dexamethasone, contrary to natural adrenal steroids, a blood-brain barrier with bypass and delayed entry through the ventricular system was demonstrated [56]. Aldosterone distribution in the brain was first reported by an East German group, independently using our thaw-mount procedure [57] with quantitative follow-up in diverse brain regions [58].Ecdysteroids were first demonstrated in insect brains and related to the molding cycle[59].Brain and spinal cord maps have been provided for several hormones and species with receptor micro-autoradiography. This topic has been reviewed recently [2], which includes references not only for steroid hormones, but also for retinoic acid, glucose, 2-deoxyglucose, and the alpha-emitter 210Pb lead.

Perhaps the most important contributions of receptor micro-autoradiography came from studies with vitamin D metabolites 3H-1,25(OH)2 vitamin D3 (vitamin D for short), 3H-25(OH) vitamin D3, and 3H-24,25(OH)2 vitamin D3 during the 1980s and early 1990s. Multiple discoveries of vitamin D target tissues - unrelated to systemic calcium regulation and, unexpected by calcium doctrinists - were initially suppressed, even rejected. Substantial published receptor micro-autoradiography data preceded by more than two decades the current scientific acceptance of vitamin D’s wide role in health for prophylaxis and therapy. Receptor micro-autoradiography identified and characterized over 50 target cell populations [12] in contrast to the 5 ‘classical target tissues’ bone, intestine, kidney, liver, and parathyroid. Already in 1979 and early 1980s 1,25(OH)2 vitamin D3 nuclear receptor distribution was reported in specific cell populations in brain, spinal cord, pituitary, skin, thymus reticular cells [60], stomach, pancreas beta cells, adrenal medulla, and many others [12]. With bioassays, in several laboratories, brain and spinal cord were repeatedly - and erroneously - found negative and considered non-target tissues [44-46]. In contrast, in these and several other tissues, strong selective distribution was demonstrated with the high resolution histochemical approach. Accordingly, maps of brain and spinal cord of vitamin D sites in various mammalian und vertebrate species have been published [2]. As predicted from the brain and spinal cord target distribution [61], multiple sclerosis, Alzheimer, Parkinsonism, autism, sleep disorders, various mental diseases and dysfunctions, are now treated successfully with vitamin D [62,63].

Not only positive results, but also negative findings were startling to biochemists. In rodents, there was no nuclear concentration of 3H-1,25(OH)2 vitamin D3 in mature skeletal and smooth intestinal muscle, while nuclear concentration was demonstrated in heart atrial muscle, pyloric muscle, and myo-epithelial cells of exocrine glands [42]. Fat cells, postulated in the literature as sites of deposition of fat-soluble vitamin D and treated as such in computer models, did not reveal concentration of radiolabel, rather concentration and retention was instead noted in the lumen of blood vessels and in ground substance of

connective tissue, especially the substantia propria of mucous membranes [42]. Following the results of extensive and revealing receptor micro-autoradiography studies, the concept that calcium homeostasis is vitamin D’s main function has been challenged and reduced to it being one of its many actions, one related mainly to bone growth and repair.

Far from these Procrustean calcium confines of many decades, the amazing story turns out to be that the main function of this “vitamin” is no less than the fundamental maintenance of life, including the adaptation of vital functions to the solar environment with changing prevalence and sensitivity of organ systems.

7.1 Drug Homunculus

The distribution pattern of vitamin D obtained in preclinical animal experiments can be usefully projected on a human body diagram named “drug homunculus”. A drug homunculus is created from the detailed information about the distribution of target tissues. The concept can be applied to any drug to comprehensively and conveniently present deposition sites, i.e., target and non-target distribution, related actions, side effects, and possible toxicity. Through computer links, dose- and time-related pictures can be created, and related biochemical, functional, and clinical data can be included. More simply, a drug homunculus could inform about drug logistics only, without or with added quantification. Revealing comparisons between parent compound and analogues, antagonists, the outcome of genetic ‘null’ animals, assessment of changes related to age, endocrine status, and disease conditions could be presented. Comparisons related to the assessment of sensitivity or specificity of different imaging techniques could also be worthwhile.

A drug homunculus could become a valuable tool for ‘systems pharmacology’. Whole-body autoradiography assumes to do just that in an expedient and simplified fashion. But evidence provided in this article makes it clear that it is necessary to do the precise, detailed, specifically localized work of receptor micro-autoradiography before then aggregating the individual pieces into an accurate whole body view, as with the homunculus. High resolution information is indispensable; it cannot be gained without dissection and focus, and any focus requires reintegration. Historic and scientific analysis of the vitamin D story makes this evident.

Structures in red indicate sites of vitamin D receptors

Fig. 8: Vitamin D ‘Drug Homunculus’ of male [64].

7.2 Vitamin D and central nervous system

Brain, spinal cord, and neurons of the peripheral nervous system provide strong evidence for the need to obtain cellular resolution [2]. At a time, when biochemical methods were used to publish the absence of vitamin D [44-46], as with whole-body autoradiography [13], receptor micro-autoradiography in mammalian and vertebrate species led to the published diagram below [2,61].

Vitamin D sites of action in the central nervous system

Fig. 9: This schematic of the central nervous system indicates nuclear receptor binding sites and action of vitamin D on motor, sensory, autonomic-endocrine circuits, and on select cortical, cerebellar, and spinal neurons [40,61]. The size of dots reflects different degrees of nuclear binding and suggests selective and differential responses related to vitamin D blood levels.

