Potency assay to predict the anti-inflammatory capacity of a cell therapy product for macrophage-driven diseases: overcoming the challenges of assay development and validation.

BACKGROUND: Given the high level of product complexity and limited regulatory guidance, designing and implementing appropriate potency assays is often the most challenging part of establishing a quality control testing matrix for a cell-based medicinal product. Among the most elusive tasks are the selection of suitable read-out parameters, the development of assay designs that most closely model the pathophysiological conditions, and the validation of the methods. Here we describe these challenges and how they were addressed in developing an assay that measures the anti-inflammatory potency of mesenchymal stromal cells (MSCs) in an M1 macrophage-dominated inflammatory environment. METHODS: An in vitro inflammation model was established by coculturing skin-derived ABCB5+ MSCs with THP-1 monocyte-derived M1-polarized macrophages. Readout was the amount of interleukin 1 receptor antagonist (IL-1RA) secreted by the MSCs in the coculture, measured by an enzyme-linked immunosorbent assay. RESULTS: IL-1RA was quantified with guideline-concordant selectivity, accuracy and precision over a relevant concentration range. Consistent induction of the macrophage markers CD36 and CD80 indicated successful macrophage differentiation and M1 polarization of THP-1 cells, which was functionally confirmed by release of proinflammatory tumor necrosis factor α. Testing a wide range of MSC/macrophage ratios revealed the optimal ratio for near-maximal stimulation of MSCs to secrete IL-1RA, providing absolute maximum levels per individual MSC that can be used for future comparison with clinical efficacy. Batch release testing of 71 consecutively manufactured MSC batches showed a low overall failure rate and a high comparability between donors. CONCLUSIONS: We describe the systematic development and validation of a therapeutically relevant, straightforward, robust and reproducible potency assay to measure the immunomodulatory capacity of MSCs in M1 macrophage-driven inflammation. The insights into the challenges and how they were addressed may also be helpful to developers of potency assays related to other cellular functions and clinical indications.

Drug Regulatory-Compliant Validation of a qPCR Assay for Bioanalysis Studies of a Cell Therapy Product with a Special Focus on Matrix Interferences in a Wide Range of Organ Tissues.

Quantitative polymerase chain reaction (qPCR) has emerged as an important bioanalytical method for assessing the pharmacokinetics of human-cell-based medicinal products after xenotransplantation into immunodeficient mice. A particular challenge in bioanalytical qPCR studies is that the different tissues of the host organism can affect amplification efficiency and amplicon detection to varying degrees, and ignoring these matrix effects can easily cause a significant underestimation of the true number of target cells in a sample. Here, we describe the development and drug regulatory-compliant validation of a TaqMan® qPCR assay for the quantification of mesenchymal stromal cells in the range of 125 to 20,000 cells/200 µL lysate via the amplification of a human-specific, highly repetitive α-satellite DNA sequence of the chromosome 17 centromere region HSSATA17. An assessment of matrix effects in 14 different mouse tissues and blood revealed a wide range of spike recovery rates across the different tissue types, from 11 to 174%. Based on these observations, we propose performing systematic spike-and-recovery experiments during assay validation and correcting for the effects of the different tissue matrices on cell quantification in subsequent bioanalytical studies by multiplying the back-calculated cell number by tissue-specific factors derived from the inverse of the validated percent recovery rate.

Kinetics of Wound Development and Healing Suggests a Skin-Stabilizing Effect of Allogeneic ABCB5+ Mesenchymal Stromal Cell Treatment in Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis (RDEB) is a rare, inherited, and currently incurable skin blistering disorder characterized by cyclically recurring wounds coexisting with chronic non-healing wounds. In a recent clinical trial, three intravenous infusions of skin-derived ABCB5+ mesenchymal stromal cells (MSCs) to 14 patients with RDEB improved the healing of wounds that were present at baseline. Since in RDEB even minor mechanical forces perpetually provoke the development of new or recurrent wounds, a post-hoc analysis of patient photographs was performed to specifically assess the effects of ABCB5+ MSCs on new or recurrent wounds by evaluating 174 wounds that occurred after baseline. During 12 weeks of systemic treatment with ABCB5+ MSCs, the number of newly occurring wounds declined. When compared to the previously reported healing responses of the wounds present at baseline, the newly occurring wounds healed faster, and a greater portion of healed wounds remained stably closed. These data suggest a previously undescribed skin-stabilizing effect of treatment with ABCB5+ MSCs and support repeated dosing of ABCB5+ MSCs in RDEB to continuously slow the wound development and accelerate the healing of new or recurrent wounds before they become infected or progress to a chronic, difficult-to-heal stage.

