S.G. Ayre. Medical Hypotheses 29:283-291, 1989
The Human Immunodeficiency Virus (HIV) is the causative agent of AIDS and this has been found to be neurotropic. For this reason the development of an effective strategy for the delivery of antiviral drugs across the blood-brain barrier is of paramount importance in the treatment of HIV infection. There are insulin receptors on the capillary endothelial cells making up the blood-brain barrier (BBB) and it is proposed that these may play a role, along with exogenously administered insulin, in enhancing the transport of drug molecules across the BBB. Evidence is presented showing that insulin may be used as a pharmacologic adjunct in the therapy of HIV infection by allowing for higher concentrations of antiviral drugs to be obtained within the CNS using lower total doses of drug. This would enhance the drug’s therapeutic effectiveness while simultaneously obviating potential dose-related side-effects.
The search for more effective therapies to treat disease has traditionally focused on the development of new drugs. Newer approaches are looking instead for ways of altering the pharmacokinetics of established drugs, particularly with respect to improving the ability of these to penetrate cell membranes at the end-organ/tissue level. The blood-brain barrier (BBB) is a special conformation of cellular membranes that has at times served to confound effective drug therapy for intra-CNS conditions, most notably tertiary neurosyphilis. Presently, the BBB is once again posing a problem with AIDS and HIV infection of the CNS. Because of the integral role played by intra-CNS HIV infection in the pathogenesis of AIDS, and because of the magnitude of the threatened epidemic with the condition, the development of an effective strategy for overcoming the therapeutic impasse of the BBB has become one of the paramount concerns of contemporary medical science.
Physiologically, the cell membranes of the body’s tissues possess a variety of mechanisms through which substances on one side of a biomembrane may be selectively transported across to the other side. The evolution of these mechanisms has given tissues an intelligence with which to maintain their integrity and their autonomy, ordering and separating cellular contents from the surroundings. It must be acknowledged that much of allopathic pharmacology has evolved in concert with the growth of our understanding in the field of physiology. With our pressing need to find ways for drugs to cross the blood-brain barrier; we would do well once again to follow the lead of physiology and take a good look at some of these intelligently contrived mechanisms of membrane transport.
Insulin transport through biomembranes
The hormone insulin is recognized as having actions that affect the trans-membrane transport of different substances, particularly glucose, into numerous different kinds of cells. Insulin is a large polypeptide molecule with a molecular weight of 5808. It consists of an A chain and a B chain, connected together by two disulfide bridges. The hormone is made in the beta cells of the pancreas, and the stimulus for its secretion into the blood stream is a rise in the blood glucose concentration. Its actions on liver, adipose tissue, and skeletal muscle have all been studied in great detail, and it is now recognized that insulin also affects a wide variety of tissues in addition to just these three. (1)
Apart from the membrane transport of glucose, insulin also regulates the transport of some amino acids, some fatty acids, potassium, magnesium, and certain other monosaccharides. Furthermore, it mediates the formation of macromolecules in cells which are used in cell structure, energy stores, and regulation of many cell functions. It stimulates glycogenolysis, lipogenesis, proteogenesis, and nucleic acid synthesis. It also increases glucose oxidation and magnesium-activated sodium-potassium ATPase activity. (1)
There is a single mechanism involved in the initiation of all these biological effects, and this is the interaction of the hormone with its specific receptor. The insulin receptor consists of two alpha subunits (Mr 135 000) and two beta subunits (Mr 95 000) which are linked together by disulfide bonds. The alpha unit is predominantly located on the outer surface of the cell membrane, and the insulin binding domain is located here. The transmembrane beta subunit contains tyrosine kinase activity on its cytoplasmic domain that results in rapid receptor autophosphorylation. Activation of the kinase towards exogenous substrates is apparently preceeded by this insulin-dependent autophosphorylation reaction of the beta subunit. Action on other cellular substrates ultimately leads to the expression of the full range of insulin actions at the cellular level. (2)
After insulin binds to the receptor with activation of the kinase, followed by receptor autophosphorylation, the insulin-receptor complex is endocytosed into The cell cytoplasm. This phenomenon accounts for the down-regulation of insulin receptor activity that ensues following insulin stimulation. With this endocytosis, a variety of events may then take place. Insulin dissociates from the receptor and, following fusion of the endocytotic vessicle with cellular lysosomes, it is degraded by lysosomal enzymes. The free receptor may itself be degraded by the lysosomal enzymes, or it may recycle back to the surface of the cell membrane. Finally, the free phosphorylated receptor may proceed to activate other substrates in the cytoplasm or within cellular organelles (Golgi apparatus, nucleus, etc?) to produce the plethora of changes described above. (3)