7.3 Vitamin D regulation of the digestive system

Vitamin D can also be considered a regulatory hormone of the digestive system, much like its historically appreciated role for systemic calcium, in addition to similar roles for endocrine, reproductive, immune, and other systems [65]. In the digestive system, the abundance of vitamin D nuclear targets identified and characterized with receptor micro-autoradiography include specific cell populations of all salivary glands, teeth, epithelia of the oral cavity and esophagus, gastric gland isthmus cells, entero-endocrine cells of the antrum, pyloric muscle cells, absorptive and proliferating epithelium of the small and large intestine, beta cells of pancreatic islets, as well as cytoplasmic accumulation of radiolabel in litoral cells of liver sinusoids, probably Ito cells. Even in the brain, neurons of the central amygdale and related septal region, which are involved in gastric functions [66], also show nuclear concentration. Such detailed information of distribution, provides clues for function and a basis for follow-up on prophylaxis and potential therapies.

Fig. 10: This schematic reviews vitamin D target tissues involved in digestive functions [42]. It includes brain target neurons in the central amygdala and septal nucleus of stria terminalis, corresponding to experimental data [66].

8. Conclusions

This review demonstrates the importance of tissue distribution studies at both the organ and, in particular, the cellular level as a basis for understanding drug actions. Both low and high resolution is essential. Autoradiography with radiolabeled compounds is an attractive, proven imaging procedure for the distribution of drugs. Various methods have been used over the course of almost a century. Two approaches remain of particular interest and are critically reviewed here: whole-body autoradiography and receptor micro-autoradiography.

The advantages of whole-body macro-autoradiography have led to its widespread use. It is quite uniform in its present applications, ‘expedient’ in execution, and able to yield both qualitative and quantitative data. The time- and dose-related information provided in the whole body pictures can be

informative and conveniently used as a guide. However, there are significant limitations of resolution and sensitivity that are not widely acknowledged or understood, but should be considered in the interpretation of data. With whole-body autoradiography, no cellular information can be obtained. The tissue structure is not preserved, but rather destroyed by the slow whole body freezing process and tissue slices are thick (40-20 μm). Signal recognition requires a high dose, which excludes low-dose drug distribution studies and produces false negatives for important ‘low dose-high specificity’ sites of receptor binding. Likewise, competition studies for distinguishing specific target sites from unspecific sites of deposition are not possible. Therefore, any cases that require cell biological information or that include target cell populations embedded in non-target tissues necessitate something more than whole-body macro-autoradiography.

Receptor micro-autoradiography has been available for over 40 years. An array of results has been provided that not only validates its utility, but information has been gained that would have been impossible or difficult to obtain otherwise. New vistas and avenues have been opened for any of the compounds studied [2]. However, the execution of receptor micro-autoradiography - as is the case with any high resolution, high sensitivity method – requires a higher degree of attention to detail and experimental experience compared to ‘expedient’ low resolution methods. As history has shown, the choice of method has a direct bearing on the formulation of concepts, and restrictive choice can lead even the most avid scientist astray [12,65]. Because of the specific requirements, the premium on precision, and the demand on skilled and experienced personnel, routine application of high resolution methods may narrow to those willing to make the effort [13,35].

Many drugs, old ones included, are poorly understood with respect to their distribution and multiple targets. This used to be the case with vitamin D under the calcium doctrine before information became available from receptor micro-autoradiography [12,60] – and there are still vitamin D questions to be explored (e.g. which targets are associated with enzymes for local hydroxylation of the precursor molecules, is there a relation to the calcium sensing receptor [68], etc. Drug use could be improved, and side effects ameliorated, if target tissues were better identified. With a willingness to apply receptor micro-autoradiography, responses to lingering questions may finally be forthcoming. For example, how different is the distribution between low and high doses? How does the cellular and tissue distribution relate to differing low and high dose effects, including when they are opposite to each other – as under the Arndt-Schultz rule of hormesis [64,69]. How constant or adaptive are expressions of receptors and targets under varying conditions of age, disease, etc.? Does a newly developed drug reach its expected targets? Does the drug also reach distribution sites that are unexpected, particularly ones that could cause potential side effects, be related to toxicity, or open new avenues?

Some may query whether receptor micro-autoradiography can be simplified, digitized, or even replaced by new spectrometric methods. This question has been discussed [2]. While improvements of methods may always be possible, there has been no alternate success in matching the sensitivity of nuclear emulsion for radiation detection and storage, whether through digitizing the radiation signals or otherwise [69]. The thaw-mounting of thin 4 μm section with the nuclear emulsion provides exceptionally close contact between radiation source and detector. This, together with the preservation

of cellular structure and of unaltered ‘in vivo’ drug deposition, account for the unique information possible with receptor micro-autoradiography

Current evidence suggests that some spectrometric applications can provide information similar to that obtained with whole body autoradiography [70,71]. Spectrometric methods can be useful for the distribution and characterization of large and covalently bound molecules that withstand histological tissue preparation without translocation. However, diffusible compounds, including most drugs, require the same precautions featured in receptor micro-autoradiography. Autoradiographic and spectrometric methods are likely to coexist and provide complementary information in the future, depending on the type of compound. Autoradiography with radiolabeled compounds continues to be useful for the foreseeable future and essential for drug investigations at the cellular level. As demonstrated by the evidence in this article and validated by its record of discovery, innovation and paradigm shift, autoradiography with radiolabeled compounds provides information that is difficult or impossible to achieve with other methods. Receptor micro-autoradiography remains the best method for cellular tissue imaging of drug distribution.

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No conflict of Interest