ABCB5+ mesenchymal stromal cells facilitate complete and durable wound closure in recessive dystrophic epidermolysis bullosa.

BACKGROUND AND AIMS: Recessive dystrophic epidermolysis bullosa (RDEB) is a hereditary, rare, devastating and life-threatening skin fragility disorder with a high unmet medical need. In a recent international, single-arm clinical trial, treatment of 16 patients (aged 6-36 years) with three intravenous infusions of 2 × 106 immunomodulatory ABCB5+ dermal mesenchymal stromal cells (MSCs)/kg on days 0, 17 and 35 reduced disease activity, itch and pain. A post-hoc analysis was undertaken to assess the potential effects of treatment with ABCB5+ MSCs on the overall skin wound healing in patients suffering from RDEB. METHODS: Documentary photographs of the affected body regions taken on days 0, 17, 35 and at 12 weeks were evaluated regarding proportion, temporal course and durability of wound closure as well as development of new wounds. RESULTS: Of 168 baseline wounds in 14 patients, 109 (64.9%) wounds had closed at week 12, of which 63.3% (69 wounds) had closed already by day 35 or day 17. Conversely, 74.2% of the baseline wounds that had closed by day 17 or day 35 remained closed until week 12. First-closure ratio within 12 weeks was 75.6%. The median rate of newly developing wounds decreased significantly (P = 0.001) by 79.3%. CONCLUSIONS: Comparison of the findings with published data from placebo arms and vehicle-treated wounds in controlled clinical trials suggests potential capability of ABCB5+ MSCs to facilitate wound closure, prolongate wound recurrence and decelerate formation of new wounds in RDEB. Beyond suggesting therapeutic efficacy for ABCB5+ MSCs, the analysis might stimulate researchers who develop therapies for RDEB and other skin fragility disorders to not only assess closure of preselected target wounds but pay attention to the patients' dynamic and diverse overall wound presentation as well as to the durability of achieved wound closure and the development of new wounds. TRIAL REGISTRATION: Clinicaltrials.gov NCT03529877; EudraCT 2018-001009-98.

Translational development of ABCB5+ dermal mesenchymal stem cells for therapeutic induction of angiogenesis in non-healing diabetic foot ulcers.

BACKGROUND: While rapid healing of diabetic foot ulcers (DFUs) is highly desirable to avoid infections, amputations and life-threatening complications, DFUs often respond poorly to standard treatment. GMP-manufactured skin-derived ABCB5+ mesenchymal stem cells (MSCs) might provide a new adjunctive DFU treatment, based on their remarkable skin wound homing and engraftment potential, their ability to adaptively respond to inflammatory signals, and their wound healing-promoting efficacy in mouse wound models and human chronic venous ulcers. METHODS: The angiogenic potential of ABCB5+ MSCs was characterized with respect to angiogenic factor expression at the mRNA and protein level, in vitro endothelial trans-differentiation and tube formation potential, and perfusion-restoring capacity in a mouse hindlimb ischemia model. Finally, the efficacy and safety of ABCB5+ MSCs for topical adjunctive treatment of chronic, standard therapy-refractory, neuropathic plantar DFUs were assessed in an open-label single-arm clinical trial. RESULTS: Hypoxic incubation of ABCB5+ MSCs led to posttranslational stabilization of the hypoxia-inducible transcription factor 1α (HIF-1α) and upregulation of HIF-1α mRNA levels. HIF-1α pathway activation was accompanied by upregulation of vascular endothelial growth factor (VEGF) transcription and increase in VEGF protein secretion. Upon culture in growth factor-supplemented medium, ABCB5+ MSCs expressed the endothelial-lineage marker CD31, and after seeding on gel matrix, ABCB5+ MSCs demonstrated formation of capillary-like structures comparable with human umbilical vein endothelial cells. Intramuscularly injected ABCB5+ MSCs to mice with surgically induced hindlimb ischemia accelerated perfusion recovery as measured by laser Doppler blood perfusion imaging and enhanced capillary proliferation and vascularization in the ischemic muscles. Adjunctive topical application of ABCB5+ MSCs onto therapy-refractory DFUs elicited median wound surface area reductions from baseline of 59% (full analysis set, n = 23), 64% (per-protocol set, n = 20) and 67% (subgroup of responders, n = 17) at week 12, while no treatment-related adverse events were observed. CONCLUSIONS: The present observations identify GMP-manufactured ABCB5+ dermal MSCs as a potential, safe candidate for adjunctive therapy of otherwise incurable DFUs and justify the conduct of a larger, randomized controlled trial to validate the clinical efficacy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03267784, Registered 30 August 2017, https://clinicaltrials.gov/ct2/show/NCT03267784.