The most commonly recognized action of insulin is that of lowering blood glucose. This is accomplished via a process of facilitated diffusion across cell membranes. It is hypothesized that the mechanism of this facilitated diffusion involves the translocation of a glucose transport protein from the cytoplasm out to the cell membrane. This translocation process involves the fusion of intracytoplasmic vesicles with the membrane of the cell. These vesicles contain the glucose transport protein in their enclosing membranes. Once exteriorized on the cell surface, the transport proteins serve as channels for glucose to enter the cell. This particular protein has been identified as a 40 000 molecular weight moiety (4) found by centrifugation to be associated with the Golgi rich fraction. The process of translocation is reversible via endocytosis of the membrane fragment containing the transport proteins, reconstituting the intracytoplasmic vessicles. The whole activity of the glucose transport protein is dependent on metabolic energy, and independent of protein synthesis. (5) The precise nature of the signal through which insulin turns this process on and off remains to be elucidated.
Insulin receptors are widely distributed in mammalian organisms with there being from 100 to 100 000 receptors per cell in different tissues. Rarely are there any cells having no receptors at all. (6) A number of malignant neoplastic tissues have also been found to have a plentiful supply of insulin receptors, (7—9) perhaps reflecting cancer cell metabolism and the need that malignant cells have for glucose. Insulin may also play a role here in the stimulation of cancer cell growth, (10, 11) and a number of different cancers have been found to actually produce and secrete their own insulin. (12—19) Investigation of many of the actions of insulin on insulin receptors in numerous species have demonstrated that the properties of insulin receptors in mammalian tissues are remarkably similar, irrespective of cell type. (1, 6, 20) This being so, it may be anticipated that what the activated insulin /insulin receptor complex does in one tissue, it will do in all. This would of course be dependent on there being the necessary metabolic machinery within a particular tissue to react to insulin activation. Not all tissues are similarly endowed in this regard.
Insulin and the brain
Brain is a tissue which does have insulin receptors, but which does not have the same insulin-dependent glucose transport mechanism common to many other of the body’s tissues. Insulin receptors are found both on the capillary endothelium of the BBB, as well as on the glial elements within the substance of the brain. These receptors do not seem to play any role, in conjunction with insulin, in the transmembrane transport of the glucose which is so essential for proper brain metabolism. The capillary endothelium of the BBB has its own unique transport system for glucose, as well as a number of other nutrient transport systems for substances such as choline, adenine, adenosine, lactate, glutamate, phenylalanine, and arginine. (21) The composition of the scant interstitial fluid of the brain is carefully controlled by the very selective functioning of the BBB. Having access to this space, across the BBB, substances then have free access to the brain cells.
The glucose transport system in brain responds to chronic changes in blood glucose levels, and there is some interesting clinical correlation for this. The system is up-regulated during prolonged periods of hypoglycemia (22) which can explain why some patients with chronic hypoglycemia or insulinomas may not have symptoms of brain glucopenia at blood glucose concentrations of less than 50 mg%. In a like fashion, the brain glucose transport system is down-regulated during prolonged periods of hyperglycemia such as can occur with poorly controlled diabetes. (23) When such patients are brought under rapid control with insulin therapy, because of this down-regulation of the BBB glucose transporter, they may develop symptoms of hypoglycemia even though the blood sugar level is in the normal range. (24)
Glucose transport across the EBB is insulin-independent, and yet insulin receptors are found on the same BBB capillary endothelium which carries the glucose transport system. This insulin transport system is just one of a number of peptide transport systems found on the BBB. Others carry the insulin-like growth factors I and II, and transferrin. (21) The blood-brain barrier insulin receptor is a glycoprotein having structural characteristics typical of the insulin receptor in peripheral tissues. It may be part of a combined endocytosis-exocytosis (transcytosis) system for the transport of the peptide through the BBB in man. A transcytosis of insulin through the human BBB would allow for distribution of circulating insulin into brain interstitial space and insulin action on brain cells. (25)
The role of insulin in the regulation of brain function continues to be a major unsolved problem in insulin physiology. Evidence to date shows that it seems to be primarily involved with brain growth and development, and this seems to be more important in the newborn mammalian brain. (26) Research continues in efforts to elucidate this question in its entirety. It has been through such research, looking to find the extent of insulin’s role in brain physiology, that some interesting possibilities have come to light concerning the pharmacologic applications insulin may have in clinical situations other than the management of type I diabetes mellitus. Here again, our understanding of physiology has come to shed new light on heretofore unrecognized possibilities for the pharmacologic management of disease.