Skin-Derived ABCB5+ Mesenchymal Stem Cells for High-Medical-Need Inflammatory Diseases: From Discovery to Entering Clinical Routine.

The ATP-binding cassette superfamily member ABCB5 identifies a subset of skin-resident mesenchymal stem cells (MSCs) that exhibit potent immunomodulatory and wound healing-promoting capacities along with superior homing ability. The ABCB5+ MSCs can be easily accessed from discarded skin samples, expanded, and delivered as a highly homogenous medicinal product with standardized potency. A range of preclinical studies has suggested therapeutic efficacy of ABCB5+ MSCs in a variety of currently uncurable skin and non-skin inflammatory diseases, which has been substantiated thus far by distinct clinical trials in chronic skin wounds or recessive dystrophic epidermolysis bullosa. Therefore, skin-derived ABCB5+ MSCs have the potential to provide a breakthrough at the forefront of MSC-based therapies striving to fulfill current unmet medical needs. The most recent milestones in this regard are the approval of a phase III pivotal trial of ABCB5+ MSCs for treatment of recessive dystrophic and junctional epidermolysis bullosa by the US Food and Drug Administration, and national market access of ABCB5+ MSCs (AMESANAR®) for therapy-refractory chronic venous ulcers under the national hospital exemption pathway in Germany.

Allogeneic ABCB5+ mesenchymal stem cells for treatment-refractory chronic venous ulcers: a phase I/IIa clinical trial.

A significant number of chronic venous ulcers (CVUs) fail to heal despite of guideline-conform standard of care. Skin-derived ABCB5+ mesenchymal stem cells (MSCs) can dampen the sustained IL-1ß-driven inflammation present in chronic wounds. Based on their wound healing-facilitating effects in a mouse CVU model and an autologous first-in-human study, ABCB5+ MSCs have emerged as a potential candidate for cell-based advanced therapy of non-healing CVUs. In the present interventional, multicenter, single-arm, phase I/IIa clinical trial, subjects whose CVU had emerged as standard therapy-resistant received one or two topical applications of 1×106 allogeneic ABCB5+ MSCs/cm2 wound area in addition to standard treatment. Out of 83 treatment-emergent adverse events, only three were judged related to the cell product; they were mild or moderate and recovered without sequelae. Wound size markedly decreased from baseline to week 12, resulting in a median wound size reduction of 76% (full analysis set, N=31), 78% (per-protocol set, N=27) and 87% (subset of responders; n=21). In conclusion, the study treatment was well tolerated and safe. The treatment elicited a profound wound size reduction within 12 weeks, identifying ABCB5+ MSCs as a potential candidate for adjunctive therapy of otherwise incurable CVUs. These results justify the conduct of a larger, randomized, controlled trial to confirm clinical efficacy.

Clinical trial of ABCB5+ mesenchymal stem cells for recessive dystrophic epidermolysis bullosa.

BACKGROUNDRecessive dystrophic epidermolysis bullosa (RDEB) is a rare, devastating, and life-threatening inherited skin fragility disorder that comes about due to a lack of functional type VII collagen, for which no effective therapy exists. ABCB5+ dermal mesenchymal stem cells (ABCB5+ MSCs) possess immunomodulatory, inflammation-dampening, and tissue-healing capacities. In a Col7a1-/- mouse model of RDEB, treatment with ABCB5+ MSCs markedly extended the animals' lifespans.METHODSIn this international, multicentric, single-arm, phase I/IIa clinical trial, 16 patients (aged 4-36 years) enrolled into 4 age cohorts received 3 i.v. infusions of 2 × 106 ABCB5+ MSCs/kg on days 0, 17, and 35. Patients were followed up for 12 weeks regarding efficacy and 12 months regarding safety.RESULTSAt 12 weeks, statistically significant median (IQR) reductions in the Epidermolysis Bullosa Disease Activity and Scarring Index activity (EBDASI activity) score of 13.0% (2.9%-30%; P = 0.049) and the Instrument for Scoring Clinical Outcome of Research for Epidermolysis Bullosa clinician (iscorEB­c) score of 18.2% (1.9%-39.8%; P = 0.037) were observed. Reductions in itch and pain numerical rating scale scores were greatest on day 35, amounting to 37.5% (0.0%-42.9%; P = 0.033) and 25.0% (-8.4% to 46.4%; P = 0.168), respectively. Three adverse events were considered related to the cell product: 1 mild lymphadenopathy and 2 hypersensitivity reactions. The latter 2 were serious but resolved without sequelae shortly after withdrawal of treatment.CONCLUSIONThis trial demonstrates good tolerability, manageable safety, and potential efficacy of i.v. ABCB5+ MSCs as a readily available disease-modifying therapy for RDEB and provides a rationale for further clinical evaluation.TRIAL REGISTRATIONClinicaltrials.gov NCT03529877; EudraCT 2018-001009-98.FUNDINGThe trial was sponsored by RHEACELL GmbH & Co. KG. Contributions by NYF and MHF to this work were supported by the NIH/National Eye Institute (NEI) grants RO1EY025794 and R24EY028767.

Process development and safety evaluation of ABCB5+ limbal stem cells as advanced-therapy medicinal product to treat limbal stem cell deficiency.

BACKGROUND: While therapeutic success of the limbal tissue or cell transplantation to treat severe cases of limbal stem cell (LSC) deficiency (LSCD) strongly depends on the percentage of LSCs within the transplanted cells, prospective LSC enrichment has been hampered by the intranuclear localization of the previously reported LSC marker p63. The recent identification of the ATP-binding cassette transporter ABCB5 as a plasma membrane-spanning marker of LSCs that are capable of restoring the cornea and the development of an antibody directed against an extracellular loop of the ABCB5 molecule stimulated us to develop a novel treatment strategy based on the utilization of in vitro expanded allogeneic ABCB5+ LSCs derived from human cadaveric limbal tissue. METHODS: We developed and validated a Good Manufacturing Practice- and European Pharmacopeia-conform production and quality-control process, by which ABCB5+ LSCs are derived from human corneal rims, expanded ex vivo, isolated as homogenous cell population, and manufactured as an advanced-therapy medicinal product (ATMP). This product was tested in a preclinical study program investigating the cells' engraftment potential, biodistribution behavior, and safety. RESULTS: ABCB5+ LSCs were reliably expanded and manufactured as an ATMP that contains comparably high percentages of cells expressing transcription factors critical for LSC stemness maintenance (p63) and corneal epithelial differentiation (PAX6). Preclinical studies confirmed local engraftment potential of the cells and gave no signals of toxicity and tumorgenicity. These findings were sufficient for the product to be approved by the German Paul Ehrlich Institute and the U.S. Food & Drug Administration to be tested in an international multicenter phase I/IIa clinical trial (NCT03549299) to evaluate the safety and therapeutic efficacy in patients with LSCD. CONCLUSION: Building upon these data in conjunction with the previously shown cornea-restoring capacity of human ABCB5+ LSCs in animal models of LSCD, we provide an advanced allogeneic LSC-based treatment strategy that shows promise for replenishment of the patient's LSC pool, recreation of a functional barrier against invading conjunctival cells and restoration of a transparent, avascular cornea.

Ex vivo-expanded highly pure ABCB5+ mesenchymal stromal cells as Good Manufacturing Practice-compliant autologous advanced therapy medicinal product for clinical use: process validation and first in-human data.