Insulin as a pharmacologic adjunct
It is entirely possible for insulin to have applications in the medical management of diseases apart from just diabetes mellitus. In tissues possessing insulin receptors, such as brain, insulin can potentiate the pharmacologic actions of drugs that may be administered in conjunction with insulin and a hypertonic glucose solution. The drug potentiation is a function of the increased intracellular concentration of the drug that can be obtained due to the synergistic action of insulin on target cell membranes. (In brain, this means the membranes of cells comprising the BBB). With this use of insulin in the non-diabetic context, the hypoglycemia produced is considered a side effect of therapy, and this is managed by the concomitant administration of a hypertonic glucose solution. To date, there have been no published reports of any extensive clinical experience employing this concept in this country. However, there has been conducted a good deal of basic scientific research that has direct bearing on the hypothesis being developed here.
Specifically with respect. to brain, extensive work has been done in this area by Pardridge. He has stated, “The presence of specific peptide receptor transport systems in the blood-brain barrier suggests a new strategy for peptide delivery to brain. Coupling peptides or even enzymes to insulin results in the uptake of the chimeric peptide by cells via the insulin receptor-mediated uptake system”. (27)
Poznansky et al published a report in Science which did not deal directly with brain, but offered factual evidence concerning the potential role of insulin as a carrier for enzyme and drug therapy. In their paper they state, “insulin is shown to be effective in delivering insulin-albumin conjugates to cells and tissues rich in insulin receptors (skeletal muscle). The complex is transported into the cells by a process that resembles receptor-mediated endocytosis. The enzyme-albumin-insulin complex retains its enzymatic activity and its ability to bind antibodies to insulin”. (28) in similar published reports in the literature, there are statements to the effect that “the fragment A (of diphtheria toxin conjugated to insulin) underwent endocytosis through insulin receptors (into rat fibroblasts)”, (29) and “Insulin carries the psoralen into the (human lymphocyte) cell via (insulin) receptor-mediated endocytosis”. (30)
There is further evidence for insulin potentiation of drugs that differs somewhat from the above cited reports. This has to do with the increased uptake of free drug by cells with insulin receptors, rather than the uptake of insulin-drug chimeric complexes. The experiments supplying this additional evidence were performed using breast cancer cells in vitro. It is established that breast, colon, and melanoma cancer cells have insulin receptors on their cell membranes. (7—9 ) Interestingly, in one paper discussing breast cancer cells in this particular context, it was reported that, “Most (90%) of the tumors demonstrated significant binding of insulin, as did 80% of non-malignant tissues. Autoradiographic studies indicated that insulin bound dominantly to tumor cells rather than to fat and fibrous tissue contained within tumors”. (7) It is possible that the insulin-potentiation anticancer drug therapy might be made more concentrated and selective were this observation found to be true of malignant cells in general.
The report about insulin potentiation in breast cancer cells in vitro involved methotrexate. The work done by Alabaster et a! found that, “Insulin is shown to increase the cytotoxic effect of methotrexate up to ten thousand-fold in vitro”. (31) The authors were of the opinion that this increased cytotoxicity was due to metabolic modification by insulin of the cancer cells rendering the cancer cells more sensitive to the methotrexate. However, in a later related paper by Schilsky et al, another explanation for the observed results was put forward. These authors stated, “Insulin has significant effects on the intramembrane methotrexate transport system of MCF-7 (human breast cancer) cells. Enhanced cytotoxicity may be related to an increased capacity of the cells to accumulate free intracellular methotrexate. Insulin-induced changes in cellular lipid synthesis and perhaps in membrane lipid profile could result in changes in membrane fluidity and enhanced methotrexae transport.” (32)
As to the important question of operative mechanisms here, these foregoing statements about changes in cellular lipid synthesis, membrane lipid profile, and membrane fluidity may well prove to be the significant factors in this whole insulin-potentiation phenomenon. These matters have been fully adressed in some absorbing work by Eibl at the Max Planck Institute in Goningen, West Germany. Looking at the properties of phospholipids as functional constituents of biomembranes in quite some detail, Eibl succeeded in developing new theoretical and practical models for the synthesis of biologically active phospholipids. Manipulating the chemical structure and physical properties of phospholipids makes it possible to alter phase transitions of fluidity in the membranes that come to incorporate these compounds, and to thereby influence and control biological membrane processes. (33) Given the widespread biochemical alterations that insulin induces in cells —including phospholipid synthesis — it is possible that insulin’s drug potentiating effect on cell membranes may operate through some similar mechanism as that described by Eibl.
Animal experimentation with rats served to prove the significance of Eibl’s theoretical considerations. Commenting on this aspect of the work, the author states, “Alkyl glycerides can modify the properties of biological membranes quickly and reversibly to increase the permeation of active compounds. An important example is the improved transport of cytostatic drugs across the blood-brain barrier in the presence of 1-pentylglycerol”. (33) Apart from the successful results of this experiment which measured the improved brain-uptake-index (BUI) of these drugs when given along with 1-pentylglycerol, an important new fact may have also been unearthed here. This has to do with some conventional understanding on the passage of substances through the BBB.
In 1974, (34) Oldendorf first described what has become conventional medical wisdom, that lipophilicity is the decisive parameter determining transport through the blood-brain barrier. According to the work of Eibl however, this is only true for a limited range of molecular weights, which appears to be something less than Mr 600. This new proposition could provide an alternative interpretation for some observations reported in another paper on the “Restrictive Transport of a Lipid-Soluble Peptide (Cyclosporin) Through the Blood-Brain Barrier” by Cefalu and Pardridge. This report concludes that “the BBB transport of cyclosporin is markedly restricted owing to the combined effects of binding by serum proteins and a paradoxically low permeability of the BBB to the (highly lipid-soluble) peptide”. (35) It is interesting to note that the molecular weight of cyclosporin is 1202.
A good deal of support for the insulin-potentiation hypothesis emerges from the above collected evidence. The following is a formal statement of this.
Through some unidentified interaction with its specific receptors on brain capillary endothelial cells, insulin facilitates the passage of drug molecules across the blood-brain barrier into the substance of the brain where these molecules may have access to the brain cells. A similar interaction between insulin and receptors on other insulin-sensitive tissues in the body may likewise facilitate the trans-membrane passage of drug molecules from the extracellular to the intracellular compartment of the cells of these other tissues. By producing increased intracellular drug concentrations in this way, insulin may be used as a pharmacologic adjunct to potentiate conventional drug therapy for disease.
It is said of hypotheses that they need not necessarily be true; they need only work. Further basic scientific research, followed by placebo-controlled clinical trials, will be necessary before the truth or workability of this concept can be made evident, or the whole discarded as invalid. In the absence of any such scientific observations, all that now exists are these ideas, and the questions these may generate in the enquiring mind. The following questions are relevant to the hypothesis, and may serve to open up further avenues of questioning:
1)Is it a chimeric drug-insulin molecule which gets internalized via the membrane insulin receptor?
2) Do drugs get adsorbed onto glucose molecules and then get transported into the intracellular milieu via the glucose transport protein?
3) Is there some other insulin-dependent active transport system acting to facilitate the entry of drugs into cells?
4) Does insulin cause some change in the phospholipids and fluid characteristics of cell membranes thus facilitating passage of certain molecular weight substances through these membranes?