BACKGROUND AIM: Mesenchymal stromal cells (MSCs) hold promise for the treatment of tissue damage and injury. However, MSCs comprise multiple subpopulations with diverse properties, which could explain inconsistent therapeutic outcomes seen among therapeutic attempts. Recently, the adenosine triphosphate-binding cassette transporter ABCB5 has been shown to identify a novel dermal immunomodulatory MSC subpopulation. METHODS: The authors have established a validated Good Manufacturing Practice (GMP)-compliant expansion and manufacturing process by which ABCB5+ MSCs can be isolated from skin tissue and processed to generate a highly functional homogeneous cell population manufactured as an advanced therapy medicinal product (ATMP). This product has been approved by the German competent regulatory authority to be tested in a clinical trial to treat therapy-resistant chronic venous ulcers. RESULTS: As of now, 12 wounds in nine patients have been treated with 5 × 105 autologous ABCB5+ MSCs per cm2 wound area, eliciting a median wound size reduction of 63% (range, 32-100%) at 12 weeks and early relief of pain. CONCLUSIONS: The authors describe here their GMP- and European Pharmacopoeia-compliant production and quality control process, report on a pre-clinical dose selection study and present the first in-human results. Together, these data substantiate the idea that ABCB5+ MSCs manufactured as ATMPs could deliver a clinically relevant wound closure strategy for patients with chronic therapy-resistant wounds.

Process data of allogeneic ex vivo-expanded ABCB5+ mesenchymal stromal cells for human use: off-the-shelf GMP-manufactured donor-independent ATMP.

BACKGROUND: Human dermal mesenchymal stromal cells (MSCs) expressing the ATP-binding cassette (ABC) efflux transporter ABCB5 represent an easily accessible MSC population that, based on preclinical and first-in-human data, holds significant promise to treat a broad spectrum of conditions associated not only with skin-related but also systemic inflammatory and/or degenerative processes. METHODS: We have developed a validated Good Manufacturing Practice-compliant expansion and manufacturing process by which ABCB5+ MSCs derived from surgical discard skin tissues are processed to an advanced-therapy medicinal product (ATMP) for clinical use. Enrichment for ABCB5+ MSCs is achieved in a three-step process involving plastic adherence selection, expansion in a highly efficient MSC-selecting medium, and immunomagnetic isolation of the ABCB5+ cells from the mixed culture. RESULTS: Product Quality Review data covering 324 cell expansions, 728 ABCB5+ MSC isolations, 66 ABCB5+ MSC batches, and 85 final drug products reveal high process robustness and reproducible, reliable quality of the manufactured cell therapy product. CONCLUSION: We have successfully established an expansion and manufacturing process that enables the generation of homogenous ABCB5+ MSC populations of proven biological activity manufactured as a standardized, donor-independent, highly pure, and highly functional off-the-shelf available ATMP, which is currently tested in multiple clinical trials.

In vivo safety profile and biodistribution of GMP-manufactured human skin-derived ABCB5-positive mesenchymal stromal cells for use in clinical trials.

BACKGROUND AIMS: Human dermal ABCB5-expressing mesenchymal stromal cells (ABCB5+ MSCs) represent a promising candidate for stem cell-based therapy of various currently uncurable diseases in several fields of regenerative medicine. We have developed and validated a method to isolate, from human skin samples, and expand ABCB5+ MSCs that meet the guideline criteria of the International Society for Cellular Therapy. We are able to process these cells into a Good Manufacturing Practice-conforming, MSC-based advanced-therapy medicinal product. METHODS: To support the development of ABCB5+ MSCs for potential therapeutic topical, intramuscular and intravenous administration, we have tested our product in a series of Good Laboratory Practice-compliant nonclinical in-vivo studies addressing all relevant aspects of biosafety, including potential long-term persistence and proliferation, distribution to nontarget tissues, differentiation into undesired cell types, ectopic tissue formation, tumor formation and local tissue reaction. RESULTS: (i) Subcutaneous application of 1 × 107 ABCB5+ MSCs/animal and intravenous application of 2 × 106 ABCB5+ MSCs/animal, respectively, to immunocompromised mice did not result in safety-relevant biodistribution, persistence or proliferation of the cells; (ii) three monthly subcutaneous injections of ABCB5+ MSCs at doses ranging from 1 × 105 to 1 × 107 cells/animal and three biweekly intravenous injections of 2 × 106 ABCB5+ MSCs/animal, respectively, to immunocompromised mice were nontoxic and revealed no tumorigenic potential; and (iii) intramuscular injection of 5 × 106 ABCB5+ MSCs/animal to immunocompromised mice was locally well tolerated. DISCUSSION: The present preclinical in vivo data demonstrate the local and systemic safety and tolerability of a novel advanced-therapy medicinal product based on human skin-derived ABCB5+ MSCs.

Secretion from acinar cells of the exocrine pancreas: role of enteropancreatic reflexes and cholecystokinin.