5) Are there some other metabolic factors stimulated by insulin within the cell cytoplasm — totally unrelated to drug-distribution kinetics — that act in some way to potentiate pharmacologic activity?
Clinical applications of insulin-potentiation in the management of HIV infection
In December 1985, there first appeared a series of articles in the New England Journal of Medicine documenting the fact that the causative organism of AIDS, the Human Immunodeficiency Virus (HIV), was neurotropic. (36, 37) Commenting on this fact in an editorial appearing in this same edition of NEJM, Dr. Paul Black articulated the major concern about this finding most incisively when he wrote, “The special features of persistent infection in the brain, coupled with the difficulty of bringing adequate concentrations of drugs to this locus across the blood-brain-barrier, will make eradication of HTLV-III (HIV) infection of the central nervous system very difficult, if not impossible”. 38 The incidence of HIV involvement of the CNS in individuals who are simply HIV positive and/or those with symptomatic HIV infection has not as yet been established. As with many of the numbers given in discussions on AIDS, what we must make do with are estimates. In this particular context, the numbers vary from a low of 30% to a high of 100% of individuals who may have CNS involvement along with their HIV seropositivity. In symptomatic patients, it seems that the latter may be the more accurate number.
The involvement of the CNS in HIV infection may be playing a devastating role in the pathogenesis of this condition. Underscoring this possibility is the following evidence from some clinical trials with suramin and the French agent, HPA-23 performed on AIDS and ARC atients at NIH. Both of these studies reported transient clearing of virus from the peripheral circulation of these patients, with a reappearance of viremia following withdrawal of the treating agents. (39, 40) Neither suramin nor HPA-23 is reported as being able to cross the blood-brain-barrier, and it is entirely possible that the HIV sequestered within the brain, behind the blood-brain barrier, was responsible for the observed reappearance of viremia in these cases. It is recognized that cells of the monocyte/macrophage line can be infected with HIV, and call act as vectors of spread to the brain. It is these same cells that may just as well serve to carry virus the other way — from the CNS to the blood. There is still much to be determined here, but there is a strong possibility that intra-CNS infection with HIV plays a significant role in the pathogenesis of progressive symptomatic disease in human subjects. It may just be responsible in large part for the resistance that AIDS has shown to definitive therapy, and insulin-potentiation of drug therapy here may just prove to be a most valuable discovery to counter this resistance.
The antiviral agent azidothymidine (AZT), now known as zidovudine (Retrovir), was found to significantly improve the clinical course of certain patients with symptomatic HIV infection. (41) Subsequent widespread use of AZT on HIV infected patients in this country has continued to bear out this initial clinical impression, although the drug is by no means the definitive answer for treating HIV infection. There is a prolongation of survival, but patients still die of their disease. The dose-related hematologic toxicity of AZT has also prohibited its continued use in a significant proportion of patients.
Much of the success of AZT has been attributed to its ability to cross the blood-brain barrier. In point of fact, the blood-brain barrier transport of AZT has been inferred from determinations of AZT levels in the cerebrospinal fluid (CSF) of human patients taking the drug. According to the available literature, (41, 42) no brain-uptake-index studies have been done on AZT to conclusively prove that all or part of the drug found in the CSF actually got there via passage through the BBB, into brain interstitial space and thence into the CSF. There is reason to argue that this assumption may not be as completely valid as it appears.
One very interesting aspect of the excellent work done on the BBB by Pardridgc has been the focus he draws on the two barrier systems that separate the brain interstitial space from the systemic intravascular space. There is the blood-brain barrier, and then there is the blood-çSF barrier, comprised of the choroid plexus and several other related structures, together referred to as the circumventricular organs (CVO). (27) The relative capillary surface area of the two is 5000 to 1, with the BBB having the much larger proportion. The BBB capillaries are closed, having tight junctions and scant pinocytosis, while the capillaries of the CVO’s on the other hand are relatively porous, with fenestrations and active pinocytosis. There would thus be much less impedance to the passage of material from the blood into the CSF via the blood-CSF barrier than there would be through the BBB. In fact, about half of the CSF fluid made per day arises from the CVO’s, and the other half arises from the endothelia comprising the BBB. That amount of substance getting into the CSF via the blood-CSF route would have access to only that portion of the brain substance in direct contact with the CSF.