Although the molecular machinery and mechanism of cell secretion in acinar cells of the exocrine pancreas is well documented and clear, only recently has the pharmacophysiology of pancreatic exocrine secretion come to light. Therefore, we focus in this article on the current understanding of the pharmacophysiology of pancreatic exocrine secretion. The pancreatic secretory response to ingestion of a meal is mediated via a complex interplay of neural, humoral and paracrine mediators. A major role in the control of the intestinal phase of pancreatic secretion is attributed to vago-vagal enteropancreatic reflexes. In the scheme of this control mechanism, afferents originating in the duodenal mucosa, and efferents mediating central input on the pancreatic ganglia, activate intrapancreatic postganglionic neurons. Experiments utilizing specific receptor antagonists demonstrate the involvement of both muscarinic M1 and M3 receptors expressed in pancreatic acinar cells. Cholecystokinin (CCK), originally implicated in the humoral secretion of pancreatic enzymes, through a direct action on acinar CCK receptors, is also essential to the enteropancreatic reflex mechanism. CCK stimulation of the exocrine pancreatic secretion through excitation of sensory afferents of the enteropancreatic reflexes, is a paracrine mode of CCK action, and is probably the only one in humans and the predominant one in rats. In dogs, however, CCK acts on the pancreas via both the humoral and a paracrine route. More recent experiments suggest further possible sites of CCK action. Additionally, at the brain stem, vago-vagal enteropancreatic reflexes may be modulated by input from higher brain centres, particularly the hypothalamic-cholinergic system in the tonic stimulation of preganglionic neurons of the dorsal motor nucleus of the vagus projecting into the pancreas.

Enteropancreatic reflexes mediating the pancreatic enzyme response to nutrients.

The observation that in dogs electrical stimulation of the vagus nerve elicited a strong secretory activity of the pancreas, prompted I. P. Pavlov in 1888 to conclude that the pancreatic secretory response to nutrients is mediated by enteropancreatic reflexes involving the vagus nerves. It took, however, more than 90 years until by studying the latency of pancreatic amylase response to exogenous and endogenous stimuli for the first time experimental evidence was provided for the actual existence of cholinergic vago-vagal enteropancreatic reflexes. Follow-up studies, based on stepwise extrinsic denervation of the pancreas, ruled out possible splanchnic pathways for enteropancreatic reflexes. In more recent years, experiments utilizing specific antagonists demonstrated a physiological role for both cholinergic M1 and cholecystokinin (CCK) receptors within the enteropancreatic reflex. At least a significant portion of the cholinergic fibres of the enteropancreatic reflex end on muscarinic receptors of the subtype M1. CCK, the most important hormone stimulating pancreatic enzyme secretion, appears to act at least in part on CCK receptors located on vagal afferent nerves, which in turn elicit a vago-vagal reflex, implying that CCK exerts its effect on the pancreas at least in part through vago-vagal reflexes. Furthermore, pharmacological blockade of CCK receptors totally abolished the early pancreatic amylase response to intestinal nutrients, suggesting that the activation of (probably vagal) CCK receptors is essential to run the enteropancreatic reflex.

Control of pancreatic exocrine secretion via muscarinic receptors: which subtype(s) are involved? A review.

The present report gives an overview of the experimental, pharmacological and molecular investigations that have been undertaken during the past two decades to characterize and identify the muscarinic receptor subtype(s) involved in the cholinergic control of pancreatic exocrine secretion in humans and different animal species. The results published in the literature clearly indicate that both M1 and M3 receptors contribute to the regulation of pancreatic enzyme secretion, although contradictory conclusions have been drawn from secretory studies using specific M1 and M3 receptor antagonists in vivo and in vitro. Binding studies using specific M1 and M3 receptor antagonists have supported the existence of both M1 and M3 receptors on pancreatic acinar cells, which was confirmed by the demonstration of specific mRNA for both receptor subtypes in rat pancreatic acinar cells. In addition, experimental evidence exists that nonacinar (possibly presynaptic) M1 receptors also contribute to the control of pancreatic enzyme secretion. The role of the different muscarinic receptor subtypes in the control of pancreatic fluid and bicarbonate output, however, still needs to be clarified. Future research should cover the evaluation of the relative contribution of the different receptor subtypes to the regulation of pancreatic exocrine function, the localization of the receptors involved as well as possible species differences.