Because of these distinctions, it is not accurate to infer that the finding of some substance in the CSF is an indication that that substance has crossed the BBB, nor that the substance, if it is a drug, has thus had access to the brain cells to act therapeutically. It is possible that some proportion of the reported concentrations of AZT in the CSF may indeed reflect drug passage through the blood-CSF barrier, rather than the more clinically significant blood-brain barrier passage. From this different perspective, it is compelling to consider what impact insulin-potentiation might have on the actual intra-CNS concentration of a drug like AZT, and what kind of a differential this might produce as far as clinical results are concerned were it used this way in AIDS treatment.
There are clinical precedents for using insulin in the manner being proposed in this present discussion. The clinical work in question was not done in this country, but in Mexico, and a description of the insulin-potentiation concept based on this work appeared in the literature in June of 1986. (43) From an historical perspective, it is interesting to note that the first documented applications of insulin-potentiation therapy (IPT) involved the treatment of another renowned sexually transmitted disease with CNS involvement, this being syphilis. This work was done more than fifty years ago.
Prior to the clinical work here, animal experimentation had demonstrated the greatly enhanced uptake of mercury and arsenic salts by the brains of a group of dogs under the influence of insulin, as compared to their non-insulin-treated controls.
(44) Mercury and arsenic were conventional antisyphilitic treatment in this era before the advent of antibiotics. During the summer of 1937, a clinical study was undertaken at the Austin State Hospital in Austin, Texas, to evaluate the treatment of neurosyphilis using mercury and arsenic salts in conjunction with insulin. Ten patients with serologic and clinical evidence of tertiary neurosyphilis were treated. Nine of these ten patients completed the course of treatment, and six out of these nine experienced reversals of the Wasserman and Lange’s colloidal gold reactions. These six patients also experienced complete clinical recoveries from their illness. The rest of the patients experienced partial improvement in clinical status.
In 1985, the understanding of HIV infection became significantly altered with the discovery that its causative organism was neurotropic. From the unique perspective of IPT, the similarities in pathogenesis between AIDS and neurosyphilis were seen to be striking. In November of 1986, the first opportunity of treating a severely ill AIDS patient (CDC Group IV-A) with IPT presented itself. Within three weeks of treatment in Mexico City, the patient was in clinical remission, able to resume his normal activities of daily living. He has remained well up to this present time, almost two years after initiation of treatment. The primary treating agent in this case was ribavirin (Vilona,
Clinical trials with ribavirin have been designed and carried out in the United States. A number of these studies are continuing at present. No comment is being made here on the safety and/or efficacy of this agent as used in these trials, however it would seem acceptable to acknowledge that no breakthrough has been achieved here. The oral dosages of ribavirin given in these trials are either 600 mg b.i.d. or 800 mg b.i.d. This makes for total weekly doses of 8400 mg or 11200 mg of drug, respectively. By way of indicating what value IPT may have had in these two anecdotal cases mentioned above, these patients both received 30 mg of ribavirin intravenously on the day of the insulin treatment, which is customarily given once a week, and then 200 mg q.d. orally on the other days of the week. The total weekly dose here amounts to 1230 mg.
It is reiterated here that these descriptive accounts are not meant to be construed as commentary on the safety and/or efficacy of ribavirin in the treatment of HIV infection. These accounts are only intended to demonstrate the potential value of’ IPT as a pharmacologic modality that may be used to potentiate the actions of other drugs. As an aside it is interesting to note that therapeutic levels of ribavirin within the CSF are reported to occur only after 4 to 7 weeks of chronic oral therapy with the drug. (45) The patients mentioned above however showed a response within the first week after treatment, and were in clinical remission after only three weeks of treatment.
The failure of medical management in HIV infection rests in large part on the fact that the virus gets into the brain and is there protected against pharmacologic attack by the BBB. Published reports on the distribution of antiviral agents within the substance of the brain fail to give complete consideration to the anatomy of the brain, its connections with the circulatory system, and the synthesis of the CSF. It is possible that insulin may truly enhance the BBB passage of antiviral agents into the substance of the brain, and produce a significantly improved clinical response in the treatment of symptomatic HIV infection.
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